Research ArticleNeem (Azadirachta indica A Juss) Oil to TackleEnteropathogenic Escherichia coli

Paola Del Serrone1 Chiara Toniolo2 and Marcello Nicoletti2

1Agricultural Research Council (CRA) Animal Production Research Centre (CRA PCM) Via Salaria 31 Km 2670000015 Monterotondo Italy2Department of Environmental Biology University of Rome Sapienza Piazzale Aldo Moro 5 00185 Rome Italy

Correspondence should be addressed to Paola Del Serrone paoladelserroneentecrait

Received 12 July 2014 Revised 22 September 2014 Accepted 7 October 2014

Academic Editor Madhab K Chattopadhyay

Copyright copy 2015 Paola Del Serrone et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Neem (Azadirachta indicaA Juss) oil (NO) was assayed against forty-eight isolates of Escherichia coli by standardised disc diffusiontest and microdilution test By molecular biology characterization fourteen isolates resulted in diarrheagenic E coli with sixteenprimer pairs that specifically amplify unique sequences of virulence genes and of 16S rRNA The NO showed biological activityagainst all isolates The bacterial growth inhibition zone by disc diffusion method (100120583L NO) ranged between 950 plusmn 070 and3000 plusmn 100mmThe antibacterial activity was furthermore determined at lower NO concentrations (1 10ndash1 10000) The percentof growth reduction ranged between 2371plusmn 100 and 9970plusmn 153Thehighest bacterial growth reductionwas 1 10NOconcentrationwith 50 120583L of bacterial suspension (ca 1 times 106 CFUmL) There is significant difference between the antibacterial activities againstpathogenic and nonpathogenic E coli as well as NO and ciprofloxacin activities Viable cells after the different NO concentrationtreatments were checked by molecular biology assay using PMA dye On the basis of the obtained results NO counteracts E coliand also influences the virulence of E coli viable cells after NO treatment The NO metabolomic composition was obtained usingfingerprint HPTLC

1 Introduction

Zoonotic food- and waterborne pathogens began resistantto antibiotics It is now evident that antimicrobial resistanceis an environmental problem Detectable antibiotic residuesare present in waste water from water treatment plants [1]and antibiotic-resistant bacteria can be isolated from groundwater and soil [2 3]The cause of contaminationmay be interalia the consequence of farming practices Use of antibioticsas growth promoters or for prophylaxis in farm animalsselects resistant strains of enterobacteria in gastrointestinaltract These resistant strains have been also isolated fromfood and consequently this represents themain way to spreadin the human gastrointestinal tract [4 5] The increasingincidence of foodborne diseases coupled with the resultantsocial and economic implications causes a constant strivingto produce safer feed and food as to develop new naturalantimicrobial agents [6ndash8]

Meat contamination by pathogen bacteria may have greathealth consequence and high impact on consumers Themost known cases are related to HUS hemolytic uremicsyndrome that was first recognized in 1982 in USA andCanada with outbreaks associated with fast food restaurantsPeople experienced gastroenteritis with bloody diarrhoeacaused by the lining of their microbiota In 1993 a multistateoutbreak generated international interest in this diseasepopularized by the name ldquohamburger diseaserdquo Hamburgerdisease is based on association with the consumption ofground beef patties containing a pathogen Escherichia coliThis should not be confused with the related benign E colithat is in the gut of every mammal Many strains of E coliare part of the nonpathogenic facultative flora of intestinaltract of humans and other mammals However some of theminduce diseases of the gastrointestinal and urinary tracts ormay affect the central nervous system [9]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 343610 10 pageshttpdxdoiorg1011552015343610

2 BioMed Research International

On the basis of their pathogenetic mechanism diar-rheagenic E coli strains include ETEC (enterotoxigenic Ecoli) EIEC (enteroinvasive E coli) EHEC (enterohemor-rhagic E coli) EPEC (enteropathogenic E coli) EAEC(enteroaggregative E coli) and DAEC (diffusely adherentE coli) All of them cause serious economic losses infarm animal herds and are widespread in newborns [10] indeveloped and developing countries There is a wide rangeof transmission possibilities of these pathogens includingdirect contact food drinks environment and others [11]Epidemiology and clinical symptoms of the disease aresimilar in various animal species but the majority of strainsare species-specific They differ particularly in the type ofthe expressed surface ldquoadherencerdquo antigen (adhesin or pilus)These microorganisms produce two main types of virulencefactors that is adhesins and enterotoxins

In this work a collection of E coli isolates was consideredThey were different in geographical origin and source ofisolation and showed different pathogenetic characteristics

Consumers look for meat products of upgraded sensoryquality and increased functional and nutritional propertiesas well as guaranteed safety but yet less processing and feweradditives or ldquotechnologicalrdquo interventions Plant derivedextracts or phytocomplex as effective antimicrobial agentsoffer an alternative to synthetic food additives

Neem (Azadirachta indica A Juss) is considered one ofthe most promising trees of the 21st century for its greatpotential in pest management environment protection andmedicine [12] Neem oil (NO) is the most important derivedproduct with a great market worldwide It contains about onehundred biologically active compounds The most famousconstituents are several nortriterpenes named limonoidsthat is azadirachtin nimbin nimbidin and nimbolidebesides the predominant oily constituents NO is the mostcommercially relevant product obtained from the seeds Theneem cake is remaining after the extraction process

In our previous studies the antibacterial activity of NOagainst E coli was investigated [13 14] The antibacterialactivity resulted highest in comparison to the neem cakeextract against meat spoilage microorganisms The aim ofthe present work was to evaluate NO capability to cope withplastic genome of E coli

2 Materials and Methods

21 Bacterial Strains and Growth Conditions Forty-eightstrains of E coli were considered Among them seventeen(FLC isolates) were from microorganismrsquos collection of theFodder andDairy ProductionsResearchCentre of Lodi (CRAFLC) of CRA They were isolated from milk and cheese Allstrains were typed both phenotypically and genotypicallyPhenotyping was made by the PhenePlate system for E coli(PhP-EC PhPPlate Microplate Techniques AB StockholmSweden) and genetic characterization by RAPD PCR tech-nique [15]

Seventeen CVVI isolates were from microorganismrsquoscollection of the Institute of Veterinary Research and Devel-opment of Central Vietnam VietnamThesemicroorganismswere isolated from faeces of calves affected by diarrhoea

Ten NL isolates were from microorganismrsquos collection ofthe Department of Bacteriology of Wageningen UR Live-stock Research Wageningen University amp Research CentreNetherlands They were isolated from faeces of piglets andcalves They are antigenically different and detectable usingspecific monoclonal antibodies towards different fimbriaantigens by in vitro agglutination test [16] Four referencestrains were also considered (DSMZ and ATCC isolates)They were from international culture collections

The cultivationassay medium for E coli was Minca +1 Iso Vitalex AgarBroth (Sifin Berlin Germany) Bacterialcultures for antibacterial testing were prepared by pickingcolony from 24-hour-old plates and suspending them in thebroth medium (5mL) Cultures were grown aerobically for18 h at 37∘C and 100 rpm For antibacterial activity assay 1mLof each culture was diluted to 105ndash106 CFUmLThe referencestrains were grown on media and at the growth conditions asreported on products sheets

22 Plant Extract A commercial neem oil produced byNeem Italia (Manerba (BS) Italy) was used as test startingmaterial (035 azadirachtin A) Total composition of theneem oil was checked by high performance thin layer chro-matography [17]

Neem oil was diluted in Tween 80 (1 1 VV VWR PBIInternational MI Italy) under agitation and sterilised byfiltration through a 022120583m Millipore express filter (Millex-GP Bedford OH USA) before use in the experiment

23 HPTLC Assay

231 HPTLC System and Materials The HPTLC system(CAMAG Muttenz Switzerland) consisted of (i) Linomat5 sample applicator using 100120583L syringes connected to anitrogen tank (ii) ADC 2 chamber containing twin troughchamber 20times 10 cm (iii) immersion device III (iv) TLC PlateHeater III (v) TLC visualizer (vi) TLC scanner 3 linked towinCATS software

Solvents for extraction and HPLC grade solvents werepurchased from Sigma-Aldrich andCarlo Erba (Milan Italy)Glass plates 20 cm times 10 cm with glass-backed layers silicagel 60 (2 120583m thickness) were from Merck (Darmstadt Ger-many) Before use plates were prewashed with methanol anddried for 3min at 100∘C Standards used in theHPTLC analy-sis were isolated from neem cake (ie salannin azadirachtinA and unsaturated and saturated lipids) in previous research[18] and data concerning isolation and identification arenot reported but they are available per request Limonoidsstandards concentration was 2mM

24 Sample Application Filtered solutions were applied withnitrogen flow Operating conditions were syringe deliveryspeed 10 s 120583Lminus1 (100 nL sminus1) injection volume 2 120583L bandwidth 6mm distance from bottom 15mm

25 Development The HPTLC plates were developed intoluene AcOEt 7 3 (vv) as mobile phase (Figure 1) in theautomatic and reproducibly developing chamber ADC 2

BioMed Research International 3

1 2

(a)

1 2

(b)

Figure 1 HPTLC analysis of neem oil EtOAc extract Mobile phasetoluene AcOEt 7 3 (vv) Visualization plate (a) (on the left) whitelight upper and lower plate (b) (on the right) UV lamp at 366 nmDerivatization 119901-anisaldheyde Track 1 neem oil track 2 salannin

saturated with the same mobile phase for 20min at roomtemperature The developing solvents (ie type of solventsand ratios) were carefully optimized before the analyses Thelength of the chromatogram run was 80mm from the pointof application The developed layers were allowed to dry inair for 5min derivatized with a selected solution including119901-anisaldheyde (15mL 119901-anisaldheyde 25mL H

2SO4 and

1mL AcOH in 37mL EtOH) dried in the open air andthen dipped into Macrogol reagent (1 g polyethylene glycol400 in 20mL of dichloromethane) Finally the plates werewarmed for 5min at 120∘C before inspection All treatedplates were inspected by a CAMAG TLC visualizer undera UV light at 254 or 366 nm or under reflectance andtransmissionwhite light (WRT) respectively before and afterderivatization

26 Molecular Biology Characterization of the E coli IsolatesTwo primer pairs that amplify specific E coli 16S rRNAsequences and fourteen primer pairs that specifically amplifytarget gene coding for virulence factors (adhesins and toxins)were employed to characterize the E coli isolates consideredin this study (Table 1) The PCR reaction mixtures andconditions are those as reported in the literature (Table 1)

The amplification productsrsquo sizes coordinates and acces-sion numbers of each primer pair are shown in Table 2Amplified products (7120583L) were analyzed by electrophoresisin 2 or 3 agarose gels buffered in 05x TBE (TBE buffer90mM tris(hydroxymethyl)aminomethane 90mM boricacid and 3mM ethylenediaminetetraacetate Na salt pH 83Sigma-AldrichMilano Italy) against a 50 bp 100 bp and 1 Kbladder used as size marker (Invitrogen Milano Italia) andvisualized by UV light at 260 nm (Fotodine 3-3102 Celbio

Milano Italy) after staining with ethidium bromide (38-diamino-5-ethyl-6-phenylphenanthridinium bromide EtBrSigma-Aldrich Milano Italy)

27 Assessment of Antibacterial Activity The antibacterialactivity of the NO was assayed using standardized discdiffusion agar and microdilution methods Disc diffusionmethod was carried out according to the standardmethod byBauer et al [19] Bacteria cultures adjusted to 05 McFarlandstandard were used to lawn Muller Hinton agar plates evenlyusing a sterile swabThe agar plates were dried for 15minutesThe discs impregnated with NO (100 120583L) were placed on theagar surface Each test plate comprises three discs The discswere placed equidistant to each other Muller Hinton agarplates were set also up with positive control which is theantibiotic ciprofloxacin (CFX) (100 120583Lwtv) (hydrochloridemonohydrate 1mgmL Bayer Milano Italy) and Tween 80(TWN) (VWR International PBI Srl Milano Italy 1mgmL)as negative control The plates were then incubated at 37∘Cfor 18 h After the incubation the plates and those consideredas controls were examined for inhibition zone The inhi-bition zones were then measured using calipers and wererecordedThe plates were done in triplicate for each bacterialisolate and the experiment was performed twice The resultswere recorded as mean plusmn SD of the duplicate experimentDifferences between means of data were compared by LSDcalculated using the SAS

The antibacterial activity of NO was also evaluated usingmicrodilution method in conventional sterile polystyrenemicroplates (Corning Euroclone SpA Milan Italy) Eachwell of the microplate was filled with 100 120583L of sterile suitableliquid media for each bacterial isolate considered 50 120583L ofinoculums and amounts of extract at lower concentrations(1 10ndash1 10000) were added Control treatment without NOwas used in the experiment The microplates were incu-bated at 37∘C for 24 h Bacterial growth was determined byOD reading at 630 nm10mm pathlength with an ELISAmicroplate reader (DynatechML-3000 Pina de Ebro Spain)Bacterial cell concentration was transformed to cellsmLusing the reference curve equation

The reference curve was constructed by diluting at 1 100each bacterial isolate Counting the number of bacterial cellsof an aliquot of this dilution was done using a Neubauerchamber (Celeromics Vedano al Lambro MI Italy) Finallycell concentrations were transformed to a percentage ofbacterial inhibition The percentage of bacterial growthreduction (GR)was estimated using as reference the controltreatment (119879 = without extract) as

GR = 119862 minus 119879119862times 100 (1)

Three replicates were considered The results wererecorded as mean plusmn SD of the duplicate experiment Dif-ferences between means of data were compared by leastsignificant difference (LSD) calculated using the SAS

4 BioMed Research International

Table 1 Primer pairs used to specifically amplify target gene coding for virulence factors (1ndash9 = toxins 9ndash15 fimbriae) of E coli and 16S rRNA(16-17)

Target gene coding forvirulence factors Oligonucleotide sequences of primers Reference

(1) LT F 51015840-ATT TAC GGC GTT ACT ATC CTC-31015840R 51015840-TTT TGG TCT CGG TCA GAT ATG-31015840 [25]

(2) Sta F 51015840-TCC GTG AAA CAA CAT GAC GG-31015840R 51015840-ATA ACA TCC AGC ACA GGC AG-31015840 [26]

(3) STb F 51015840-GCC TAT GCA TCT ACA CAA TC-31015840R 51015840-TGA GAA ATG GAC AAT GTC CG-31015840 [26]

(4) Stx1all F 51015840-CGC TGA ATG TCA TTC GCT CTG C-31015840R 51015840-CGT GGT ATA GCT ACT GTC ACC-31015840 [27]

(5) Stx2all F 51015840-CTT CGG TAT CCT ATT CCC GG-31015840R 51015840-CTG CTG TGA CAG TGA CAA AAC GC-31015840 [27]

(6) Stx2e F 5-ATG AAG AAG ATG TTT ATA GCG-31015840R 51015840-TCA GTT AAA CTT CAC CTG GGC-31015840 [25]

(7) EAST1 F 51015840-CCA TCA ACA CAG TAT ATC CGA-31015840R 51015840-GGT CGC GAG TGA CGG CTT TGT-31015840 [28]

(8) eae F 51015840-GGA ACG GCA GAG GTT AAT CTGCAG-31015840R 51015840-GGC GCT CAT CAT AGT CTTTC-31015840 [27]

(9) hlyA F 51015840-AGCTGCAAGTGCGGGTCTG-31015840R 51015840-TACGGGTTATGCCTGCAAGTTCAC-31015840 [29]

(10) F4 (K88) F 51015840-GCT GCA TCT GCT GCA TCT GGTATG G-31015840R 51015840-CCA CTG AGT GCT GGTAGT TAC AGC C-31015840 [30]

(11) F5 (K99) F 51015840-TGC GAC TAC CAA TGC TTC TG-31015840R 51015840-TAT CCA CCA TTA GAC GGA GC-31015840 [26]

(12) F6 (P987) F 51015840-TCT GCT CTT AAA GCT ACT GG-31015840R 51015840-AAC TCC ACC GTT TGT ATC AG-31015840 [25]

(13) F17 F 51015840-GGG CTG ACA GAG GAG GTG GGGC-31015840R 51015840-CCC GGC GAC AAC TTC ATCACC GG-31015840 [30]

(14) F18 F 51015840-GTG AAA AGA CTA GTG TTT ATT TC-31015840R 51015840-CTT GTA AGT AAC CGC GTA AGC-31015840 [31]

(15) F41 F 51015840-GAG GGA CTT TCA TCT TTT AG-31015840R 51015840-AGT CCA TTC CAT TTA TAG GC-31015840 [26]

(16) E16SI F 51015840-CCCCCTGGACGAAGACTCAC-31015840R 51015840-ACCGCTGGCAACAAAGGATA -31015840 [29]

(17) E16SII F 51015840-AGAGTTTGATGGCTCAG-31015840R 51015840-GGACTACCAGGGTATCTAAT-31015840 [31]

3 Results and Discussion

31 Molecular Biology Characterization of the E coli Iso-lates The molecular biology characterization of the forty-eight E coli isolates showed that fourteen isolates werediarrheagenic E coli They were ten E coli isolated fromfeces of calves and piglets and four from calves collectedrespectively in Netherlands and Central Vietnam Theirvirulence characteristics are reported in Table 3

32 HPTLC Assay The NO metabolomic fingerprint showscharacteristic sequence ofmetabolites according to the polar-ity of constituents The identification of the raw materialwas assured by the presence of salannin (Rf = 042) whichis a typical maker of neem In comparison with the spotof azadirachtin (Rf = 023) salannin appears as the mainlimonoid spot Spots concerning lipids are present at Rf

values at ca 080 due to unsaturated fatty acids and fattyalcohols and at Rf ca 050 due to saturated and unsatu-rated triglycerides The most interesting feature of the plateconcerns the presence of compounds with high fluorescentreaction at between Rf 055 and 066 which are perfectlyvisible at 366 nm after derivatization with 119901-anisaldheydeThese spots can be attributed to compounds with highconjugated unsaturation in polycyclic aromatic structuresvery different from those of the nortriterpenes limonoids sofar considered responsible for the activity Therefore morestudies are necessary to decide about the importance ofantibacterial activity of these substances in the phytocomplex

33 NO Antibacterial Activity The results obtained showthat NO has a broad spectrum of antibacterial activitiesagainst the tested E coli isolates As shown in Table 4 theantibacterial activity was evaluated based on the diameters

BioMed Research International 5

Table 2 List of primer pairsrsquo amplification products coordinates and accession numbers

Target gene coding forvirulence factors Amplicon (bp) Primer coordinates Accession number

(1) LT 281 27ndash47 287ndash307 S60731(2) STa 244 267ndash286 492ndash510 M58746(3) STb 279 515ndash534 773ndash793 AY028790(4) Stx1all 302 113ndash134 394ndash414 M17358(5) Stx2all 516 50ndash69 543ndash565 M59432(6) Stx2e 264 1176ndash1196 1419ndash1439 M36727(7) EAST1 111 2ndash24 94ndash114 S81691(8) eae 775 1441ndash1460 2193ndash2215 AF022236(9) hylA 569 867-885 1435ndash1412 X79839(10) F4 (K88) 792 31ndash54 798ndash822 M29374(11) F5 (K99) 450 45ndash64 475ndash494 M35282(12) F6 (P987) 333 193ndash212 506ndash525 M35257(13) F17 411 289ndash310 677ndash699 AF055313(14) F18 510 1ndash23 490ndash510 M61713(15) F41 431 154ndash173 565ndash584 X14354(16) E16SI 401 1628-170 2063ndash2082 AB035924(17) E16SII 798 8-27 798ndash805 J01859

Table 3 Molecular characterisation of enteropathogenic E coli and reference strains considered in this study

E coli isolate collectionrsquos designation Surface antigen Toxins Fimbriae(1) CVVI K10B nd STb LT EAST1 F4(2) CVVI KH10 nd STa STb F18(3) NLK99 O8K85K99 nr1 F5(4) NLP987 O64 K 9877 STa+ F6(5) CVVI E12b nd STa F5 F41(6) CVVI E10 nd STa F5 F41(7) NLK99-1 O8 K25 K99 nr F5(8) NLK99-3 O101 K28 K99 nr F5(9) NLK99-5 O9 K30 K99 nr F5(10) NLK99-7 O101 K32 K99 nr F5(11) NLK99-9 O9 K35 K99 nr F5(12) NLK99-11 O9 K37 K99 nr F5(13) NLK99-15 O20 K K99 nr F5(14) NLK99-19 O101 K K99 nr F5(15) DSMZ8696 O55 H6 nr Nr(16) DSMZ9025 mdash mdash mdash(17) DSMZ10973 O6 nr nr(18) ATCC33559 mdash mdash mdashCVVI Central Vietnam Veterinary Institute NL Department of Bacteriology and Animal Science University of Wageningen Netherlands DSMZ Leibniz-Institut DSMZmdashDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ATCC American Type Culture Collection

of clear inhibition zone surrounding the paper discs soakedwith 100 120583L of neem oil The NO average GIZ mm rangefrom 950 plusmn 070 to 3000 plusmn 100 The NO GIZ varies betweenenteropathogenic and nonenteropathogenic E coli beingrespectively 2433plusmn 058ndash3000plusmn 100 and 950plusmn 070ndash2153plusmn153 It is significantly (119875 lt 005) different with respect to theantibiotic activity However the E coli isolate FLC1167 (frommilk) resulted to be less susceptible and the E coli isolate

NLP097F5 (from piglet feces) the most susceptible to NOtreatment (100 120583L) among all tested bacteria using the discdiffusion method

The CFX GIZ range is 000 plusmn 000ndash3265 plusmn 75 Theenteropathogenic E coli resulted to be resistant or lesssusceptible to CFX than the nonenteropathogenic E colishowing a GIZ range of respectively 000 plusmn 000ndash1824plusmn 168mm and 2164 plusmn 094ndash3265 plusmn 75mm The isolates

6 BioMed Research International

Table 4 Antibacterial activity of neem oil (NO) against forty-eight Escherichia coli isolates revealed as growth inhibition zone (mm)

E coli isolates Growth inhibition zone (mm)lowast

NO (100120583L ) TWN (100120583L) WTR (100120583L) CFX (100 120583L)(1) FLC 1056 1133 plusmn 058 b mdash mdash 3041 plusmn 020 a(2) FLC 1247 1613 plusmn 115 b mdash mdash 3052 plusmn 107 a(3) FLC 1059 1583 plusmn 113 b mdash mdash 2962 plusmn 100 a(4) FLC 1243 1900 plusmn 100 b mdash mdash 3153 plusmn 067 a(5) FLC 1048 1233 plusmn 058 b mdash mdash 2942 plusmn 058 a(6) FLC 1167 950 plusmn 070 b mdash mdash 3061 plusmn 121 a(7) FLC 1249 1333 plusmn 058 b mdash mdash 2961 plusmn 111 a(8) FLC 1055 1453 plusmn 125 b mdash mdash 3175 plusmn 082 a(9) FLC 1054 1623 plusmn 118 b mdash mdash 3141 plusmn 076 a(10) FLC 1085 1800 plusmn 100 b mdash mdash 3053 plusmn 117 a(11) FLC 1244 1533 plusmn 048 b mdash mdash 2886 plusmn 100 a(12) FLC 1165 1950 plusmn 070 b mdash mdash 3133 plusmn 067 a(13) FLC 1086 1133 plusmn 058 b mdash mdash 2982 plusmn 048 a(14) FLC 1053 1453 plusmn 115 b mdash mdash 3265 plusmn 139 a(15) FLC 1095 1683 plusmn 118 b mdash mdash 2905 plusmn 122 a(16) FLC 1219 1070 plusmn 100 b mdash mdash 3275 plusmn 055 a(17) FLC 1235 1323 plusmn 088 b mdash mdash 3015 plusmn 055 a(18) DSM8696 1350 plusmn 050 b mdash mdash 2621 plusmn 100 a(19) DSM9025 1333 plusmn 058 b mdash mdash 2164 plusmn 094 a(20) DSM10973 1353 plusmn 125 b mdash mdash 2914 plusmn 175 a(21) ATCC33559 1383 plusmn 118 b mdash mdash 3212 plusmn 109 a(22) CVVI E210 1300 plusmn 100 a mdash mdash 2583 plusmn 165 a(23) CVVI E173 1223 plusmn 058 b mdash mdash 3235 plusmn 149 a(24) CVVI E12b 2750 plusmn 050 b mdash mdash 1125 plusmn 068 a(25) CVVI E16 1433 plusmn 088 b mdash mdash 2754 plusmn 145 a(26) CVVI E320 2153 plusmn 135 b mdash mdash 2875 plusmn 186 a(27) CVVI E130 1183 plusmn 178 b mdash mdash 2964 plusmn 087 a(28) CVVI E48 1000 plusmn 140 a mdash mdash 2931 plusmn 027 a(29) CVVI KH10 2633 plusmn 053 b mdash mdash 1534 plusmn 066 a(30) CVVI K10B 2750 plusmn 056 b mdash mdash 0(31) CVVI E298 1133 plusmn 048 b mdash mdash 2390 plusmn 169 a(32) CVVI E273 1353 plusmn 175 b mdash mdash 2959 plusmn 177 b(33) CVVI K436 1314 plusmn 168 b mdash mdash 3021 plusmn 138 a(34) CVVI E98 1100 plusmn 100 a mdash mdash 2691 plusmn 156 a(35) CVVI E77 1433 plusmn 058 b mdash mdash 2393 plusmn 059 a(36) CVVI E148 1350 plusmn 050 b mdash mdash 2871 plusmn 087 a(37) CVVI E10 2433 plusmn 058 b mdash mdash 1035 plusmn 111 a(38) CVVI E215 1653 plusmn 115 b mdash mdash 2641 plusmn 140 a(39) NLK99F5 2983 plusmn 118 b mdash mdash 16 21 plusmn 089 a(40) NLP987F5 3000 plusmn 100 a mdash mdash 1321 plusmn 115 a(41) NLK99-1lowast 2173 plusmn 135 b mdash mdash 1534 plusmn 137 a(42) NLK99-3lowast 2833 plusmn 150 b mdash mdash 0(43) NLK99-5lowast 2500 plusmn 110 a mdash mdash 954 plusmn 111 a(44) NLK99-7lowast 2633 plusmn 058 b mdash mdash 1425 plusmn 111 a(45) NLK99-9lowast 2750 plusmn 050 b mdash mdash 1126 plusmn 178 a(46) NLK99-11lowast 2953 plusmn 125 b mdash mdash 1624 plusmn 168 a(47) NLK99-15lowast 2883 plusmn 138 b mdash mdash 1635 plusmn 111 a(48) NLK99-19lowast 2500 plusmn 170 a mdash mdash 1553 plusmn 084 aThree paper discs per plate and three plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Valuesin a row followed by different lowercased letters are significantly different at 119875 le 005

BioMed Research International 7

CVVIK10B (from calve feces) and NLK99-3lowast (from calvefeces) both revealed resistance to CFX No GIZ was detectedin plates treated with negative controls (TWN andWTR)

As shown in Table 5 the percent bacterial GR revealed at100 120583L 10 120583L 1 120583L and 01 120583L NO concentrations was in therange 2371plusmn 100ndash9970plusmn 153 2161plusmn 056ndash9163plusmn 008 1758plusmn 133ndash6957 plusmn 000 and 1118 plusmn 089ndash6758 plusmn 089

There is a significant difference of antibacterial activ-ity among the isolates and the NO concentrations tested(Table 5) The highest percent bacterial GRs were detected at100 120583L NO and they concerned mainly enteropathogenic Ecoli isolates Amplicons of the expected sizes from virulencegenes of enteropathogenic E coli isolates were not detectedwhen bacterial viable cells were checked in samples treatedwith 100 120583L and 10 120583L NO concentrations On the contraryamplicons of the expected size were revealed in the samesamples using primer pair numbers 16 and 17 as reported inTable 1 that specifically amplify unique sequences of E coli16S rRNA

The antibiotic activity of ciprofloxacin is to bind andinhibit bacterial topoisomerase types II and IV thus beingable to interfere with the bacterial processes of replicationtranscription and DNA repair

An increasing ciprofloxacin resistance of E coli isolateswas reported [20] according several epidemiological studiesE coli the most commonly isolated bacterium in clini-cal samples from patients affected by different severity ofdiarrheal symptoms shows high antibiotic resistance [2122] Diarrheagenic E coli considered in the experimentshowed a resistance or less susceptibility to ciprofloxacinin comparison with the other nonenteropathogenic isolatestested

The viable cells of the fourteen diarrheagenic E coliwere checked after NO treatment with primer pairs listedin Table 1 and PMA dye The dye propidium monoazide(PMA Biotium Inc Hayward CA USA) is a photoreactivedye with high affinity for DNA The dye intercalates intoDNA and forms a covalent linkage upon exposure to intensevisible light It is cellmembrane impermeableWhen a samplecomprising both live and dead bacteria is treated with PMAonly dead cells are susceptible to DNA modification dueto their compromised cell membranes [23 24] Thereforeselective detection of the sole live cells is achieved

The fourteen ciprofloxacin resistantless susceptible diar-rheagenic E coli seem to lose their virulence after NO treat-ment because amplicons were obtained only with the primerpairs numbers 16 and 17 (Table 1) This let us suppose thatantibacterial activity acts on adhesion factor and membraneand its permeability with possible loss of extrachromosomalDNA

4 Conclusions and Future Implications

Studies of new antimicrobials from plant derived extractsand agroindustrial byproducts as antimicrobials andpreservatives are important issues in applied microbiologyand biotechnology for both implementing and improvingeffective alternative technologies to tackle antimicrobial

resistance The potential use of plant natural antimicrobialswould require amendments of several different legal textsinvolving areas such as food additives food packaging andhygiene Anyway the applications could concern either thenatural preservation in the food industries or an accessibleand safe alternative to synthetic antimicrobial drugs

Abbreviations

ATCC American Type Culture CollectionCFX Antibiotic hydrochloride monohydrateCRA Agricultural Research Council ItalyCVVI Central Vietnam Veterinary InstituteDAEC Diffusely adherent E coliDSMZ Leibniz-Institut DSMZmdashDeutsche

Sammlung von Mikroorganismen undZellkulturen GmbH

EAEC Enteroaggregative E coliEHEC Enterohemorrhagic E coliEIEC Enteroinvasive E coliELISA Enzyme-linked immunosorbent assayEPEC Enteropathogenic E coliETEC Enterotoxigenic E coliGIZ Growth inhibition zoneHPTLC High performance thin layer

chromatographyHUS Hemolytic uremic syndromeLSD Least significant differenceNCE Neem cake extractNL Department of Bacteriology and Animal

Science University of WageningenNetherlands

NO Neem oilOD Optical densityPMA Propidium monoazideRAPD PCR Random amplified polymorphic DNA

polymerase chain reactionRf Ratio between the migration distance of

substance and the migration distance ofsolvent front

SAS Statistical Analysis System (SAS InstituteInc Cary NC USA)

SD Standard deviationTWN Tween 80WTR Sterile distilled water

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Paola Del Serrone carried out research concept and designcollection andor assembly of biological assay data collectionandor assembly of molecular biology assay data data analy-sis and interpretation statistical analysis writing the papercritical revision of the paper and the final approval of the

8 BioMed Research International

Table 5 Bacterial growth reduction () at 24 h in liquidmediumwith different concentrations ofNO using as reference the control treatment(without NO)

E coli isolates Percent growth reduction zone ()NO (100120583L) NO (10120583L) NO (1 120583L) NO (01 120583L)

(1) FLC 1056 3925 plusmn 143 c 3561 plusmn 100 b 2167 plusmn 133 a 1131 plusmn 208 a(2) FLC 1247 2551 plusmn 115 c 2470 plusmn 100 b 2188 plusmn 133 a 1186 plusmn 100 a(3) FLC 1059 3151 plusmn 115 c 2570 plusmn 100 b 2458 plusmn 133 a 1486 plusmn 100 a(4) FLC 1243 3890 plusmn 100 d 2879 plusmn 100 c 2940 plusmn 000 b 1668 plusmn 120 a(5) FLC 1048 2560 plusmn 153 d 2373 plusmn 208 b 2369 plusmn 200 b 1283 plusmn 173 a(6) FLC 1167 2371 plusmn 100 b 2161 plusmn 058 a 2127 plusmn 000 a 1118 plusmn 089 a(7) FLC 1249 2965 plusmn 153 b 2861 plusmn 100 a 2877 plusmn 133 a 1851 plusmn 208 a(8) FLC 1055 3451 plusmn 115 c 2970 plusmn 100 c 2438 plusmn 133 a 1486 plusmn 100 a(9) FLC 1054 3151 plusmn 115 d 2970 plusmn 100 c 25 78 plusmn 133 a 1486 plusmn 100 a(10) FLC 1085 3990 plusmn 100 c 2879 plusmn 100 b 2810 plusmn 000 b 1558 plusmn 120 a(11) FLC 1244 3570 plusmn 153 d 2873 plusmn 208 b 2859 plusmn 200 b 1263 plusmn 173 a(12) FLC 1165 3971 plusmn 100 c 2961 plusmn 058 c 2917 plusmn 000 b 1648 plusmn 089 a(13) FLC 1086 3955 plusmn 153 c 2861 plusmn 100 c 2787 plusmn 133 b 1221 plusmn 208 a(14) FLC 1053 3451 plusmn 115 c 8970 plusmn 100 c 2758 plusmn 133 b 1486 plusmn 100 a(15) FLC 1095 3151 plusmn 115 c 2970 plusmn 100 b 2518 plusmn 133 a 1486 plusmn 100 a(16) FLC 1219 3895 plusmn 100 b 2879 plusmn 100 b 2810 plusmn 000 b 1668 plusmn 120 a(17) FLC 1235 3670 plusmn 153 d 2773 plusmn 208 c 2779 plusmn 200 b 1283 plusmn 173 a(18) DSM8696 3971 plusmn 100 c 2961 plusmn 058 c 2967 plusmn 000 b 1758 plusmn 089 a(19) DSM9025 3965 plusmn 153 c 2861 plusmn 100 c 2617 plusmn 133 b 1810 plusmn 208 a(20) DSM10973 3451 plusmn 115 c 2970 plusmn 100 c 2328 plusmn 133 b 1486 plusmn 100 a(21) ATCC33559 3151 plusmn 115 d 2970 plusmn 100 c 2158 plusmn 133 b 1486 plusmn 100 a(22) CVVI E210 8890 plusmn 100 c 8879 plusmn 100 c 6920 plusmn 000 b 1478 plusmn 120 a(23) CVVI E173 3650 plusmn 153 d 2773 plusmn 208 c 2799 plusmn 200 b 2283 plusmn 173 a(24) CVVI E126 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 4458 plusmn 089 a(25) CVVI E16 3855 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 1151 plusmn 208 a(26) CVVI E320 3851 plusmn 115 c 2970 plusmn 100 c 2718 plusmn 133 b 1486 plusmn 100 a(27) CVVI E130 3071 plusmn 115 c 2970 plusmn 100 c 2768 plusmn 133 a 1446 plusmn 100 a(28) CVVI E48 3860 plusmn 100 c 2879 plusmn 100 c 2920 plusmn 000 a 1668 plusmn 120 a(29) CVVI KH10 9960 plusmn 153 d 8173 plusmn 208 c 6839 plusmn 200 b 6233 plusmn 173 a(30) CVVI K10B 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 6058 plusmn 089 a(31) CVVI E298 3975 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 2011 plusmn 208 a(32) CVVI E273 3451 plusmn 115 c 2970 plusmn 100 c 2658 plusmn 133 b 2286 plusmn 100 a(33) CVVI K436 3185 plusmn 115 d 2970 plusmn 100 c 2718 plusmn 133 b 1586 plusmn 100 a(34) CVVI E98 3793 plusmn 100 c 2879 plusmn 100 c 2950 plusmn 000 b 1668 plusmn 120 a(35) CVVI E77 3369 plusmn 153 d 2173 plusmn 208 c 2729 plusmn 200 b 1983 plusmn 173 a(36) CVVI E148 3971 plusmn 100 c 2961 plusmn 058 c 2957 plusmn 000 b 2258 plusmn 089 a(37) CVVI E10 8965 plusmn 153 c 8861 plusmn 100 c 6167 plusmn 133 b 5079 plusmn 208 a(38) CVVI E215 3451 plusmn 115 c 2970 plusmn 100 c 1758 plusmn 133 b 2186 plusmn 100 a(39) NLK99F5 9151 plusmn 115 d 8970 plusmn 100 c 6768 plusmn 133 b 6186 plusmn 100 a(40) NL12BF5 8890 plusmn 100 c 8879 plusmn 100 c 6960 plusmn 000 b 6368 plusmn 120 a(41) NLK99-1lowast 9770 plusmn 153 d 8173 plusmn 208 c 6869 plusmn 200 b 6283 plusmn 173 a(42) NLK99-3lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 a(43) NLK99-5lowast 8965 plusmn 153 c 7861 plusmn 100 c 6767 plusmn 133 b 5081 plusmn 208 a(44) NLK99-7lowast 8451 plusmn 115 c 7970 plusmn 100 c 6758 plusmn 133 b 4486 plusmn 100 a(45) NLK99-9lowast 9151 plusmn 115 c 7970 plusmn 100 c 6728 plusmn 113 b 6486 plusmn 100 a(46) NLK99-11lowast 8890 plusmn 100 c 7879 plusmn 100 c 6960 plusmn 000 b 6668 plusmn 120 a(47) NLK99-15lowast 9970 plusmn 153 d 9163 plusmn 028 c 6869 plusmn 200 b 6283 plusmn 173 a(48) NLK99-19lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 aThree plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Values in a row followed by differentlowercased letters are significantly different at 119875 le 005

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

On the basis of their pathogenetic mechanism diar-rheagenic E coli strains include ETEC (enterotoxigenic Ecoli) EIEC (enteroinvasive E coli) EHEC (enterohemor-rhagic E coli) EPEC (enteropathogenic E coli) EAEC(enteroaggregative E coli) and DAEC (diffusely adherentE coli) All of them cause serious economic losses infarm animal herds and are widespread in newborns [10] indeveloped and developing countries There is a wide rangeof transmission possibilities of these pathogens includingdirect contact food drinks environment and others [11]Epidemiology and clinical symptoms of the disease aresimilar in various animal species but the majority of strainsare species-specific They differ particularly in the type ofthe expressed surface ldquoadherencerdquo antigen (adhesin or pilus)These microorganisms produce two main types of virulencefactors that is adhesins and enterotoxins

In this work a collection of E coli isolates was consideredThey were different in geographical origin and source ofisolation and showed different pathogenetic characteristics

Consumers look for meat products of upgraded sensoryquality and increased functional and nutritional propertiesas well as guaranteed safety but yet less processing and feweradditives or ldquotechnologicalrdquo interventions Plant derivedextracts or phytocomplex as effective antimicrobial agentsoffer an alternative to synthetic food additives

Neem (Azadirachta indica A Juss) is considered one ofthe most promising trees of the 21st century for its greatpotential in pest management environment protection andmedicine [12] Neem oil (NO) is the most important derivedproduct with a great market worldwide It contains about onehundred biologically active compounds The most famousconstituents are several nortriterpenes named limonoidsthat is azadirachtin nimbin nimbidin and nimbolidebesides the predominant oily constituents NO is the mostcommercially relevant product obtained from the seeds Theneem cake is remaining after the extraction process

In our previous studies the antibacterial activity of NOagainst E coli was investigated [13 14] The antibacterialactivity resulted highest in comparison to the neem cakeextract against meat spoilage microorganisms The aim ofthe present work was to evaluate NO capability to cope withplastic genome of E coli

2 Materials and Methods

21 Bacterial Strains and Growth Conditions Forty-eightstrains of E coli were considered Among them seventeen(FLC isolates) were from microorganismrsquos collection of theFodder andDairy ProductionsResearchCentre of Lodi (CRAFLC) of CRA They were isolated from milk and cheese Allstrains were typed both phenotypically and genotypicallyPhenotyping was made by the PhenePlate system for E coli(PhP-EC PhPPlate Microplate Techniques AB StockholmSweden) and genetic characterization by RAPD PCR tech-nique [15]

Seventeen CVVI isolates were from microorganismrsquoscollection of the Institute of Veterinary Research and Devel-opment of Central Vietnam VietnamThesemicroorganismswere isolated from faeces of calves affected by diarrhoea

Ten NL isolates were from microorganismrsquos collection ofthe Department of Bacteriology of Wageningen UR Live-stock Research Wageningen University amp Research CentreNetherlands They were isolated from faeces of piglets andcalves They are antigenically different and detectable usingspecific monoclonal antibodies towards different fimbriaantigens by in vitro agglutination test [16] Four referencestrains were also considered (DSMZ and ATCC isolates)They were from international culture collections

The cultivationassay medium for E coli was Minca +1 Iso Vitalex AgarBroth (Sifin Berlin Germany) Bacterialcultures for antibacterial testing were prepared by pickingcolony from 24-hour-old plates and suspending them in thebroth medium (5mL) Cultures were grown aerobically for18 h at 37∘C and 100 rpm For antibacterial activity assay 1mLof each culture was diluted to 105ndash106 CFUmLThe referencestrains were grown on media and at the growth conditions asreported on products sheets

22 Plant Extract A commercial neem oil produced byNeem Italia (Manerba (BS) Italy) was used as test startingmaterial (035 azadirachtin A) Total composition of theneem oil was checked by high performance thin layer chro-matography [17]

Neem oil was diluted in Tween 80 (1 1 VV VWR PBIInternational MI Italy) under agitation and sterilised byfiltration through a 022120583m Millipore express filter (Millex-GP Bedford OH USA) before use in the experiment

23 HPTLC Assay

231 HPTLC System and Materials The HPTLC system(CAMAG Muttenz Switzerland) consisted of (i) Linomat5 sample applicator using 100120583L syringes connected to anitrogen tank (ii) ADC 2 chamber containing twin troughchamber 20times 10 cm (iii) immersion device III (iv) TLC PlateHeater III (v) TLC visualizer (vi) TLC scanner 3 linked towinCATS software

Solvents for extraction and HPLC grade solvents werepurchased from Sigma-Aldrich andCarlo Erba (Milan Italy)Glass plates 20 cm times 10 cm with glass-backed layers silicagel 60 (2 120583m thickness) were from Merck (Darmstadt Ger-many) Before use plates were prewashed with methanol anddried for 3min at 100∘C Standards used in theHPTLC analy-sis were isolated from neem cake (ie salannin azadirachtinA and unsaturated and saturated lipids) in previous research[18] and data concerning isolation and identification arenot reported but they are available per request Limonoidsstandards concentration was 2mM

24 Sample Application Filtered solutions were applied withnitrogen flow Operating conditions were syringe deliveryspeed 10 s 120583Lminus1 (100 nL sminus1) injection volume 2 120583L bandwidth 6mm distance from bottom 15mm

25 Development The HPTLC plates were developed intoluene AcOEt 7 3 (vv) as mobile phase (Figure 1) in theautomatic and reproducibly developing chamber ADC 2

BioMed Research International 3

1 2

(a)

1 2

(b)

Figure 1 HPTLC analysis of neem oil EtOAc extract Mobile phasetoluene AcOEt 7 3 (vv) Visualization plate (a) (on the left) whitelight upper and lower plate (b) (on the right) UV lamp at 366 nmDerivatization 119901-anisaldheyde Track 1 neem oil track 2 salannin

saturated with the same mobile phase for 20min at roomtemperature The developing solvents (ie type of solventsand ratios) were carefully optimized before the analyses Thelength of the chromatogram run was 80mm from the pointof application The developed layers were allowed to dry inair for 5min derivatized with a selected solution including119901-anisaldheyde (15mL 119901-anisaldheyde 25mL H

2SO4 and

1mL AcOH in 37mL EtOH) dried in the open air andthen dipped into Macrogol reagent (1 g polyethylene glycol400 in 20mL of dichloromethane) Finally the plates werewarmed for 5min at 120∘C before inspection All treatedplates were inspected by a CAMAG TLC visualizer undera UV light at 254 or 366 nm or under reflectance andtransmissionwhite light (WRT) respectively before and afterderivatization

26 Molecular Biology Characterization of the E coli IsolatesTwo primer pairs that amplify specific E coli 16S rRNAsequences and fourteen primer pairs that specifically amplifytarget gene coding for virulence factors (adhesins and toxins)were employed to characterize the E coli isolates consideredin this study (Table 1) The PCR reaction mixtures andconditions are those as reported in the literature (Table 1)

The amplification productsrsquo sizes coordinates and acces-sion numbers of each primer pair are shown in Table 2Amplified products (7120583L) were analyzed by electrophoresisin 2 or 3 agarose gels buffered in 05x TBE (TBE buffer90mM tris(hydroxymethyl)aminomethane 90mM boricacid and 3mM ethylenediaminetetraacetate Na salt pH 83Sigma-AldrichMilano Italy) against a 50 bp 100 bp and 1 Kbladder used as size marker (Invitrogen Milano Italia) andvisualized by UV light at 260 nm (Fotodine 3-3102 Celbio

Milano Italy) after staining with ethidium bromide (38-diamino-5-ethyl-6-phenylphenanthridinium bromide EtBrSigma-Aldrich Milano Italy)

27 Assessment of Antibacterial Activity The antibacterialactivity of the NO was assayed using standardized discdiffusion agar and microdilution methods Disc diffusionmethod was carried out according to the standardmethod byBauer et al [19] Bacteria cultures adjusted to 05 McFarlandstandard were used to lawn Muller Hinton agar plates evenlyusing a sterile swabThe agar plates were dried for 15minutesThe discs impregnated with NO (100 120583L) were placed on theagar surface Each test plate comprises three discs The discswere placed equidistant to each other Muller Hinton agarplates were set also up with positive control which is theantibiotic ciprofloxacin (CFX) (100 120583Lwtv) (hydrochloridemonohydrate 1mgmL Bayer Milano Italy) and Tween 80(TWN) (VWR International PBI Srl Milano Italy 1mgmL)as negative control The plates were then incubated at 37∘Cfor 18 h After the incubation the plates and those consideredas controls were examined for inhibition zone The inhi-bition zones were then measured using calipers and wererecordedThe plates were done in triplicate for each bacterialisolate and the experiment was performed twice The resultswere recorded as mean plusmn SD of the duplicate experimentDifferences between means of data were compared by LSDcalculated using the SAS

The antibacterial activity of NO was also evaluated usingmicrodilution method in conventional sterile polystyrenemicroplates (Corning Euroclone SpA Milan Italy) Eachwell of the microplate was filled with 100 120583L of sterile suitableliquid media for each bacterial isolate considered 50 120583L ofinoculums and amounts of extract at lower concentrations(1 10ndash1 10000) were added Control treatment without NOwas used in the experiment The microplates were incu-bated at 37∘C for 24 h Bacterial growth was determined byOD reading at 630 nm10mm pathlength with an ELISAmicroplate reader (DynatechML-3000 Pina de Ebro Spain)Bacterial cell concentration was transformed to cellsmLusing the reference curve equation

The reference curve was constructed by diluting at 1 100each bacterial isolate Counting the number of bacterial cellsof an aliquot of this dilution was done using a Neubauerchamber (Celeromics Vedano al Lambro MI Italy) Finallycell concentrations were transformed to a percentage ofbacterial inhibition The percentage of bacterial growthreduction (GR)was estimated using as reference the controltreatment (119879 = without extract) as

GR = 119862 minus 119879119862times 100 (1)

Three replicates were considered The results wererecorded as mean plusmn SD of the duplicate experiment Dif-ferences between means of data were compared by leastsignificant difference (LSD) calculated using the SAS

4 BioMed Research International

Table 1 Primer pairs used to specifically amplify target gene coding for virulence factors (1ndash9 = toxins 9ndash15 fimbriae) of E coli and 16S rRNA(16-17)

Target gene coding forvirulence factors Oligonucleotide sequences of primers Reference

(1) LT F 51015840-ATT TAC GGC GTT ACT ATC CTC-31015840R 51015840-TTT TGG TCT CGG TCA GAT ATG-31015840 [25]

(2) Sta F 51015840-TCC GTG AAA CAA CAT GAC GG-31015840R 51015840-ATA ACA TCC AGC ACA GGC AG-31015840 [26]

(3) STb F 51015840-GCC TAT GCA TCT ACA CAA TC-31015840R 51015840-TGA GAA ATG GAC AAT GTC CG-31015840 [26]

(4) Stx1all F 51015840-CGC TGA ATG TCA TTC GCT CTG C-31015840R 51015840-CGT GGT ATA GCT ACT GTC ACC-31015840 [27]

(5) Stx2all F 51015840-CTT CGG TAT CCT ATT CCC GG-31015840R 51015840-CTG CTG TGA CAG TGA CAA AAC GC-31015840 [27]

(6) Stx2e F 5-ATG AAG AAG ATG TTT ATA GCG-31015840R 51015840-TCA GTT AAA CTT CAC CTG GGC-31015840 [25]

(7) EAST1 F 51015840-CCA TCA ACA CAG TAT ATC CGA-31015840R 51015840-GGT CGC GAG TGA CGG CTT TGT-31015840 [28]

(8) eae F 51015840-GGA ACG GCA GAG GTT AAT CTGCAG-31015840R 51015840-GGC GCT CAT CAT AGT CTTTC-31015840 [27]

(9) hlyA F 51015840-AGCTGCAAGTGCGGGTCTG-31015840R 51015840-TACGGGTTATGCCTGCAAGTTCAC-31015840 [29]

(10) F4 (K88) F 51015840-GCT GCA TCT GCT GCA TCT GGTATG G-31015840R 51015840-CCA CTG AGT GCT GGTAGT TAC AGC C-31015840 [30]

(11) F5 (K99) F 51015840-TGC GAC TAC CAA TGC TTC TG-31015840R 51015840-TAT CCA CCA TTA GAC GGA GC-31015840 [26]

(12) F6 (P987) F 51015840-TCT GCT CTT AAA GCT ACT GG-31015840R 51015840-AAC TCC ACC GTT TGT ATC AG-31015840 [25]

(13) F17 F 51015840-GGG CTG ACA GAG GAG GTG GGGC-31015840R 51015840-CCC GGC GAC AAC TTC ATCACC GG-31015840 [30]

(14) F18 F 51015840-GTG AAA AGA CTA GTG TTT ATT TC-31015840R 51015840-CTT GTA AGT AAC CGC GTA AGC-31015840 [31]

(15) F41 F 51015840-GAG GGA CTT TCA TCT TTT AG-31015840R 51015840-AGT CCA TTC CAT TTA TAG GC-31015840 [26]

(16) E16SI F 51015840-CCCCCTGGACGAAGACTCAC-31015840R 51015840-ACCGCTGGCAACAAAGGATA -31015840 [29]

(17) E16SII F 51015840-AGAGTTTGATGGCTCAG-31015840R 51015840-GGACTACCAGGGTATCTAAT-31015840 [31]

3 Results and Discussion

31 Molecular Biology Characterization of the E coli Iso-lates The molecular biology characterization of the forty-eight E coli isolates showed that fourteen isolates werediarrheagenic E coli They were ten E coli isolated fromfeces of calves and piglets and four from calves collectedrespectively in Netherlands and Central Vietnam Theirvirulence characteristics are reported in Table 3

32 HPTLC Assay The NO metabolomic fingerprint showscharacteristic sequence ofmetabolites according to the polar-ity of constituents The identification of the raw materialwas assured by the presence of salannin (Rf = 042) whichis a typical maker of neem In comparison with the spotof azadirachtin (Rf = 023) salannin appears as the mainlimonoid spot Spots concerning lipids are present at Rf

values at ca 080 due to unsaturated fatty acids and fattyalcohols and at Rf ca 050 due to saturated and unsatu-rated triglycerides The most interesting feature of the plateconcerns the presence of compounds with high fluorescentreaction at between Rf 055 and 066 which are perfectlyvisible at 366 nm after derivatization with 119901-anisaldheydeThese spots can be attributed to compounds with highconjugated unsaturation in polycyclic aromatic structuresvery different from those of the nortriterpenes limonoids sofar considered responsible for the activity Therefore morestudies are necessary to decide about the importance ofantibacterial activity of these substances in the phytocomplex

33 NO Antibacterial Activity The results obtained showthat NO has a broad spectrum of antibacterial activitiesagainst the tested E coli isolates As shown in Table 4 theantibacterial activity was evaluated based on the diameters

BioMed Research International 5

Table 2 List of primer pairsrsquo amplification products coordinates and accession numbers

Target gene coding forvirulence factors Amplicon (bp) Primer coordinates Accession number

(1) LT 281 27ndash47 287ndash307 S60731(2) STa 244 267ndash286 492ndash510 M58746(3) STb 279 515ndash534 773ndash793 AY028790(4) Stx1all 302 113ndash134 394ndash414 M17358(5) Stx2all 516 50ndash69 543ndash565 M59432(6) Stx2e 264 1176ndash1196 1419ndash1439 M36727(7) EAST1 111 2ndash24 94ndash114 S81691(8) eae 775 1441ndash1460 2193ndash2215 AF022236(9) hylA 569 867-885 1435ndash1412 X79839(10) F4 (K88) 792 31ndash54 798ndash822 M29374(11) F5 (K99) 450 45ndash64 475ndash494 M35282(12) F6 (P987) 333 193ndash212 506ndash525 M35257(13) F17 411 289ndash310 677ndash699 AF055313(14) F18 510 1ndash23 490ndash510 M61713(15) F41 431 154ndash173 565ndash584 X14354(16) E16SI 401 1628-170 2063ndash2082 AB035924(17) E16SII 798 8-27 798ndash805 J01859

Table 3 Molecular characterisation of enteropathogenic E coli and reference strains considered in this study

E coli isolate collectionrsquos designation Surface antigen Toxins Fimbriae(1) CVVI K10B nd STb LT EAST1 F4(2) CVVI KH10 nd STa STb F18(3) NLK99 O8K85K99 nr1 F5(4) NLP987 O64 K 9877 STa+ F6(5) CVVI E12b nd STa F5 F41(6) CVVI E10 nd STa F5 F41(7) NLK99-1 O8 K25 K99 nr F5(8) NLK99-3 O101 K28 K99 nr F5(9) NLK99-5 O9 K30 K99 nr F5(10) NLK99-7 O101 K32 K99 nr F5(11) NLK99-9 O9 K35 K99 nr F5(12) NLK99-11 O9 K37 K99 nr F5(13) NLK99-15 O20 K K99 nr F5(14) NLK99-19 O101 K K99 nr F5(15) DSMZ8696 O55 H6 nr Nr(16) DSMZ9025 mdash mdash mdash(17) DSMZ10973 O6 nr nr(18) ATCC33559 mdash mdash mdashCVVI Central Vietnam Veterinary Institute NL Department of Bacteriology and Animal Science University of Wageningen Netherlands DSMZ Leibniz-Institut DSMZmdashDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ATCC American Type Culture Collection

of clear inhibition zone surrounding the paper discs soakedwith 100 120583L of neem oil The NO average GIZ mm rangefrom 950 plusmn 070 to 3000 plusmn 100 The NO GIZ varies betweenenteropathogenic and nonenteropathogenic E coli beingrespectively 2433plusmn 058ndash3000plusmn 100 and 950plusmn 070ndash2153plusmn153 It is significantly (119875 lt 005) different with respect to theantibiotic activity However the E coli isolate FLC1167 (frommilk) resulted to be less susceptible and the E coli isolate

NLP097F5 (from piglet feces) the most susceptible to NOtreatment (100 120583L) among all tested bacteria using the discdiffusion method

The CFX GIZ range is 000 plusmn 000ndash3265 plusmn 75 Theenteropathogenic E coli resulted to be resistant or lesssusceptible to CFX than the nonenteropathogenic E colishowing a GIZ range of respectively 000 plusmn 000ndash1824plusmn 168mm and 2164 plusmn 094ndash3265 plusmn 75mm The isolates

6 BioMed Research International

Table 4 Antibacterial activity of neem oil (NO) against forty-eight Escherichia coli isolates revealed as growth inhibition zone (mm)

E coli isolates Growth inhibition zone (mm)lowast

NO (100120583L ) TWN (100120583L) WTR (100120583L) CFX (100 120583L)(1) FLC 1056 1133 plusmn 058 b mdash mdash 3041 plusmn 020 a(2) FLC 1247 1613 plusmn 115 b mdash mdash 3052 plusmn 107 a(3) FLC 1059 1583 plusmn 113 b mdash mdash 2962 plusmn 100 a(4) FLC 1243 1900 plusmn 100 b mdash mdash 3153 plusmn 067 a(5) FLC 1048 1233 plusmn 058 b mdash mdash 2942 plusmn 058 a(6) FLC 1167 950 plusmn 070 b mdash mdash 3061 plusmn 121 a(7) FLC 1249 1333 plusmn 058 b mdash mdash 2961 plusmn 111 a(8) FLC 1055 1453 plusmn 125 b mdash mdash 3175 plusmn 082 a(9) FLC 1054 1623 plusmn 118 b mdash mdash 3141 plusmn 076 a(10) FLC 1085 1800 plusmn 100 b mdash mdash 3053 plusmn 117 a(11) FLC 1244 1533 plusmn 048 b mdash mdash 2886 plusmn 100 a(12) FLC 1165 1950 plusmn 070 b mdash mdash 3133 plusmn 067 a(13) FLC 1086 1133 plusmn 058 b mdash mdash 2982 plusmn 048 a(14) FLC 1053 1453 plusmn 115 b mdash mdash 3265 plusmn 139 a(15) FLC 1095 1683 plusmn 118 b mdash mdash 2905 plusmn 122 a(16) FLC 1219 1070 plusmn 100 b mdash mdash 3275 plusmn 055 a(17) FLC 1235 1323 plusmn 088 b mdash mdash 3015 plusmn 055 a(18) DSM8696 1350 plusmn 050 b mdash mdash 2621 plusmn 100 a(19) DSM9025 1333 plusmn 058 b mdash mdash 2164 plusmn 094 a(20) DSM10973 1353 plusmn 125 b mdash mdash 2914 plusmn 175 a(21) ATCC33559 1383 plusmn 118 b mdash mdash 3212 plusmn 109 a(22) CVVI E210 1300 plusmn 100 a mdash mdash 2583 plusmn 165 a(23) CVVI E173 1223 plusmn 058 b mdash mdash 3235 plusmn 149 a(24) CVVI E12b 2750 plusmn 050 b mdash mdash 1125 plusmn 068 a(25) CVVI E16 1433 plusmn 088 b mdash mdash 2754 plusmn 145 a(26) CVVI E320 2153 plusmn 135 b mdash mdash 2875 plusmn 186 a(27) CVVI E130 1183 plusmn 178 b mdash mdash 2964 plusmn 087 a(28) CVVI E48 1000 plusmn 140 a mdash mdash 2931 plusmn 027 a(29) CVVI KH10 2633 plusmn 053 b mdash mdash 1534 plusmn 066 a(30) CVVI K10B 2750 plusmn 056 b mdash mdash 0(31) CVVI E298 1133 plusmn 048 b mdash mdash 2390 plusmn 169 a(32) CVVI E273 1353 plusmn 175 b mdash mdash 2959 plusmn 177 b(33) CVVI K436 1314 plusmn 168 b mdash mdash 3021 plusmn 138 a(34) CVVI E98 1100 plusmn 100 a mdash mdash 2691 plusmn 156 a(35) CVVI E77 1433 plusmn 058 b mdash mdash 2393 plusmn 059 a(36) CVVI E148 1350 plusmn 050 b mdash mdash 2871 plusmn 087 a(37) CVVI E10 2433 plusmn 058 b mdash mdash 1035 plusmn 111 a(38) CVVI E215 1653 plusmn 115 b mdash mdash 2641 plusmn 140 a(39) NLK99F5 2983 plusmn 118 b mdash mdash 16 21 plusmn 089 a(40) NLP987F5 3000 plusmn 100 a mdash mdash 1321 plusmn 115 a(41) NLK99-1lowast 2173 plusmn 135 b mdash mdash 1534 plusmn 137 a(42) NLK99-3lowast 2833 plusmn 150 b mdash mdash 0(43) NLK99-5lowast 2500 plusmn 110 a mdash mdash 954 plusmn 111 a(44) NLK99-7lowast 2633 plusmn 058 b mdash mdash 1425 plusmn 111 a(45) NLK99-9lowast 2750 plusmn 050 b mdash mdash 1126 plusmn 178 a(46) NLK99-11lowast 2953 plusmn 125 b mdash mdash 1624 plusmn 168 a(47) NLK99-15lowast 2883 plusmn 138 b mdash mdash 1635 plusmn 111 a(48) NLK99-19lowast 2500 plusmn 170 a mdash mdash 1553 plusmn 084 aThree paper discs per plate and three plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Valuesin a row followed by different lowercased letters are significantly different at 119875 le 005

BioMed Research International 7

CVVIK10B (from calve feces) and NLK99-3lowast (from calvefeces) both revealed resistance to CFX No GIZ was detectedin plates treated with negative controls (TWN andWTR)

As shown in Table 5 the percent bacterial GR revealed at100 120583L 10 120583L 1 120583L and 01 120583L NO concentrations was in therange 2371plusmn 100ndash9970plusmn 153 2161plusmn 056ndash9163plusmn 008 1758plusmn 133ndash6957 plusmn 000 and 1118 plusmn 089ndash6758 plusmn 089

There is a significant difference of antibacterial activ-ity among the isolates and the NO concentrations tested(Table 5) The highest percent bacterial GRs were detected at100 120583L NO and they concerned mainly enteropathogenic Ecoli isolates Amplicons of the expected sizes from virulencegenes of enteropathogenic E coli isolates were not detectedwhen bacterial viable cells were checked in samples treatedwith 100 120583L and 10 120583L NO concentrations On the contraryamplicons of the expected size were revealed in the samesamples using primer pair numbers 16 and 17 as reported inTable 1 that specifically amplify unique sequences of E coli16S rRNA

The antibiotic activity of ciprofloxacin is to bind andinhibit bacterial topoisomerase types II and IV thus beingable to interfere with the bacterial processes of replicationtranscription and DNA repair

An increasing ciprofloxacin resistance of E coli isolateswas reported [20] according several epidemiological studiesE coli the most commonly isolated bacterium in clini-cal samples from patients affected by different severity ofdiarrheal symptoms shows high antibiotic resistance [2122] Diarrheagenic E coli considered in the experimentshowed a resistance or less susceptibility to ciprofloxacinin comparison with the other nonenteropathogenic isolatestested

The viable cells of the fourteen diarrheagenic E coliwere checked after NO treatment with primer pairs listedin Table 1 and PMA dye The dye propidium monoazide(PMA Biotium Inc Hayward CA USA) is a photoreactivedye with high affinity for DNA The dye intercalates intoDNA and forms a covalent linkage upon exposure to intensevisible light It is cellmembrane impermeableWhen a samplecomprising both live and dead bacteria is treated with PMAonly dead cells are susceptible to DNA modification dueto their compromised cell membranes [23 24] Thereforeselective detection of the sole live cells is achieved

The fourteen ciprofloxacin resistantless susceptible diar-rheagenic E coli seem to lose their virulence after NO treat-ment because amplicons were obtained only with the primerpairs numbers 16 and 17 (Table 1) This let us suppose thatantibacterial activity acts on adhesion factor and membraneand its permeability with possible loss of extrachromosomalDNA

4 Conclusions and Future Implications

Studies of new antimicrobials from plant derived extractsand agroindustrial byproducts as antimicrobials andpreservatives are important issues in applied microbiologyand biotechnology for both implementing and improvingeffective alternative technologies to tackle antimicrobial

resistance The potential use of plant natural antimicrobialswould require amendments of several different legal textsinvolving areas such as food additives food packaging andhygiene Anyway the applications could concern either thenatural preservation in the food industries or an accessibleand safe alternative to synthetic antimicrobial drugs

Abbreviations

ATCC American Type Culture CollectionCFX Antibiotic hydrochloride monohydrateCRA Agricultural Research Council ItalyCVVI Central Vietnam Veterinary InstituteDAEC Diffusely adherent E coliDSMZ Leibniz-Institut DSMZmdashDeutsche

Sammlung von Mikroorganismen undZellkulturen GmbH

EAEC Enteroaggregative E coliEHEC Enterohemorrhagic E coliEIEC Enteroinvasive E coliELISA Enzyme-linked immunosorbent assayEPEC Enteropathogenic E coliETEC Enterotoxigenic E coliGIZ Growth inhibition zoneHPTLC High performance thin layer

chromatographyHUS Hemolytic uremic syndromeLSD Least significant differenceNCE Neem cake extractNL Department of Bacteriology and Animal

Science University of WageningenNetherlands

NO Neem oilOD Optical densityPMA Propidium monoazideRAPD PCR Random amplified polymorphic DNA

polymerase chain reactionRf Ratio between the migration distance of

substance and the migration distance ofsolvent front

SAS Statistical Analysis System (SAS InstituteInc Cary NC USA)

SD Standard deviationTWN Tween 80WTR Sterile distilled water

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Paola Del Serrone carried out research concept and designcollection andor assembly of biological assay data collectionandor assembly of molecular biology assay data data analy-sis and interpretation statistical analysis writing the papercritical revision of the paper and the final approval of the

8 BioMed Research International

Table 5 Bacterial growth reduction () at 24 h in liquidmediumwith different concentrations ofNO using as reference the control treatment(without NO)

E coli isolates Percent growth reduction zone ()NO (100120583L) NO (10120583L) NO (1 120583L) NO (01 120583L)

(1) FLC 1056 3925 plusmn 143 c 3561 plusmn 100 b 2167 plusmn 133 a 1131 plusmn 208 a(2) FLC 1247 2551 plusmn 115 c 2470 plusmn 100 b 2188 plusmn 133 a 1186 plusmn 100 a(3) FLC 1059 3151 plusmn 115 c 2570 plusmn 100 b 2458 plusmn 133 a 1486 plusmn 100 a(4) FLC 1243 3890 plusmn 100 d 2879 plusmn 100 c 2940 plusmn 000 b 1668 plusmn 120 a(5) FLC 1048 2560 plusmn 153 d 2373 plusmn 208 b 2369 plusmn 200 b 1283 plusmn 173 a(6) FLC 1167 2371 plusmn 100 b 2161 plusmn 058 a 2127 plusmn 000 a 1118 plusmn 089 a(7) FLC 1249 2965 plusmn 153 b 2861 plusmn 100 a 2877 plusmn 133 a 1851 plusmn 208 a(8) FLC 1055 3451 plusmn 115 c 2970 plusmn 100 c 2438 plusmn 133 a 1486 plusmn 100 a(9) FLC 1054 3151 plusmn 115 d 2970 plusmn 100 c 25 78 plusmn 133 a 1486 plusmn 100 a(10) FLC 1085 3990 plusmn 100 c 2879 plusmn 100 b 2810 plusmn 000 b 1558 plusmn 120 a(11) FLC 1244 3570 plusmn 153 d 2873 plusmn 208 b 2859 plusmn 200 b 1263 plusmn 173 a(12) FLC 1165 3971 plusmn 100 c 2961 plusmn 058 c 2917 plusmn 000 b 1648 plusmn 089 a(13) FLC 1086 3955 plusmn 153 c 2861 plusmn 100 c 2787 plusmn 133 b 1221 plusmn 208 a(14) FLC 1053 3451 plusmn 115 c 8970 plusmn 100 c 2758 plusmn 133 b 1486 plusmn 100 a(15) FLC 1095 3151 plusmn 115 c 2970 plusmn 100 b 2518 plusmn 133 a 1486 plusmn 100 a(16) FLC 1219 3895 plusmn 100 b 2879 plusmn 100 b 2810 plusmn 000 b 1668 plusmn 120 a(17) FLC 1235 3670 plusmn 153 d 2773 plusmn 208 c 2779 plusmn 200 b 1283 plusmn 173 a(18) DSM8696 3971 plusmn 100 c 2961 plusmn 058 c 2967 plusmn 000 b 1758 plusmn 089 a(19) DSM9025 3965 plusmn 153 c 2861 plusmn 100 c 2617 plusmn 133 b 1810 plusmn 208 a(20) DSM10973 3451 plusmn 115 c 2970 plusmn 100 c 2328 plusmn 133 b 1486 plusmn 100 a(21) ATCC33559 3151 plusmn 115 d 2970 plusmn 100 c 2158 plusmn 133 b 1486 plusmn 100 a(22) CVVI E210 8890 plusmn 100 c 8879 plusmn 100 c 6920 plusmn 000 b 1478 plusmn 120 a(23) CVVI E173 3650 plusmn 153 d 2773 plusmn 208 c 2799 plusmn 200 b 2283 plusmn 173 a(24) CVVI E126 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 4458 plusmn 089 a(25) CVVI E16 3855 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 1151 plusmn 208 a(26) CVVI E320 3851 plusmn 115 c 2970 plusmn 100 c 2718 plusmn 133 b 1486 plusmn 100 a(27) CVVI E130 3071 plusmn 115 c 2970 plusmn 100 c 2768 plusmn 133 a 1446 plusmn 100 a(28) CVVI E48 3860 plusmn 100 c 2879 plusmn 100 c 2920 plusmn 000 a 1668 plusmn 120 a(29) CVVI KH10 9960 plusmn 153 d 8173 plusmn 208 c 6839 plusmn 200 b 6233 plusmn 173 a(30) CVVI K10B 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 6058 plusmn 089 a(31) CVVI E298 3975 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 2011 plusmn 208 a(32) CVVI E273 3451 plusmn 115 c 2970 plusmn 100 c 2658 plusmn 133 b 2286 plusmn 100 a(33) CVVI K436 3185 plusmn 115 d 2970 plusmn 100 c 2718 plusmn 133 b 1586 plusmn 100 a(34) CVVI E98 3793 plusmn 100 c 2879 plusmn 100 c 2950 plusmn 000 b 1668 plusmn 120 a(35) CVVI E77 3369 plusmn 153 d 2173 plusmn 208 c 2729 plusmn 200 b 1983 plusmn 173 a(36) CVVI E148 3971 plusmn 100 c 2961 plusmn 058 c 2957 plusmn 000 b 2258 plusmn 089 a(37) CVVI E10 8965 plusmn 153 c 8861 plusmn 100 c 6167 plusmn 133 b 5079 plusmn 208 a(38) CVVI E215 3451 plusmn 115 c 2970 plusmn 100 c 1758 plusmn 133 b 2186 plusmn 100 a(39) NLK99F5 9151 plusmn 115 d 8970 plusmn 100 c 6768 plusmn 133 b 6186 plusmn 100 a(40) NL12BF5 8890 plusmn 100 c 8879 plusmn 100 c 6960 plusmn 000 b 6368 plusmn 120 a(41) NLK99-1lowast 9770 plusmn 153 d 8173 plusmn 208 c 6869 plusmn 200 b 6283 plusmn 173 a(42) NLK99-3lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 a(43) NLK99-5lowast 8965 plusmn 153 c 7861 plusmn 100 c 6767 plusmn 133 b 5081 plusmn 208 a(44) NLK99-7lowast 8451 plusmn 115 c 7970 plusmn 100 c 6758 plusmn 133 b 4486 plusmn 100 a(45) NLK99-9lowast 9151 plusmn 115 c 7970 plusmn 100 c 6728 plusmn 113 b 6486 plusmn 100 a(46) NLK99-11lowast 8890 plusmn 100 c 7879 plusmn 100 c 6960 plusmn 000 b 6668 plusmn 120 a(47) NLK99-15lowast 9970 plusmn 153 d 9163 plusmn 028 c 6869 plusmn 200 b 6283 plusmn 173 a(48) NLK99-19lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 aThree plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Values in a row followed by differentlowercased letters are significantly different at 119875 le 005

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

1 2

(a)

1 2

(b)

Figure 1 HPTLC analysis of neem oil EtOAc extract Mobile phasetoluene AcOEt 7 3 (vv) Visualization plate (a) (on the left) whitelight upper and lower plate (b) (on the right) UV lamp at 366 nmDerivatization 119901-anisaldheyde Track 1 neem oil track 2 salannin

saturated with the same mobile phase for 20min at roomtemperature The developing solvents (ie type of solventsand ratios) were carefully optimized before the analyses Thelength of the chromatogram run was 80mm from the pointof application The developed layers were allowed to dry inair for 5min derivatized with a selected solution including119901-anisaldheyde (15mL 119901-anisaldheyde 25mL H

2SO4 and

1mL AcOH in 37mL EtOH) dried in the open air andthen dipped into Macrogol reagent (1 g polyethylene glycol400 in 20mL of dichloromethane) Finally the plates werewarmed for 5min at 120∘C before inspection All treatedplates were inspected by a CAMAG TLC visualizer undera UV light at 254 or 366 nm or under reflectance andtransmissionwhite light (WRT) respectively before and afterderivatization

26 Molecular Biology Characterization of the E coli IsolatesTwo primer pairs that amplify specific E coli 16S rRNAsequences and fourteen primer pairs that specifically amplifytarget gene coding for virulence factors (adhesins and toxins)were employed to characterize the E coli isolates consideredin this study (Table 1) The PCR reaction mixtures andconditions are those as reported in the literature (Table 1)

The amplification productsrsquo sizes coordinates and acces-sion numbers of each primer pair are shown in Table 2Amplified products (7120583L) were analyzed by electrophoresisin 2 or 3 agarose gels buffered in 05x TBE (TBE buffer90mM tris(hydroxymethyl)aminomethane 90mM boricacid and 3mM ethylenediaminetetraacetate Na salt pH 83Sigma-AldrichMilano Italy) against a 50 bp 100 bp and 1 Kbladder used as size marker (Invitrogen Milano Italia) andvisualized by UV light at 260 nm (Fotodine 3-3102 Celbio

Milano Italy) after staining with ethidium bromide (38-diamino-5-ethyl-6-phenylphenanthridinium bromide EtBrSigma-Aldrich Milano Italy)

27 Assessment of Antibacterial Activity The antibacterialactivity of the NO was assayed using standardized discdiffusion agar and microdilution methods Disc diffusionmethod was carried out according to the standardmethod byBauer et al [19] Bacteria cultures adjusted to 05 McFarlandstandard were used to lawn Muller Hinton agar plates evenlyusing a sterile swabThe agar plates were dried for 15minutesThe discs impregnated with NO (100 120583L) were placed on theagar surface Each test plate comprises three discs The discswere placed equidistant to each other Muller Hinton agarplates were set also up with positive control which is theantibiotic ciprofloxacin (CFX) (100 120583Lwtv) (hydrochloridemonohydrate 1mgmL Bayer Milano Italy) and Tween 80(TWN) (VWR International PBI Srl Milano Italy 1mgmL)as negative control The plates were then incubated at 37∘Cfor 18 h After the incubation the plates and those consideredas controls were examined for inhibition zone The inhi-bition zones were then measured using calipers and wererecordedThe plates were done in triplicate for each bacterialisolate and the experiment was performed twice The resultswere recorded as mean plusmn SD of the duplicate experimentDifferences between means of data were compared by LSDcalculated using the SAS

The antibacterial activity of NO was also evaluated usingmicrodilution method in conventional sterile polystyrenemicroplates (Corning Euroclone SpA Milan Italy) Eachwell of the microplate was filled with 100 120583L of sterile suitableliquid media for each bacterial isolate considered 50 120583L ofinoculums and amounts of extract at lower concentrations(1 10ndash1 10000) were added Control treatment without NOwas used in the experiment The microplates were incu-bated at 37∘C for 24 h Bacterial growth was determined byOD reading at 630 nm10mm pathlength with an ELISAmicroplate reader (DynatechML-3000 Pina de Ebro Spain)Bacterial cell concentration was transformed to cellsmLusing the reference curve equation

The reference curve was constructed by diluting at 1 100each bacterial isolate Counting the number of bacterial cellsof an aliquot of this dilution was done using a Neubauerchamber (Celeromics Vedano al Lambro MI Italy) Finallycell concentrations were transformed to a percentage ofbacterial inhibition The percentage of bacterial growthreduction (GR)was estimated using as reference the controltreatment (119879 = without extract) as

GR = 119862 minus 119879119862times 100 (1)

Three replicates were considered The results wererecorded as mean plusmn SD of the duplicate experiment Dif-ferences between means of data were compared by leastsignificant difference (LSD) calculated using the SAS

4 BioMed Research International

Table 1 Primer pairs used to specifically amplify target gene coding for virulence factors (1ndash9 = toxins 9ndash15 fimbriae) of E coli and 16S rRNA(16-17)

Target gene coding forvirulence factors Oligonucleotide sequences of primers Reference

(1) LT F 51015840-ATT TAC GGC GTT ACT ATC CTC-31015840R 51015840-TTT TGG TCT CGG TCA GAT ATG-31015840 [25]

(2) Sta F 51015840-TCC GTG AAA CAA CAT GAC GG-31015840R 51015840-ATA ACA TCC AGC ACA GGC AG-31015840 [26]

(3) STb F 51015840-GCC TAT GCA TCT ACA CAA TC-31015840R 51015840-TGA GAA ATG GAC AAT GTC CG-31015840 [26]

(4) Stx1all F 51015840-CGC TGA ATG TCA TTC GCT CTG C-31015840R 51015840-CGT GGT ATA GCT ACT GTC ACC-31015840 [27]

(5) Stx2all F 51015840-CTT CGG TAT CCT ATT CCC GG-31015840R 51015840-CTG CTG TGA CAG TGA CAA AAC GC-31015840 [27]

(6) Stx2e F 5-ATG AAG AAG ATG TTT ATA GCG-31015840R 51015840-TCA GTT AAA CTT CAC CTG GGC-31015840 [25]

(7) EAST1 F 51015840-CCA TCA ACA CAG TAT ATC CGA-31015840R 51015840-GGT CGC GAG TGA CGG CTT TGT-31015840 [28]

(8) eae F 51015840-GGA ACG GCA GAG GTT AAT CTGCAG-31015840R 51015840-GGC GCT CAT CAT AGT CTTTC-31015840 [27]

(9) hlyA F 51015840-AGCTGCAAGTGCGGGTCTG-31015840R 51015840-TACGGGTTATGCCTGCAAGTTCAC-31015840 [29]

(10) F4 (K88) F 51015840-GCT GCA TCT GCT GCA TCT GGTATG G-31015840R 51015840-CCA CTG AGT GCT GGTAGT TAC AGC C-31015840 [30]

(11) F5 (K99) F 51015840-TGC GAC TAC CAA TGC TTC TG-31015840R 51015840-TAT CCA CCA TTA GAC GGA GC-31015840 [26]

(12) F6 (P987) F 51015840-TCT GCT CTT AAA GCT ACT GG-31015840R 51015840-AAC TCC ACC GTT TGT ATC AG-31015840 [25]

(13) F17 F 51015840-GGG CTG ACA GAG GAG GTG GGGC-31015840R 51015840-CCC GGC GAC AAC TTC ATCACC GG-31015840 [30]

(14) F18 F 51015840-GTG AAA AGA CTA GTG TTT ATT TC-31015840R 51015840-CTT GTA AGT AAC CGC GTA AGC-31015840 [31]

(15) F41 F 51015840-GAG GGA CTT TCA TCT TTT AG-31015840R 51015840-AGT CCA TTC CAT TTA TAG GC-31015840 [26]

(16) E16SI F 51015840-CCCCCTGGACGAAGACTCAC-31015840R 51015840-ACCGCTGGCAACAAAGGATA -31015840 [29]

(17) E16SII F 51015840-AGAGTTTGATGGCTCAG-31015840R 51015840-GGACTACCAGGGTATCTAAT-31015840 [31]

3 Results and Discussion

31 Molecular Biology Characterization of the E coli Iso-lates The molecular biology characterization of the forty-eight E coli isolates showed that fourteen isolates werediarrheagenic E coli They were ten E coli isolated fromfeces of calves and piglets and four from calves collectedrespectively in Netherlands and Central Vietnam Theirvirulence characteristics are reported in Table 3

32 HPTLC Assay The NO metabolomic fingerprint showscharacteristic sequence ofmetabolites according to the polar-ity of constituents The identification of the raw materialwas assured by the presence of salannin (Rf = 042) whichis a typical maker of neem In comparison with the spotof azadirachtin (Rf = 023) salannin appears as the mainlimonoid spot Spots concerning lipids are present at Rf

values at ca 080 due to unsaturated fatty acids and fattyalcohols and at Rf ca 050 due to saturated and unsatu-rated triglycerides The most interesting feature of the plateconcerns the presence of compounds with high fluorescentreaction at between Rf 055 and 066 which are perfectlyvisible at 366 nm after derivatization with 119901-anisaldheydeThese spots can be attributed to compounds with highconjugated unsaturation in polycyclic aromatic structuresvery different from those of the nortriterpenes limonoids sofar considered responsible for the activity Therefore morestudies are necessary to decide about the importance ofantibacterial activity of these substances in the phytocomplex

33 NO Antibacterial Activity The results obtained showthat NO has a broad spectrum of antibacterial activitiesagainst the tested E coli isolates As shown in Table 4 theantibacterial activity was evaluated based on the diameters

BioMed Research International 5

Table 2 List of primer pairsrsquo amplification products coordinates and accession numbers

Target gene coding forvirulence factors Amplicon (bp) Primer coordinates Accession number

(1) LT 281 27ndash47 287ndash307 S60731(2) STa 244 267ndash286 492ndash510 M58746(3) STb 279 515ndash534 773ndash793 AY028790(4) Stx1all 302 113ndash134 394ndash414 M17358(5) Stx2all 516 50ndash69 543ndash565 M59432(6) Stx2e 264 1176ndash1196 1419ndash1439 M36727(7) EAST1 111 2ndash24 94ndash114 S81691(8) eae 775 1441ndash1460 2193ndash2215 AF022236(9) hylA 569 867-885 1435ndash1412 X79839(10) F4 (K88) 792 31ndash54 798ndash822 M29374(11) F5 (K99) 450 45ndash64 475ndash494 M35282(12) F6 (P987) 333 193ndash212 506ndash525 M35257(13) F17 411 289ndash310 677ndash699 AF055313(14) F18 510 1ndash23 490ndash510 M61713(15) F41 431 154ndash173 565ndash584 X14354(16) E16SI 401 1628-170 2063ndash2082 AB035924(17) E16SII 798 8-27 798ndash805 J01859

Table 3 Molecular characterisation of enteropathogenic E coli and reference strains considered in this study

E coli isolate collectionrsquos designation Surface antigen Toxins Fimbriae(1) CVVI K10B nd STb LT EAST1 F4(2) CVVI KH10 nd STa STb F18(3) NLK99 O8K85K99 nr1 F5(4) NLP987 O64 K 9877 STa+ F6(5) CVVI E12b nd STa F5 F41(6) CVVI E10 nd STa F5 F41(7) NLK99-1 O8 K25 K99 nr F5(8) NLK99-3 O101 K28 K99 nr F5(9) NLK99-5 O9 K30 K99 nr F5(10) NLK99-7 O101 K32 K99 nr F5(11) NLK99-9 O9 K35 K99 nr F5(12) NLK99-11 O9 K37 K99 nr F5(13) NLK99-15 O20 K K99 nr F5(14) NLK99-19 O101 K K99 nr F5(15) DSMZ8696 O55 H6 nr Nr(16) DSMZ9025 mdash mdash mdash(17) DSMZ10973 O6 nr nr(18) ATCC33559 mdash mdash mdashCVVI Central Vietnam Veterinary Institute NL Department of Bacteriology and Animal Science University of Wageningen Netherlands DSMZ Leibniz-Institut DSMZmdashDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ATCC American Type Culture Collection

of clear inhibition zone surrounding the paper discs soakedwith 100 120583L of neem oil The NO average GIZ mm rangefrom 950 plusmn 070 to 3000 plusmn 100 The NO GIZ varies betweenenteropathogenic and nonenteropathogenic E coli beingrespectively 2433plusmn 058ndash3000plusmn 100 and 950plusmn 070ndash2153plusmn153 It is significantly (119875 lt 005) different with respect to theantibiotic activity However the E coli isolate FLC1167 (frommilk) resulted to be less susceptible and the E coli isolate

NLP097F5 (from piglet feces) the most susceptible to NOtreatment (100 120583L) among all tested bacteria using the discdiffusion method

The CFX GIZ range is 000 plusmn 000ndash3265 plusmn 75 Theenteropathogenic E coli resulted to be resistant or lesssusceptible to CFX than the nonenteropathogenic E colishowing a GIZ range of respectively 000 plusmn 000ndash1824plusmn 168mm and 2164 plusmn 094ndash3265 plusmn 75mm The isolates

6 BioMed Research International

Table 4 Antibacterial activity of neem oil (NO) against forty-eight Escherichia coli isolates revealed as growth inhibition zone (mm)

E coli isolates Growth inhibition zone (mm)lowast

NO (100120583L ) TWN (100120583L) WTR (100120583L) CFX (100 120583L)(1) FLC 1056 1133 plusmn 058 b mdash mdash 3041 plusmn 020 a(2) FLC 1247 1613 plusmn 115 b mdash mdash 3052 plusmn 107 a(3) FLC 1059 1583 plusmn 113 b mdash mdash 2962 plusmn 100 a(4) FLC 1243 1900 plusmn 100 b mdash mdash 3153 plusmn 067 a(5) FLC 1048 1233 plusmn 058 b mdash mdash 2942 plusmn 058 a(6) FLC 1167 950 plusmn 070 b mdash mdash 3061 plusmn 121 a(7) FLC 1249 1333 plusmn 058 b mdash mdash 2961 plusmn 111 a(8) FLC 1055 1453 plusmn 125 b mdash mdash 3175 plusmn 082 a(9) FLC 1054 1623 plusmn 118 b mdash mdash 3141 plusmn 076 a(10) FLC 1085 1800 plusmn 100 b mdash mdash 3053 plusmn 117 a(11) FLC 1244 1533 plusmn 048 b mdash mdash 2886 plusmn 100 a(12) FLC 1165 1950 plusmn 070 b mdash mdash 3133 plusmn 067 a(13) FLC 1086 1133 plusmn 058 b mdash mdash 2982 plusmn 048 a(14) FLC 1053 1453 plusmn 115 b mdash mdash 3265 plusmn 139 a(15) FLC 1095 1683 plusmn 118 b mdash mdash 2905 plusmn 122 a(16) FLC 1219 1070 plusmn 100 b mdash mdash 3275 plusmn 055 a(17) FLC 1235 1323 plusmn 088 b mdash mdash 3015 plusmn 055 a(18) DSM8696 1350 plusmn 050 b mdash mdash 2621 plusmn 100 a(19) DSM9025 1333 plusmn 058 b mdash mdash 2164 plusmn 094 a(20) DSM10973 1353 plusmn 125 b mdash mdash 2914 plusmn 175 a(21) ATCC33559 1383 plusmn 118 b mdash mdash 3212 plusmn 109 a(22) CVVI E210 1300 plusmn 100 a mdash mdash 2583 plusmn 165 a(23) CVVI E173 1223 plusmn 058 b mdash mdash 3235 plusmn 149 a(24) CVVI E12b 2750 plusmn 050 b mdash mdash 1125 plusmn 068 a(25) CVVI E16 1433 plusmn 088 b mdash mdash 2754 plusmn 145 a(26) CVVI E320 2153 plusmn 135 b mdash mdash 2875 plusmn 186 a(27) CVVI E130 1183 plusmn 178 b mdash mdash 2964 plusmn 087 a(28) CVVI E48 1000 plusmn 140 a mdash mdash 2931 plusmn 027 a(29) CVVI KH10 2633 plusmn 053 b mdash mdash 1534 plusmn 066 a(30) CVVI K10B 2750 plusmn 056 b mdash mdash 0(31) CVVI E298 1133 plusmn 048 b mdash mdash 2390 plusmn 169 a(32) CVVI E273 1353 plusmn 175 b mdash mdash 2959 plusmn 177 b(33) CVVI K436 1314 plusmn 168 b mdash mdash 3021 plusmn 138 a(34) CVVI E98 1100 plusmn 100 a mdash mdash 2691 plusmn 156 a(35) CVVI E77 1433 plusmn 058 b mdash mdash 2393 plusmn 059 a(36) CVVI E148 1350 plusmn 050 b mdash mdash 2871 plusmn 087 a(37) CVVI E10 2433 plusmn 058 b mdash mdash 1035 plusmn 111 a(38) CVVI E215 1653 plusmn 115 b mdash mdash 2641 plusmn 140 a(39) NLK99F5 2983 plusmn 118 b mdash mdash 16 21 plusmn 089 a(40) NLP987F5 3000 plusmn 100 a mdash mdash 1321 plusmn 115 a(41) NLK99-1lowast 2173 plusmn 135 b mdash mdash 1534 plusmn 137 a(42) NLK99-3lowast 2833 plusmn 150 b mdash mdash 0(43) NLK99-5lowast 2500 plusmn 110 a mdash mdash 954 plusmn 111 a(44) NLK99-7lowast 2633 plusmn 058 b mdash mdash 1425 plusmn 111 a(45) NLK99-9lowast 2750 plusmn 050 b mdash mdash 1126 plusmn 178 a(46) NLK99-11lowast 2953 plusmn 125 b mdash mdash 1624 plusmn 168 a(47) NLK99-15lowast 2883 plusmn 138 b mdash mdash 1635 plusmn 111 a(48) NLK99-19lowast 2500 plusmn 170 a mdash mdash 1553 plusmn 084 aThree paper discs per plate and three plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Valuesin a row followed by different lowercased letters are significantly different at 119875 le 005

BioMed Research International 7

CVVIK10B (from calve feces) and NLK99-3lowast (from calvefeces) both revealed resistance to CFX No GIZ was detectedin plates treated with negative controls (TWN andWTR)

As shown in Table 5 the percent bacterial GR revealed at100 120583L 10 120583L 1 120583L and 01 120583L NO concentrations was in therange 2371plusmn 100ndash9970plusmn 153 2161plusmn 056ndash9163plusmn 008 1758plusmn 133ndash6957 plusmn 000 and 1118 plusmn 089ndash6758 plusmn 089

There is a significant difference of antibacterial activ-ity among the isolates and the NO concentrations tested(Table 5) The highest percent bacterial GRs were detected at100 120583L NO and they concerned mainly enteropathogenic Ecoli isolates Amplicons of the expected sizes from virulencegenes of enteropathogenic E coli isolates were not detectedwhen bacterial viable cells were checked in samples treatedwith 100 120583L and 10 120583L NO concentrations On the contraryamplicons of the expected size were revealed in the samesamples using primer pair numbers 16 and 17 as reported inTable 1 that specifically amplify unique sequences of E coli16S rRNA

The antibiotic activity of ciprofloxacin is to bind andinhibit bacterial topoisomerase types II and IV thus beingable to interfere with the bacterial processes of replicationtranscription and DNA repair

An increasing ciprofloxacin resistance of E coli isolateswas reported [20] according several epidemiological studiesE coli the most commonly isolated bacterium in clini-cal samples from patients affected by different severity ofdiarrheal symptoms shows high antibiotic resistance [2122] Diarrheagenic E coli considered in the experimentshowed a resistance or less susceptibility to ciprofloxacinin comparison with the other nonenteropathogenic isolatestested

The viable cells of the fourteen diarrheagenic E coliwere checked after NO treatment with primer pairs listedin Table 1 and PMA dye The dye propidium monoazide(PMA Biotium Inc Hayward CA USA) is a photoreactivedye with high affinity for DNA The dye intercalates intoDNA and forms a covalent linkage upon exposure to intensevisible light It is cellmembrane impermeableWhen a samplecomprising both live and dead bacteria is treated with PMAonly dead cells are susceptible to DNA modification dueto their compromised cell membranes [23 24] Thereforeselective detection of the sole live cells is achieved

The fourteen ciprofloxacin resistantless susceptible diar-rheagenic E coli seem to lose their virulence after NO treat-ment because amplicons were obtained only with the primerpairs numbers 16 and 17 (Table 1) This let us suppose thatantibacterial activity acts on adhesion factor and membraneand its permeability with possible loss of extrachromosomalDNA

4 Conclusions and Future Implications

Studies of new antimicrobials from plant derived extractsand agroindustrial byproducts as antimicrobials andpreservatives are important issues in applied microbiologyand biotechnology for both implementing and improvingeffective alternative technologies to tackle antimicrobial

resistance The potential use of plant natural antimicrobialswould require amendments of several different legal textsinvolving areas such as food additives food packaging andhygiene Anyway the applications could concern either thenatural preservation in the food industries or an accessibleand safe alternative to synthetic antimicrobial drugs

Abbreviations

ATCC American Type Culture CollectionCFX Antibiotic hydrochloride monohydrateCRA Agricultural Research Council ItalyCVVI Central Vietnam Veterinary InstituteDAEC Diffusely adherent E coliDSMZ Leibniz-Institut DSMZmdashDeutsche

Sammlung von Mikroorganismen undZellkulturen GmbH

EAEC Enteroaggregative E coliEHEC Enterohemorrhagic E coliEIEC Enteroinvasive E coliELISA Enzyme-linked immunosorbent assayEPEC Enteropathogenic E coliETEC Enterotoxigenic E coliGIZ Growth inhibition zoneHPTLC High performance thin layer

chromatographyHUS Hemolytic uremic syndromeLSD Least significant differenceNCE Neem cake extractNL Department of Bacteriology and Animal

Science University of WageningenNetherlands

NO Neem oilOD Optical densityPMA Propidium monoazideRAPD PCR Random amplified polymorphic DNA

polymerase chain reactionRf Ratio between the migration distance of

substance and the migration distance ofsolvent front

SAS Statistical Analysis System (SAS InstituteInc Cary NC USA)

SD Standard deviationTWN Tween 80WTR Sterile distilled water

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Paola Del Serrone carried out research concept and designcollection andor assembly of biological assay data collectionandor assembly of molecular biology assay data data analy-sis and interpretation statistical analysis writing the papercritical revision of the paper and the final approval of the

8 BioMed Research International

Table 5 Bacterial growth reduction () at 24 h in liquidmediumwith different concentrations ofNO using as reference the control treatment(without NO)

E coli isolates Percent growth reduction zone ()NO (100120583L) NO (10120583L) NO (1 120583L) NO (01 120583L)

(1) FLC 1056 3925 plusmn 143 c 3561 plusmn 100 b 2167 plusmn 133 a 1131 plusmn 208 a(2) FLC 1247 2551 plusmn 115 c 2470 plusmn 100 b 2188 plusmn 133 a 1186 plusmn 100 a(3) FLC 1059 3151 plusmn 115 c 2570 plusmn 100 b 2458 plusmn 133 a 1486 plusmn 100 a(4) FLC 1243 3890 plusmn 100 d 2879 plusmn 100 c 2940 plusmn 000 b 1668 plusmn 120 a(5) FLC 1048 2560 plusmn 153 d 2373 plusmn 208 b 2369 plusmn 200 b 1283 plusmn 173 a(6) FLC 1167 2371 plusmn 100 b 2161 plusmn 058 a 2127 plusmn 000 a 1118 plusmn 089 a(7) FLC 1249 2965 plusmn 153 b 2861 plusmn 100 a 2877 plusmn 133 a 1851 plusmn 208 a(8) FLC 1055 3451 plusmn 115 c 2970 plusmn 100 c 2438 plusmn 133 a 1486 plusmn 100 a(9) FLC 1054 3151 plusmn 115 d 2970 plusmn 100 c 25 78 plusmn 133 a 1486 plusmn 100 a(10) FLC 1085 3990 plusmn 100 c 2879 plusmn 100 b 2810 plusmn 000 b 1558 plusmn 120 a(11) FLC 1244 3570 plusmn 153 d 2873 plusmn 208 b 2859 plusmn 200 b 1263 plusmn 173 a(12) FLC 1165 3971 plusmn 100 c 2961 plusmn 058 c 2917 plusmn 000 b 1648 plusmn 089 a(13) FLC 1086 3955 plusmn 153 c 2861 plusmn 100 c 2787 plusmn 133 b 1221 plusmn 208 a(14) FLC 1053 3451 plusmn 115 c 8970 plusmn 100 c 2758 plusmn 133 b 1486 plusmn 100 a(15) FLC 1095 3151 plusmn 115 c 2970 plusmn 100 b 2518 plusmn 133 a 1486 plusmn 100 a(16) FLC 1219 3895 plusmn 100 b 2879 plusmn 100 b 2810 plusmn 000 b 1668 plusmn 120 a(17) FLC 1235 3670 plusmn 153 d 2773 plusmn 208 c 2779 plusmn 200 b 1283 plusmn 173 a(18) DSM8696 3971 plusmn 100 c 2961 plusmn 058 c 2967 plusmn 000 b 1758 plusmn 089 a(19) DSM9025 3965 plusmn 153 c 2861 plusmn 100 c 2617 plusmn 133 b 1810 plusmn 208 a(20) DSM10973 3451 plusmn 115 c 2970 plusmn 100 c 2328 plusmn 133 b 1486 plusmn 100 a(21) ATCC33559 3151 plusmn 115 d 2970 plusmn 100 c 2158 plusmn 133 b 1486 plusmn 100 a(22) CVVI E210 8890 plusmn 100 c 8879 plusmn 100 c 6920 plusmn 000 b 1478 plusmn 120 a(23) CVVI E173 3650 plusmn 153 d 2773 plusmn 208 c 2799 plusmn 200 b 2283 plusmn 173 a(24) CVVI E126 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 4458 plusmn 089 a(25) CVVI E16 3855 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 1151 plusmn 208 a(26) CVVI E320 3851 plusmn 115 c 2970 plusmn 100 c 2718 plusmn 133 b 1486 plusmn 100 a(27) CVVI E130 3071 plusmn 115 c 2970 plusmn 100 c 2768 plusmn 133 a 1446 plusmn 100 a(28) CVVI E48 3860 plusmn 100 c 2879 plusmn 100 c 2920 plusmn 000 a 1668 plusmn 120 a(29) CVVI KH10 9960 plusmn 153 d 8173 plusmn 208 c 6839 plusmn 200 b 6233 plusmn 173 a(30) CVVI K10B 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 6058 plusmn 089 a(31) CVVI E298 3975 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 2011 plusmn 208 a(32) CVVI E273 3451 plusmn 115 c 2970 plusmn 100 c 2658 plusmn 133 b 2286 plusmn 100 a(33) CVVI K436 3185 plusmn 115 d 2970 plusmn 100 c 2718 plusmn 133 b 1586 plusmn 100 a(34) CVVI E98 3793 plusmn 100 c 2879 plusmn 100 c 2950 plusmn 000 b 1668 plusmn 120 a(35) CVVI E77 3369 plusmn 153 d 2173 plusmn 208 c 2729 plusmn 200 b 1983 plusmn 173 a(36) CVVI E148 3971 plusmn 100 c 2961 plusmn 058 c 2957 plusmn 000 b 2258 plusmn 089 a(37) CVVI E10 8965 plusmn 153 c 8861 plusmn 100 c 6167 plusmn 133 b 5079 plusmn 208 a(38) CVVI E215 3451 plusmn 115 c 2970 plusmn 100 c 1758 plusmn 133 b 2186 plusmn 100 a(39) NLK99F5 9151 plusmn 115 d 8970 plusmn 100 c 6768 plusmn 133 b 6186 plusmn 100 a(40) NL12BF5 8890 plusmn 100 c 8879 plusmn 100 c 6960 plusmn 000 b 6368 plusmn 120 a(41) NLK99-1lowast 9770 plusmn 153 d 8173 plusmn 208 c 6869 plusmn 200 b 6283 plusmn 173 a(42) NLK99-3lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 a(43) NLK99-5lowast 8965 plusmn 153 c 7861 plusmn 100 c 6767 plusmn 133 b 5081 plusmn 208 a(44) NLK99-7lowast 8451 plusmn 115 c 7970 plusmn 100 c 6758 plusmn 133 b 4486 plusmn 100 a(45) NLK99-9lowast 9151 plusmn 115 c 7970 plusmn 100 c 6728 plusmn 113 b 6486 plusmn 100 a(46) NLK99-11lowast 8890 plusmn 100 c 7879 plusmn 100 c 6960 plusmn 000 b 6668 plusmn 120 a(47) NLK99-15lowast 9970 plusmn 153 d 9163 plusmn 028 c 6869 plusmn 200 b 6283 plusmn 173 a(48) NLK99-19lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 aThree plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Values in a row followed by differentlowercased letters are significantly different at 119875 le 005

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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Signal TransductionJournal of

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Nucleic AcidsJournal of

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International Journal of

Microbiology

4 BioMed Research International

Table 1 Primer pairs used to specifically amplify target gene coding for virulence factors (1ndash9 = toxins 9ndash15 fimbriae) of E coli and 16S rRNA(16-17)

Target gene coding forvirulence factors Oligonucleotide sequences of primers Reference

(1) LT F 51015840-ATT TAC GGC GTT ACT ATC CTC-31015840R 51015840-TTT TGG TCT CGG TCA GAT ATG-31015840 [25]

(2) Sta F 51015840-TCC GTG AAA CAA CAT GAC GG-31015840R 51015840-ATA ACA TCC AGC ACA GGC AG-31015840 [26]

(3) STb F 51015840-GCC TAT GCA TCT ACA CAA TC-31015840R 51015840-TGA GAA ATG GAC AAT GTC CG-31015840 [26]

(4) Stx1all F 51015840-CGC TGA ATG TCA TTC GCT CTG C-31015840R 51015840-CGT GGT ATA GCT ACT GTC ACC-31015840 [27]

(5) Stx2all F 51015840-CTT CGG TAT CCT ATT CCC GG-31015840R 51015840-CTG CTG TGA CAG TGA CAA AAC GC-31015840 [27]

(6) Stx2e F 5-ATG AAG AAG ATG TTT ATA GCG-31015840R 51015840-TCA GTT AAA CTT CAC CTG GGC-31015840 [25]

(7) EAST1 F 51015840-CCA TCA ACA CAG TAT ATC CGA-31015840R 51015840-GGT CGC GAG TGA CGG CTT TGT-31015840 [28]

(8) eae F 51015840-GGA ACG GCA GAG GTT AAT CTGCAG-31015840R 51015840-GGC GCT CAT CAT AGT CTTTC-31015840 [27]

(9) hlyA F 51015840-AGCTGCAAGTGCGGGTCTG-31015840R 51015840-TACGGGTTATGCCTGCAAGTTCAC-31015840 [29]

(10) F4 (K88) F 51015840-GCT GCA TCT GCT GCA TCT GGTATG G-31015840R 51015840-CCA CTG AGT GCT GGTAGT TAC AGC C-31015840 [30]

(11) F5 (K99) F 51015840-TGC GAC TAC CAA TGC TTC TG-31015840R 51015840-TAT CCA CCA TTA GAC GGA GC-31015840 [26]

(12) F6 (P987) F 51015840-TCT GCT CTT AAA GCT ACT GG-31015840R 51015840-AAC TCC ACC GTT TGT ATC AG-31015840 [25]

(13) F17 F 51015840-GGG CTG ACA GAG GAG GTG GGGC-31015840R 51015840-CCC GGC GAC AAC TTC ATCACC GG-31015840 [30]

(14) F18 F 51015840-GTG AAA AGA CTA GTG TTT ATT TC-31015840R 51015840-CTT GTA AGT AAC CGC GTA AGC-31015840 [31]

(15) F41 F 51015840-GAG GGA CTT TCA TCT TTT AG-31015840R 51015840-AGT CCA TTC CAT TTA TAG GC-31015840 [26]

(16) E16SI F 51015840-CCCCCTGGACGAAGACTCAC-31015840R 51015840-ACCGCTGGCAACAAAGGATA -31015840 [29]

(17) E16SII F 51015840-AGAGTTTGATGGCTCAG-31015840R 51015840-GGACTACCAGGGTATCTAAT-31015840 [31]

3 Results and Discussion

31 Molecular Biology Characterization of the E coli Iso-lates The molecular biology characterization of the forty-eight E coli isolates showed that fourteen isolates werediarrheagenic E coli They were ten E coli isolated fromfeces of calves and piglets and four from calves collectedrespectively in Netherlands and Central Vietnam Theirvirulence characteristics are reported in Table 3

32 HPTLC Assay The NO metabolomic fingerprint showscharacteristic sequence ofmetabolites according to the polar-ity of constituents The identification of the raw materialwas assured by the presence of salannin (Rf = 042) whichis a typical maker of neem In comparison with the spotof azadirachtin (Rf = 023) salannin appears as the mainlimonoid spot Spots concerning lipids are present at Rf

values at ca 080 due to unsaturated fatty acids and fattyalcohols and at Rf ca 050 due to saturated and unsatu-rated triglycerides The most interesting feature of the plateconcerns the presence of compounds with high fluorescentreaction at between Rf 055 and 066 which are perfectlyvisible at 366 nm after derivatization with 119901-anisaldheydeThese spots can be attributed to compounds with highconjugated unsaturation in polycyclic aromatic structuresvery different from those of the nortriterpenes limonoids sofar considered responsible for the activity Therefore morestudies are necessary to decide about the importance ofantibacterial activity of these substances in the phytocomplex

33 NO Antibacterial Activity The results obtained showthat NO has a broad spectrum of antibacterial activitiesagainst the tested E coli isolates As shown in Table 4 theantibacterial activity was evaluated based on the diameters

BioMed Research International 5

Table 2 List of primer pairsrsquo amplification products coordinates and accession numbers

Target gene coding forvirulence factors Amplicon (bp) Primer coordinates Accession number

(1) LT 281 27ndash47 287ndash307 S60731(2) STa 244 267ndash286 492ndash510 M58746(3) STb 279 515ndash534 773ndash793 AY028790(4) Stx1all 302 113ndash134 394ndash414 M17358(5) Stx2all 516 50ndash69 543ndash565 M59432(6) Stx2e 264 1176ndash1196 1419ndash1439 M36727(7) EAST1 111 2ndash24 94ndash114 S81691(8) eae 775 1441ndash1460 2193ndash2215 AF022236(9) hylA 569 867-885 1435ndash1412 X79839(10) F4 (K88) 792 31ndash54 798ndash822 M29374(11) F5 (K99) 450 45ndash64 475ndash494 M35282(12) F6 (P987) 333 193ndash212 506ndash525 M35257(13) F17 411 289ndash310 677ndash699 AF055313(14) F18 510 1ndash23 490ndash510 M61713(15) F41 431 154ndash173 565ndash584 X14354(16) E16SI 401 1628-170 2063ndash2082 AB035924(17) E16SII 798 8-27 798ndash805 J01859

Table 3 Molecular characterisation of enteropathogenic E coli and reference strains considered in this study

E coli isolate collectionrsquos designation Surface antigen Toxins Fimbriae(1) CVVI K10B nd STb LT EAST1 F4(2) CVVI KH10 nd STa STb F18(3) NLK99 O8K85K99 nr1 F5(4) NLP987 O64 K 9877 STa+ F6(5) CVVI E12b nd STa F5 F41(6) CVVI E10 nd STa F5 F41(7) NLK99-1 O8 K25 K99 nr F5(8) NLK99-3 O101 K28 K99 nr F5(9) NLK99-5 O9 K30 K99 nr F5(10) NLK99-7 O101 K32 K99 nr F5(11) NLK99-9 O9 K35 K99 nr F5(12) NLK99-11 O9 K37 K99 nr F5(13) NLK99-15 O20 K K99 nr F5(14) NLK99-19 O101 K K99 nr F5(15) DSMZ8696 O55 H6 nr Nr(16) DSMZ9025 mdash mdash mdash(17) DSMZ10973 O6 nr nr(18) ATCC33559 mdash mdash mdashCVVI Central Vietnam Veterinary Institute NL Department of Bacteriology and Animal Science University of Wageningen Netherlands DSMZ Leibniz-Institut DSMZmdashDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ATCC American Type Culture Collection

of clear inhibition zone surrounding the paper discs soakedwith 100 120583L of neem oil The NO average GIZ mm rangefrom 950 plusmn 070 to 3000 plusmn 100 The NO GIZ varies betweenenteropathogenic and nonenteropathogenic E coli beingrespectively 2433plusmn 058ndash3000plusmn 100 and 950plusmn 070ndash2153plusmn153 It is significantly (119875 lt 005) different with respect to theantibiotic activity However the E coli isolate FLC1167 (frommilk) resulted to be less susceptible and the E coli isolate

NLP097F5 (from piglet feces) the most susceptible to NOtreatment (100 120583L) among all tested bacteria using the discdiffusion method

The CFX GIZ range is 000 plusmn 000ndash3265 plusmn 75 Theenteropathogenic E coli resulted to be resistant or lesssusceptible to CFX than the nonenteropathogenic E colishowing a GIZ range of respectively 000 plusmn 000ndash1824plusmn 168mm and 2164 plusmn 094ndash3265 plusmn 75mm The isolates

6 BioMed Research International

Table 4 Antibacterial activity of neem oil (NO) against forty-eight Escherichia coli isolates revealed as growth inhibition zone (mm)

E coli isolates Growth inhibition zone (mm)lowast

NO (100120583L ) TWN (100120583L) WTR (100120583L) CFX (100 120583L)(1) FLC 1056 1133 plusmn 058 b mdash mdash 3041 plusmn 020 a(2) FLC 1247 1613 plusmn 115 b mdash mdash 3052 plusmn 107 a(3) FLC 1059 1583 plusmn 113 b mdash mdash 2962 plusmn 100 a(4) FLC 1243 1900 plusmn 100 b mdash mdash 3153 plusmn 067 a(5) FLC 1048 1233 plusmn 058 b mdash mdash 2942 plusmn 058 a(6) FLC 1167 950 plusmn 070 b mdash mdash 3061 plusmn 121 a(7) FLC 1249 1333 plusmn 058 b mdash mdash 2961 plusmn 111 a(8) FLC 1055 1453 plusmn 125 b mdash mdash 3175 plusmn 082 a(9) FLC 1054 1623 plusmn 118 b mdash mdash 3141 plusmn 076 a(10) FLC 1085 1800 plusmn 100 b mdash mdash 3053 plusmn 117 a(11) FLC 1244 1533 plusmn 048 b mdash mdash 2886 plusmn 100 a(12) FLC 1165 1950 plusmn 070 b mdash mdash 3133 plusmn 067 a(13) FLC 1086 1133 plusmn 058 b mdash mdash 2982 plusmn 048 a(14) FLC 1053 1453 plusmn 115 b mdash mdash 3265 plusmn 139 a(15) FLC 1095 1683 plusmn 118 b mdash mdash 2905 plusmn 122 a(16) FLC 1219 1070 plusmn 100 b mdash mdash 3275 plusmn 055 a(17) FLC 1235 1323 plusmn 088 b mdash mdash 3015 plusmn 055 a(18) DSM8696 1350 plusmn 050 b mdash mdash 2621 plusmn 100 a(19) DSM9025 1333 plusmn 058 b mdash mdash 2164 plusmn 094 a(20) DSM10973 1353 plusmn 125 b mdash mdash 2914 plusmn 175 a(21) ATCC33559 1383 plusmn 118 b mdash mdash 3212 plusmn 109 a(22) CVVI E210 1300 plusmn 100 a mdash mdash 2583 plusmn 165 a(23) CVVI E173 1223 plusmn 058 b mdash mdash 3235 plusmn 149 a(24) CVVI E12b 2750 plusmn 050 b mdash mdash 1125 plusmn 068 a(25) CVVI E16 1433 plusmn 088 b mdash mdash 2754 plusmn 145 a(26) CVVI E320 2153 plusmn 135 b mdash mdash 2875 plusmn 186 a(27) CVVI E130 1183 plusmn 178 b mdash mdash 2964 plusmn 087 a(28) CVVI E48 1000 plusmn 140 a mdash mdash 2931 plusmn 027 a(29) CVVI KH10 2633 plusmn 053 b mdash mdash 1534 plusmn 066 a(30) CVVI K10B 2750 plusmn 056 b mdash mdash 0(31) CVVI E298 1133 plusmn 048 b mdash mdash 2390 plusmn 169 a(32) CVVI E273 1353 plusmn 175 b mdash mdash 2959 plusmn 177 b(33) CVVI K436 1314 plusmn 168 b mdash mdash 3021 plusmn 138 a(34) CVVI E98 1100 plusmn 100 a mdash mdash 2691 plusmn 156 a(35) CVVI E77 1433 plusmn 058 b mdash mdash 2393 plusmn 059 a(36) CVVI E148 1350 plusmn 050 b mdash mdash 2871 plusmn 087 a(37) CVVI E10 2433 plusmn 058 b mdash mdash 1035 plusmn 111 a(38) CVVI E215 1653 plusmn 115 b mdash mdash 2641 plusmn 140 a(39) NLK99F5 2983 plusmn 118 b mdash mdash 16 21 plusmn 089 a(40) NLP987F5 3000 plusmn 100 a mdash mdash 1321 plusmn 115 a(41) NLK99-1lowast 2173 plusmn 135 b mdash mdash 1534 plusmn 137 a(42) NLK99-3lowast 2833 plusmn 150 b mdash mdash 0(43) NLK99-5lowast 2500 plusmn 110 a mdash mdash 954 plusmn 111 a(44) NLK99-7lowast 2633 plusmn 058 b mdash mdash 1425 plusmn 111 a(45) NLK99-9lowast 2750 plusmn 050 b mdash mdash 1126 plusmn 178 a(46) NLK99-11lowast 2953 plusmn 125 b mdash mdash 1624 plusmn 168 a(47) NLK99-15lowast 2883 plusmn 138 b mdash mdash 1635 plusmn 111 a(48) NLK99-19lowast 2500 plusmn 170 a mdash mdash 1553 plusmn 084 aThree paper discs per plate and three plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Valuesin a row followed by different lowercased letters are significantly different at 119875 le 005

BioMed Research International 7

CVVIK10B (from calve feces) and NLK99-3lowast (from calvefeces) both revealed resistance to CFX No GIZ was detectedin plates treated with negative controls (TWN andWTR)

As shown in Table 5 the percent bacterial GR revealed at100 120583L 10 120583L 1 120583L and 01 120583L NO concentrations was in therange 2371plusmn 100ndash9970plusmn 153 2161plusmn 056ndash9163plusmn 008 1758plusmn 133ndash6957 plusmn 000 and 1118 plusmn 089ndash6758 plusmn 089

There is a significant difference of antibacterial activ-ity among the isolates and the NO concentrations tested(Table 5) The highest percent bacterial GRs were detected at100 120583L NO and they concerned mainly enteropathogenic Ecoli isolates Amplicons of the expected sizes from virulencegenes of enteropathogenic E coli isolates were not detectedwhen bacterial viable cells were checked in samples treatedwith 100 120583L and 10 120583L NO concentrations On the contraryamplicons of the expected size were revealed in the samesamples using primer pair numbers 16 and 17 as reported inTable 1 that specifically amplify unique sequences of E coli16S rRNA

The antibiotic activity of ciprofloxacin is to bind andinhibit bacterial topoisomerase types II and IV thus beingable to interfere with the bacterial processes of replicationtranscription and DNA repair

An increasing ciprofloxacin resistance of E coli isolateswas reported [20] according several epidemiological studiesE coli the most commonly isolated bacterium in clini-cal samples from patients affected by different severity ofdiarrheal symptoms shows high antibiotic resistance [2122] Diarrheagenic E coli considered in the experimentshowed a resistance or less susceptibility to ciprofloxacinin comparison with the other nonenteropathogenic isolatestested

The viable cells of the fourteen diarrheagenic E coliwere checked after NO treatment with primer pairs listedin Table 1 and PMA dye The dye propidium monoazide(PMA Biotium Inc Hayward CA USA) is a photoreactivedye with high affinity for DNA The dye intercalates intoDNA and forms a covalent linkage upon exposure to intensevisible light It is cellmembrane impermeableWhen a samplecomprising both live and dead bacteria is treated with PMAonly dead cells are susceptible to DNA modification dueto their compromised cell membranes [23 24] Thereforeselective detection of the sole live cells is achieved

The fourteen ciprofloxacin resistantless susceptible diar-rheagenic E coli seem to lose their virulence after NO treat-ment because amplicons were obtained only with the primerpairs numbers 16 and 17 (Table 1) This let us suppose thatantibacterial activity acts on adhesion factor and membraneand its permeability with possible loss of extrachromosomalDNA

4 Conclusions and Future Implications

Studies of new antimicrobials from plant derived extractsand agroindustrial byproducts as antimicrobials andpreservatives are important issues in applied microbiologyand biotechnology for both implementing and improvingeffective alternative technologies to tackle antimicrobial

resistance The potential use of plant natural antimicrobialswould require amendments of several different legal textsinvolving areas such as food additives food packaging andhygiene Anyway the applications could concern either thenatural preservation in the food industries or an accessibleand safe alternative to synthetic antimicrobial drugs

Abbreviations

ATCC American Type Culture CollectionCFX Antibiotic hydrochloride monohydrateCRA Agricultural Research Council ItalyCVVI Central Vietnam Veterinary InstituteDAEC Diffusely adherent E coliDSMZ Leibniz-Institut DSMZmdashDeutsche

Sammlung von Mikroorganismen undZellkulturen GmbH

EAEC Enteroaggregative E coliEHEC Enterohemorrhagic E coliEIEC Enteroinvasive E coliELISA Enzyme-linked immunosorbent assayEPEC Enteropathogenic E coliETEC Enterotoxigenic E coliGIZ Growth inhibition zoneHPTLC High performance thin layer

chromatographyHUS Hemolytic uremic syndromeLSD Least significant differenceNCE Neem cake extractNL Department of Bacteriology and Animal

Science University of WageningenNetherlands

NO Neem oilOD Optical densityPMA Propidium monoazideRAPD PCR Random amplified polymorphic DNA

polymerase chain reactionRf Ratio between the migration distance of

substance and the migration distance ofsolvent front

SAS Statistical Analysis System (SAS InstituteInc Cary NC USA)

SD Standard deviationTWN Tween 80WTR Sterile distilled water

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Paola Del Serrone carried out research concept and designcollection andor assembly of biological assay data collectionandor assembly of molecular biology assay data data analy-sis and interpretation statistical analysis writing the papercritical revision of the paper and the final approval of the

8 BioMed Research International

Table 5 Bacterial growth reduction () at 24 h in liquidmediumwith different concentrations ofNO using as reference the control treatment(without NO)

E coli isolates Percent growth reduction zone ()NO (100120583L) NO (10120583L) NO (1 120583L) NO (01 120583L)

(1) FLC 1056 3925 plusmn 143 c 3561 plusmn 100 b 2167 plusmn 133 a 1131 plusmn 208 a(2) FLC 1247 2551 plusmn 115 c 2470 plusmn 100 b 2188 plusmn 133 a 1186 plusmn 100 a(3) FLC 1059 3151 plusmn 115 c 2570 plusmn 100 b 2458 plusmn 133 a 1486 plusmn 100 a(4) FLC 1243 3890 plusmn 100 d 2879 plusmn 100 c 2940 plusmn 000 b 1668 plusmn 120 a(5) FLC 1048 2560 plusmn 153 d 2373 plusmn 208 b 2369 plusmn 200 b 1283 plusmn 173 a(6) FLC 1167 2371 plusmn 100 b 2161 plusmn 058 a 2127 plusmn 000 a 1118 plusmn 089 a(7) FLC 1249 2965 plusmn 153 b 2861 plusmn 100 a 2877 plusmn 133 a 1851 plusmn 208 a(8) FLC 1055 3451 plusmn 115 c 2970 plusmn 100 c 2438 plusmn 133 a 1486 plusmn 100 a(9) FLC 1054 3151 plusmn 115 d 2970 plusmn 100 c 25 78 plusmn 133 a 1486 plusmn 100 a(10) FLC 1085 3990 plusmn 100 c 2879 plusmn 100 b 2810 plusmn 000 b 1558 plusmn 120 a(11) FLC 1244 3570 plusmn 153 d 2873 plusmn 208 b 2859 plusmn 200 b 1263 plusmn 173 a(12) FLC 1165 3971 plusmn 100 c 2961 plusmn 058 c 2917 plusmn 000 b 1648 plusmn 089 a(13) FLC 1086 3955 plusmn 153 c 2861 plusmn 100 c 2787 plusmn 133 b 1221 plusmn 208 a(14) FLC 1053 3451 plusmn 115 c 8970 plusmn 100 c 2758 plusmn 133 b 1486 plusmn 100 a(15) FLC 1095 3151 plusmn 115 c 2970 plusmn 100 b 2518 plusmn 133 a 1486 plusmn 100 a(16) FLC 1219 3895 plusmn 100 b 2879 plusmn 100 b 2810 plusmn 000 b 1668 plusmn 120 a(17) FLC 1235 3670 plusmn 153 d 2773 plusmn 208 c 2779 plusmn 200 b 1283 plusmn 173 a(18) DSM8696 3971 plusmn 100 c 2961 plusmn 058 c 2967 plusmn 000 b 1758 plusmn 089 a(19) DSM9025 3965 plusmn 153 c 2861 plusmn 100 c 2617 plusmn 133 b 1810 plusmn 208 a(20) DSM10973 3451 plusmn 115 c 2970 plusmn 100 c 2328 plusmn 133 b 1486 plusmn 100 a(21) ATCC33559 3151 plusmn 115 d 2970 plusmn 100 c 2158 plusmn 133 b 1486 plusmn 100 a(22) CVVI E210 8890 plusmn 100 c 8879 plusmn 100 c 6920 plusmn 000 b 1478 plusmn 120 a(23) CVVI E173 3650 plusmn 153 d 2773 plusmn 208 c 2799 plusmn 200 b 2283 plusmn 173 a(24) CVVI E126 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 4458 plusmn 089 a(25) CVVI E16 3855 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 1151 plusmn 208 a(26) CVVI E320 3851 plusmn 115 c 2970 plusmn 100 c 2718 plusmn 133 b 1486 plusmn 100 a(27) CVVI E130 3071 plusmn 115 c 2970 plusmn 100 c 2768 plusmn 133 a 1446 plusmn 100 a(28) CVVI E48 3860 plusmn 100 c 2879 plusmn 100 c 2920 plusmn 000 a 1668 plusmn 120 a(29) CVVI KH10 9960 plusmn 153 d 8173 plusmn 208 c 6839 plusmn 200 b 6233 plusmn 173 a(30) CVVI K10B 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 6058 plusmn 089 a(31) CVVI E298 3975 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 2011 plusmn 208 a(32) CVVI E273 3451 plusmn 115 c 2970 plusmn 100 c 2658 plusmn 133 b 2286 plusmn 100 a(33) CVVI K436 3185 plusmn 115 d 2970 plusmn 100 c 2718 plusmn 133 b 1586 plusmn 100 a(34) CVVI E98 3793 plusmn 100 c 2879 plusmn 100 c 2950 plusmn 000 b 1668 plusmn 120 a(35) CVVI E77 3369 plusmn 153 d 2173 plusmn 208 c 2729 plusmn 200 b 1983 plusmn 173 a(36) CVVI E148 3971 plusmn 100 c 2961 plusmn 058 c 2957 plusmn 000 b 2258 plusmn 089 a(37) CVVI E10 8965 plusmn 153 c 8861 plusmn 100 c 6167 plusmn 133 b 5079 plusmn 208 a(38) CVVI E215 3451 plusmn 115 c 2970 plusmn 100 c 1758 plusmn 133 b 2186 plusmn 100 a(39) NLK99F5 9151 plusmn 115 d 8970 plusmn 100 c 6768 plusmn 133 b 6186 plusmn 100 a(40) NL12BF5 8890 plusmn 100 c 8879 plusmn 100 c 6960 plusmn 000 b 6368 plusmn 120 a(41) NLK99-1lowast 9770 plusmn 153 d 8173 plusmn 208 c 6869 plusmn 200 b 6283 plusmn 173 a(42) NLK99-3lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 a(43) NLK99-5lowast 8965 plusmn 153 c 7861 plusmn 100 c 6767 plusmn 133 b 5081 plusmn 208 a(44) NLK99-7lowast 8451 plusmn 115 c 7970 plusmn 100 c 6758 plusmn 133 b 4486 plusmn 100 a(45) NLK99-9lowast 9151 plusmn 115 c 7970 plusmn 100 c 6728 plusmn 113 b 6486 plusmn 100 a(46) NLK99-11lowast 8890 plusmn 100 c 7879 plusmn 100 c 6960 plusmn 000 b 6668 plusmn 120 a(47) NLK99-15lowast 9970 plusmn 153 d 9163 plusmn 028 c 6869 plusmn 200 b 6283 plusmn 173 a(48) NLK99-19lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 aThree plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Values in a row followed by differentlowercased letters are significantly different at 119875 le 005

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table 2 List of primer pairsrsquo amplification products coordinates and accession numbers

Target gene coding forvirulence factors Amplicon (bp) Primer coordinates Accession number

(1) LT 281 27ndash47 287ndash307 S60731(2) STa 244 267ndash286 492ndash510 M58746(3) STb 279 515ndash534 773ndash793 AY028790(4) Stx1all 302 113ndash134 394ndash414 M17358(5) Stx2all 516 50ndash69 543ndash565 M59432(6) Stx2e 264 1176ndash1196 1419ndash1439 M36727(7) EAST1 111 2ndash24 94ndash114 S81691(8) eae 775 1441ndash1460 2193ndash2215 AF022236(9) hylA 569 867-885 1435ndash1412 X79839(10) F4 (K88) 792 31ndash54 798ndash822 M29374(11) F5 (K99) 450 45ndash64 475ndash494 M35282(12) F6 (P987) 333 193ndash212 506ndash525 M35257(13) F17 411 289ndash310 677ndash699 AF055313(14) F18 510 1ndash23 490ndash510 M61713(15) F41 431 154ndash173 565ndash584 X14354(16) E16SI 401 1628-170 2063ndash2082 AB035924(17) E16SII 798 8-27 798ndash805 J01859

Table 3 Molecular characterisation of enteropathogenic E coli and reference strains considered in this study

E coli isolate collectionrsquos designation Surface antigen Toxins Fimbriae(1) CVVI K10B nd STb LT EAST1 F4(2) CVVI KH10 nd STa STb F18(3) NLK99 O8K85K99 nr1 F5(4) NLP987 O64 K 9877 STa+ F6(5) CVVI E12b nd STa F5 F41(6) CVVI E10 nd STa F5 F41(7) NLK99-1 O8 K25 K99 nr F5(8) NLK99-3 O101 K28 K99 nr F5(9) NLK99-5 O9 K30 K99 nr F5(10) NLK99-7 O101 K32 K99 nr F5(11) NLK99-9 O9 K35 K99 nr F5(12) NLK99-11 O9 K37 K99 nr F5(13) NLK99-15 O20 K K99 nr F5(14) NLK99-19 O101 K K99 nr F5(15) DSMZ8696 O55 H6 nr Nr(16) DSMZ9025 mdash mdash mdash(17) DSMZ10973 O6 nr nr(18) ATCC33559 mdash mdash mdashCVVI Central Vietnam Veterinary Institute NL Department of Bacteriology and Animal Science University of Wageningen Netherlands DSMZ Leibniz-Institut DSMZmdashDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH ATCC American Type Culture Collection

of clear inhibition zone surrounding the paper discs soakedwith 100 120583L of neem oil The NO average GIZ mm rangefrom 950 plusmn 070 to 3000 plusmn 100 The NO GIZ varies betweenenteropathogenic and nonenteropathogenic E coli beingrespectively 2433plusmn 058ndash3000plusmn 100 and 950plusmn 070ndash2153plusmn153 It is significantly (119875 lt 005) different with respect to theantibiotic activity However the E coli isolate FLC1167 (frommilk) resulted to be less susceptible and the E coli isolate

NLP097F5 (from piglet feces) the most susceptible to NOtreatment (100 120583L) among all tested bacteria using the discdiffusion method

The CFX GIZ range is 000 plusmn 000ndash3265 plusmn 75 Theenteropathogenic E coli resulted to be resistant or lesssusceptible to CFX than the nonenteropathogenic E colishowing a GIZ range of respectively 000 plusmn 000ndash1824plusmn 168mm and 2164 plusmn 094ndash3265 plusmn 75mm The isolates

6 BioMed Research International

Table 4 Antibacterial activity of neem oil (NO) against forty-eight Escherichia coli isolates revealed as growth inhibition zone (mm)

E coli isolates Growth inhibition zone (mm)lowast

NO (100120583L ) TWN (100120583L) WTR (100120583L) CFX (100 120583L)(1) FLC 1056 1133 plusmn 058 b mdash mdash 3041 plusmn 020 a(2) FLC 1247 1613 plusmn 115 b mdash mdash 3052 plusmn 107 a(3) FLC 1059 1583 plusmn 113 b mdash mdash 2962 plusmn 100 a(4) FLC 1243 1900 plusmn 100 b mdash mdash 3153 plusmn 067 a(5) FLC 1048 1233 plusmn 058 b mdash mdash 2942 plusmn 058 a(6) FLC 1167 950 plusmn 070 b mdash mdash 3061 plusmn 121 a(7) FLC 1249 1333 plusmn 058 b mdash mdash 2961 plusmn 111 a(8) FLC 1055 1453 plusmn 125 b mdash mdash 3175 plusmn 082 a(9) FLC 1054 1623 plusmn 118 b mdash mdash 3141 plusmn 076 a(10) FLC 1085 1800 plusmn 100 b mdash mdash 3053 plusmn 117 a(11) FLC 1244 1533 plusmn 048 b mdash mdash 2886 plusmn 100 a(12) FLC 1165 1950 plusmn 070 b mdash mdash 3133 plusmn 067 a(13) FLC 1086 1133 plusmn 058 b mdash mdash 2982 plusmn 048 a(14) FLC 1053 1453 plusmn 115 b mdash mdash 3265 plusmn 139 a(15) FLC 1095 1683 plusmn 118 b mdash mdash 2905 plusmn 122 a(16) FLC 1219 1070 plusmn 100 b mdash mdash 3275 plusmn 055 a(17) FLC 1235 1323 plusmn 088 b mdash mdash 3015 plusmn 055 a(18) DSM8696 1350 plusmn 050 b mdash mdash 2621 plusmn 100 a(19) DSM9025 1333 plusmn 058 b mdash mdash 2164 plusmn 094 a(20) DSM10973 1353 plusmn 125 b mdash mdash 2914 plusmn 175 a(21) ATCC33559 1383 plusmn 118 b mdash mdash 3212 plusmn 109 a(22) CVVI E210 1300 plusmn 100 a mdash mdash 2583 plusmn 165 a(23) CVVI E173 1223 plusmn 058 b mdash mdash 3235 plusmn 149 a(24) CVVI E12b 2750 plusmn 050 b mdash mdash 1125 plusmn 068 a(25) CVVI E16 1433 plusmn 088 b mdash mdash 2754 plusmn 145 a(26) CVVI E320 2153 plusmn 135 b mdash mdash 2875 plusmn 186 a(27) CVVI E130 1183 plusmn 178 b mdash mdash 2964 plusmn 087 a(28) CVVI E48 1000 plusmn 140 a mdash mdash 2931 plusmn 027 a(29) CVVI KH10 2633 plusmn 053 b mdash mdash 1534 plusmn 066 a(30) CVVI K10B 2750 plusmn 056 b mdash mdash 0(31) CVVI E298 1133 plusmn 048 b mdash mdash 2390 plusmn 169 a(32) CVVI E273 1353 plusmn 175 b mdash mdash 2959 plusmn 177 b(33) CVVI K436 1314 plusmn 168 b mdash mdash 3021 plusmn 138 a(34) CVVI E98 1100 plusmn 100 a mdash mdash 2691 plusmn 156 a(35) CVVI E77 1433 plusmn 058 b mdash mdash 2393 plusmn 059 a(36) CVVI E148 1350 plusmn 050 b mdash mdash 2871 plusmn 087 a(37) CVVI E10 2433 plusmn 058 b mdash mdash 1035 plusmn 111 a(38) CVVI E215 1653 plusmn 115 b mdash mdash 2641 plusmn 140 a(39) NLK99F5 2983 plusmn 118 b mdash mdash 16 21 plusmn 089 a(40) NLP987F5 3000 plusmn 100 a mdash mdash 1321 plusmn 115 a(41) NLK99-1lowast 2173 plusmn 135 b mdash mdash 1534 plusmn 137 a(42) NLK99-3lowast 2833 plusmn 150 b mdash mdash 0(43) NLK99-5lowast 2500 plusmn 110 a mdash mdash 954 plusmn 111 a(44) NLK99-7lowast 2633 plusmn 058 b mdash mdash 1425 plusmn 111 a(45) NLK99-9lowast 2750 plusmn 050 b mdash mdash 1126 plusmn 178 a(46) NLK99-11lowast 2953 plusmn 125 b mdash mdash 1624 plusmn 168 a(47) NLK99-15lowast 2883 plusmn 138 b mdash mdash 1635 plusmn 111 a(48) NLK99-19lowast 2500 plusmn 170 a mdash mdash 1553 plusmn 084 aThree paper discs per plate and three plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Valuesin a row followed by different lowercased letters are significantly different at 119875 le 005

BioMed Research International 7

CVVIK10B (from calve feces) and NLK99-3lowast (from calvefeces) both revealed resistance to CFX No GIZ was detectedin plates treated with negative controls (TWN andWTR)

As shown in Table 5 the percent bacterial GR revealed at100 120583L 10 120583L 1 120583L and 01 120583L NO concentrations was in therange 2371plusmn 100ndash9970plusmn 153 2161plusmn 056ndash9163plusmn 008 1758plusmn 133ndash6957 plusmn 000 and 1118 plusmn 089ndash6758 plusmn 089

There is a significant difference of antibacterial activ-ity among the isolates and the NO concentrations tested(Table 5) The highest percent bacterial GRs were detected at100 120583L NO and they concerned mainly enteropathogenic Ecoli isolates Amplicons of the expected sizes from virulencegenes of enteropathogenic E coli isolates were not detectedwhen bacterial viable cells were checked in samples treatedwith 100 120583L and 10 120583L NO concentrations On the contraryamplicons of the expected size were revealed in the samesamples using primer pair numbers 16 and 17 as reported inTable 1 that specifically amplify unique sequences of E coli16S rRNA

The antibiotic activity of ciprofloxacin is to bind andinhibit bacterial topoisomerase types II and IV thus beingable to interfere with the bacterial processes of replicationtranscription and DNA repair

An increasing ciprofloxacin resistance of E coli isolateswas reported [20] according several epidemiological studiesE coli the most commonly isolated bacterium in clini-cal samples from patients affected by different severity ofdiarrheal symptoms shows high antibiotic resistance [2122] Diarrheagenic E coli considered in the experimentshowed a resistance or less susceptibility to ciprofloxacinin comparison with the other nonenteropathogenic isolatestested

The viable cells of the fourteen diarrheagenic E coliwere checked after NO treatment with primer pairs listedin Table 1 and PMA dye The dye propidium monoazide(PMA Biotium Inc Hayward CA USA) is a photoreactivedye with high affinity for DNA The dye intercalates intoDNA and forms a covalent linkage upon exposure to intensevisible light It is cellmembrane impermeableWhen a samplecomprising both live and dead bacteria is treated with PMAonly dead cells are susceptible to DNA modification dueto their compromised cell membranes [23 24] Thereforeselective detection of the sole live cells is achieved

The fourteen ciprofloxacin resistantless susceptible diar-rheagenic E coli seem to lose their virulence after NO treat-ment because amplicons were obtained only with the primerpairs numbers 16 and 17 (Table 1) This let us suppose thatantibacterial activity acts on adhesion factor and membraneand its permeability with possible loss of extrachromosomalDNA

4 Conclusions and Future Implications

Studies of new antimicrobials from plant derived extractsand agroindustrial byproducts as antimicrobials andpreservatives are important issues in applied microbiologyand biotechnology for both implementing and improvingeffective alternative technologies to tackle antimicrobial

resistance The potential use of plant natural antimicrobialswould require amendments of several different legal textsinvolving areas such as food additives food packaging andhygiene Anyway the applications could concern either thenatural preservation in the food industries or an accessibleand safe alternative to synthetic antimicrobial drugs

Abbreviations

ATCC American Type Culture CollectionCFX Antibiotic hydrochloride monohydrateCRA Agricultural Research Council ItalyCVVI Central Vietnam Veterinary InstituteDAEC Diffusely adherent E coliDSMZ Leibniz-Institut DSMZmdashDeutsche

Sammlung von Mikroorganismen undZellkulturen GmbH

EAEC Enteroaggregative E coliEHEC Enterohemorrhagic E coliEIEC Enteroinvasive E coliELISA Enzyme-linked immunosorbent assayEPEC Enteropathogenic E coliETEC Enterotoxigenic E coliGIZ Growth inhibition zoneHPTLC High performance thin layer

chromatographyHUS Hemolytic uremic syndromeLSD Least significant differenceNCE Neem cake extractNL Department of Bacteriology and Animal

Science University of WageningenNetherlands

NO Neem oilOD Optical densityPMA Propidium monoazideRAPD PCR Random amplified polymorphic DNA

polymerase chain reactionRf Ratio between the migration distance of

substance and the migration distance ofsolvent front

SAS Statistical Analysis System (SAS InstituteInc Cary NC USA)

SD Standard deviationTWN Tween 80WTR Sterile distilled water

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Paola Del Serrone carried out research concept and designcollection andor assembly of biological assay data collectionandor assembly of molecular biology assay data data analy-sis and interpretation statistical analysis writing the papercritical revision of the paper and the final approval of the

8 BioMed Research International

Table 5 Bacterial growth reduction () at 24 h in liquidmediumwith different concentrations ofNO using as reference the control treatment(without NO)

E coli isolates Percent growth reduction zone ()NO (100120583L) NO (10120583L) NO (1 120583L) NO (01 120583L)

(1) FLC 1056 3925 plusmn 143 c 3561 plusmn 100 b 2167 plusmn 133 a 1131 plusmn 208 a(2) FLC 1247 2551 plusmn 115 c 2470 plusmn 100 b 2188 plusmn 133 a 1186 plusmn 100 a(3) FLC 1059 3151 plusmn 115 c 2570 plusmn 100 b 2458 plusmn 133 a 1486 plusmn 100 a(4) FLC 1243 3890 plusmn 100 d 2879 plusmn 100 c 2940 plusmn 000 b 1668 plusmn 120 a(5) FLC 1048 2560 plusmn 153 d 2373 plusmn 208 b 2369 plusmn 200 b 1283 plusmn 173 a(6) FLC 1167 2371 plusmn 100 b 2161 plusmn 058 a 2127 plusmn 000 a 1118 plusmn 089 a(7) FLC 1249 2965 plusmn 153 b 2861 plusmn 100 a 2877 plusmn 133 a 1851 plusmn 208 a(8) FLC 1055 3451 plusmn 115 c 2970 plusmn 100 c 2438 plusmn 133 a 1486 plusmn 100 a(9) FLC 1054 3151 plusmn 115 d 2970 plusmn 100 c 25 78 plusmn 133 a 1486 plusmn 100 a(10) FLC 1085 3990 plusmn 100 c 2879 plusmn 100 b 2810 plusmn 000 b 1558 plusmn 120 a(11) FLC 1244 3570 plusmn 153 d 2873 plusmn 208 b 2859 plusmn 200 b 1263 plusmn 173 a(12) FLC 1165 3971 plusmn 100 c 2961 plusmn 058 c 2917 plusmn 000 b 1648 plusmn 089 a(13) FLC 1086 3955 plusmn 153 c 2861 plusmn 100 c 2787 plusmn 133 b 1221 plusmn 208 a(14) FLC 1053 3451 plusmn 115 c 8970 plusmn 100 c 2758 plusmn 133 b 1486 plusmn 100 a(15) FLC 1095 3151 plusmn 115 c 2970 plusmn 100 b 2518 plusmn 133 a 1486 plusmn 100 a(16) FLC 1219 3895 plusmn 100 b 2879 plusmn 100 b 2810 plusmn 000 b 1668 plusmn 120 a(17) FLC 1235 3670 plusmn 153 d 2773 plusmn 208 c 2779 plusmn 200 b 1283 plusmn 173 a(18) DSM8696 3971 plusmn 100 c 2961 plusmn 058 c 2967 plusmn 000 b 1758 plusmn 089 a(19) DSM9025 3965 plusmn 153 c 2861 plusmn 100 c 2617 plusmn 133 b 1810 plusmn 208 a(20) DSM10973 3451 plusmn 115 c 2970 plusmn 100 c 2328 plusmn 133 b 1486 plusmn 100 a(21) ATCC33559 3151 plusmn 115 d 2970 plusmn 100 c 2158 plusmn 133 b 1486 plusmn 100 a(22) CVVI E210 8890 plusmn 100 c 8879 plusmn 100 c 6920 plusmn 000 b 1478 plusmn 120 a(23) CVVI E173 3650 plusmn 153 d 2773 plusmn 208 c 2799 plusmn 200 b 2283 plusmn 173 a(24) CVVI E126 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 4458 plusmn 089 a(25) CVVI E16 3855 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 1151 plusmn 208 a(26) CVVI E320 3851 plusmn 115 c 2970 plusmn 100 c 2718 plusmn 133 b 1486 plusmn 100 a(27) CVVI E130 3071 plusmn 115 c 2970 plusmn 100 c 2768 plusmn 133 a 1446 plusmn 100 a(28) CVVI E48 3860 plusmn 100 c 2879 plusmn 100 c 2920 plusmn 000 a 1668 plusmn 120 a(29) CVVI KH10 9960 plusmn 153 d 8173 plusmn 208 c 6839 plusmn 200 b 6233 plusmn 173 a(30) CVVI K10B 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 6058 plusmn 089 a(31) CVVI E298 3975 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 2011 plusmn 208 a(32) CVVI E273 3451 plusmn 115 c 2970 plusmn 100 c 2658 plusmn 133 b 2286 plusmn 100 a(33) CVVI K436 3185 plusmn 115 d 2970 plusmn 100 c 2718 plusmn 133 b 1586 plusmn 100 a(34) CVVI E98 3793 plusmn 100 c 2879 plusmn 100 c 2950 plusmn 000 b 1668 plusmn 120 a(35) CVVI E77 3369 plusmn 153 d 2173 plusmn 208 c 2729 plusmn 200 b 1983 plusmn 173 a(36) CVVI E148 3971 plusmn 100 c 2961 plusmn 058 c 2957 plusmn 000 b 2258 plusmn 089 a(37) CVVI E10 8965 plusmn 153 c 8861 plusmn 100 c 6167 plusmn 133 b 5079 plusmn 208 a(38) CVVI E215 3451 plusmn 115 c 2970 plusmn 100 c 1758 plusmn 133 b 2186 plusmn 100 a(39) NLK99F5 9151 plusmn 115 d 8970 plusmn 100 c 6768 plusmn 133 b 6186 plusmn 100 a(40) NL12BF5 8890 plusmn 100 c 8879 plusmn 100 c 6960 plusmn 000 b 6368 plusmn 120 a(41) NLK99-1lowast 9770 plusmn 153 d 8173 plusmn 208 c 6869 plusmn 200 b 6283 plusmn 173 a(42) NLK99-3lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 a(43) NLK99-5lowast 8965 plusmn 153 c 7861 plusmn 100 c 6767 plusmn 133 b 5081 plusmn 208 a(44) NLK99-7lowast 8451 plusmn 115 c 7970 plusmn 100 c 6758 plusmn 133 b 4486 plusmn 100 a(45) NLK99-9lowast 9151 plusmn 115 c 7970 plusmn 100 c 6728 plusmn 113 b 6486 plusmn 100 a(46) NLK99-11lowast 8890 plusmn 100 c 7879 plusmn 100 c 6960 plusmn 000 b 6668 plusmn 120 a(47) NLK99-15lowast 9970 plusmn 153 d 9163 plusmn 028 c 6869 plusmn 200 b 6283 plusmn 173 a(48) NLK99-19lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 aThree plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Values in a row followed by differentlowercased letters are significantly different at 119875 le 005

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

Table 4 Antibacterial activity of neem oil (NO) against forty-eight Escherichia coli isolates revealed as growth inhibition zone (mm)

E coli isolates Growth inhibition zone (mm)lowast

NO (100120583L ) TWN (100120583L) WTR (100120583L) CFX (100 120583L)(1) FLC 1056 1133 plusmn 058 b mdash mdash 3041 plusmn 020 a(2) FLC 1247 1613 plusmn 115 b mdash mdash 3052 plusmn 107 a(3) FLC 1059 1583 plusmn 113 b mdash mdash 2962 plusmn 100 a(4) FLC 1243 1900 plusmn 100 b mdash mdash 3153 plusmn 067 a(5) FLC 1048 1233 plusmn 058 b mdash mdash 2942 plusmn 058 a(6) FLC 1167 950 plusmn 070 b mdash mdash 3061 plusmn 121 a(7) FLC 1249 1333 plusmn 058 b mdash mdash 2961 plusmn 111 a(8) FLC 1055 1453 plusmn 125 b mdash mdash 3175 plusmn 082 a(9) FLC 1054 1623 plusmn 118 b mdash mdash 3141 plusmn 076 a(10) FLC 1085 1800 plusmn 100 b mdash mdash 3053 plusmn 117 a(11) FLC 1244 1533 plusmn 048 b mdash mdash 2886 plusmn 100 a(12) FLC 1165 1950 plusmn 070 b mdash mdash 3133 plusmn 067 a(13) FLC 1086 1133 plusmn 058 b mdash mdash 2982 plusmn 048 a(14) FLC 1053 1453 plusmn 115 b mdash mdash 3265 plusmn 139 a(15) FLC 1095 1683 plusmn 118 b mdash mdash 2905 plusmn 122 a(16) FLC 1219 1070 plusmn 100 b mdash mdash 3275 plusmn 055 a(17) FLC 1235 1323 plusmn 088 b mdash mdash 3015 plusmn 055 a(18) DSM8696 1350 plusmn 050 b mdash mdash 2621 plusmn 100 a(19) DSM9025 1333 plusmn 058 b mdash mdash 2164 plusmn 094 a(20) DSM10973 1353 plusmn 125 b mdash mdash 2914 plusmn 175 a(21) ATCC33559 1383 plusmn 118 b mdash mdash 3212 plusmn 109 a(22) CVVI E210 1300 plusmn 100 a mdash mdash 2583 plusmn 165 a(23) CVVI E173 1223 plusmn 058 b mdash mdash 3235 plusmn 149 a(24) CVVI E12b 2750 plusmn 050 b mdash mdash 1125 plusmn 068 a(25) CVVI E16 1433 plusmn 088 b mdash mdash 2754 plusmn 145 a(26) CVVI E320 2153 plusmn 135 b mdash mdash 2875 plusmn 186 a(27) CVVI E130 1183 plusmn 178 b mdash mdash 2964 plusmn 087 a(28) CVVI E48 1000 plusmn 140 a mdash mdash 2931 plusmn 027 a(29) CVVI KH10 2633 plusmn 053 b mdash mdash 1534 plusmn 066 a(30) CVVI K10B 2750 plusmn 056 b mdash mdash 0(31) CVVI E298 1133 plusmn 048 b mdash mdash 2390 plusmn 169 a(32) CVVI E273 1353 plusmn 175 b mdash mdash 2959 plusmn 177 b(33) CVVI K436 1314 plusmn 168 b mdash mdash 3021 plusmn 138 a(34) CVVI E98 1100 plusmn 100 a mdash mdash 2691 plusmn 156 a(35) CVVI E77 1433 plusmn 058 b mdash mdash 2393 plusmn 059 a(36) CVVI E148 1350 plusmn 050 b mdash mdash 2871 plusmn 087 a(37) CVVI E10 2433 plusmn 058 b mdash mdash 1035 plusmn 111 a(38) CVVI E215 1653 plusmn 115 b mdash mdash 2641 plusmn 140 a(39) NLK99F5 2983 plusmn 118 b mdash mdash 16 21 plusmn 089 a(40) NLP987F5 3000 plusmn 100 a mdash mdash 1321 plusmn 115 a(41) NLK99-1lowast 2173 plusmn 135 b mdash mdash 1534 plusmn 137 a(42) NLK99-3lowast 2833 plusmn 150 b mdash mdash 0(43) NLK99-5lowast 2500 plusmn 110 a mdash mdash 954 plusmn 111 a(44) NLK99-7lowast 2633 plusmn 058 b mdash mdash 1425 plusmn 111 a(45) NLK99-9lowast 2750 plusmn 050 b mdash mdash 1126 plusmn 178 a(46) NLK99-11lowast 2953 plusmn 125 b mdash mdash 1624 plusmn 168 a(47) NLK99-15lowast 2883 plusmn 138 b mdash mdash 1635 plusmn 111 a(48) NLK99-19lowast 2500 plusmn 170 a mdash mdash 1553 plusmn 084 aThree paper discs per plate and three plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Valuesin a row followed by different lowercased letters are significantly different at 119875 le 005

BioMed Research International 7

CVVIK10B (from calve feces) and NLK99-3lowast (from calvefeces) both revealed resistance to CFX No GIZ was detectedin plates treated with negative controls (TWN andWTR)

As shown in Table 5 the percent bacterial GR revealed at100 120583L 10 120583L 1 120583L and 01 120583L NO concentrations was in therange 2371plusmn 100ndash9970plusmn 153 2161plusmn 056ndash9163plusmn 008 1758plusmn 133ndash6957 plusmn 000 and 1118 plusmn 089ndash6758 plusmn 089

There is a significant difference of antibacterial activ-ity among the isolates and the NO concentrations tested(Table 5) The highest percent bacterial GRs were detected at100 120583L NO and they concerned mainly enteropathogenic Ecoli isolates Amplicons of the expected sizes from virulencegenes of enteropathogenic E coli isolates were not detectedwhen bacterial viable cells were checked in samples treatedwith 100 120583L and 10 120583L NO concentrations On the contraryamplicons of the expected size were revealed in the samesamples using primer pair numbers 16 and 17 as reported inTable 1 that specifically amplify unique sequences of E coli16S rRNA

The antibiotic activity of ciprofloxacin is to bind andinhibit bacterial topoisomerase types II and IV thus beingable to interfere with the bacterial processes of replicationtranscription and DNA repair

An increasing ciprofloxacin resistance of E coli isolateswas reported [20] according several epidemiological studiesE coli the most commonly isolated bacterium in clini-cal samples from patients affected by different severity ofdiarrheal symptoms shows high antibiotic resistance [2122] Diarrheagenic E coli considered in the experimentshowed a resistance or less susceptibility to ciprofloxacinin comparison with the other nonenteropathogenic isolatestested

The viable cells of the fourteen diarrheagenic E coliwere checked after NO treatment with primer pairs listedin Table 1 and PMA dye The dye propidium monoazide(PMA Biotium Inc Hayward CA USA) is a photoreactivedye with high affinity for DNA The dye intercalates intoDNA and forms a covalent linkage upon exposure to intensevisible light It is cellmembrane impermeableWhen a samplecomprising both live and dead bacteria is treated with PMAonly dead cells are susceptible to DNA modification dueto their compromised cell membranes [23 24] Thereforeselective detection of the sole live cells is achieved

The fourteen ciprofloxacin resistantless susceptible diar-rheagenic E coli seem to lose their virulence after NO treat-ment because amplicons were obtained only with the primerpairs numbers 16 and 17 (Table 1) This let us suppose thatantibacterial activity acts on adhesion factor and membraneand its permeability with possible loss of extrachromosomalDNA

4 Conclusions and Future Implications

Studies of new antimicrobials from plant derived extractsand agroindustrial byproducts as antimicrobials andpreservatives are important issues in applied microbiologyand biotechnology for both implementing and improvingeffective alternative technologies to tackle antimicrobial

resistance The potential use of plant natural antimicrobialswould require amendments of several different legal textsinvolving areas such as food additives food packaging andhygiene Anyway the applications could concern either thenatural preservation in the food industries or an accessibleand safe alternative to synthetic antimicrobial drugs

Abbreviations

ATCC American Type Culture CollectionCFX Antibiotic hydrochloride monohydrateCRA Agricultural Research Council ItalyCVVI Central Vietnam Veterinary InstituteDAEC Diffusely adherent E coliDSMZ Leibniz-Institut DSMZmdashDeutsche

Sammlung von Mikroorganismen undZellkulturen GmbH

EAEC Enteroaggregative E coliEHEC Enterohemorrhagic E coliEIEC Enteroinvasive E coliELISA Enzyme-linked immunosorbent assayEPEC Enteropathogenic E coliETEC Enterotoxigenic E coliGIZ Growth inhibition zoneHPTLC High performance thin layer

chromatographyHUS Hemolytic uremic syndromeLSD Least significant differenceNCE Neem cake extractNL Department of Bacteriology and Animal

Science University of WageningenNetherlands

NO Neem oilOD Optical densityPMA Propidium monoazideRAPD PCR Random amplified polymorphic DNA

polymerase chain reactionRf Ratio between the migration distance of

substance and the migration distance ofsolvent front

SAS Statistical Analysis System (SAS InstituteInc Cary NC USA)

SD Standard deviationTWN Tween 80WTR Sterile distilled water

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Paola Del Serrone carried out research concept and designcollection andor assembly of biological assay data collectionandor assembly of molecular biology assay data data analy-sis and interpretation statistical analysis writing the papercritical revision of the paper and the final approval of the

8 BioMed Research International

Table 5 Bacterial growth reduction () at 24 h in liquidmediumwith different concentrations ofNO using as reference the control treatment(without NO)

E coli isolates Percent growth reduction zone ()NO (100120583L) NO (10120583L) NO (1 120583L) NO (01 120583L)

(1) FLC 1056 3925 plusmn 143 c 3561 plusmn 100 b 2167 plusmn 133 a 1131 plusmn 208 a(2) FLC 1247 2551 plusmn 115 c 2470 plusmn 100 b 2188 plusmn 133 a 1186 plusmn 100 a(3) FLC 1059 3151 plusmn 115 c 2570 plusmn 100 b 2458 plusmn 133 a 1486 plusmn 100 a(4) FLC 1243 3890 plusmn 100 d 2879 plusmn 100 c 2940 plusmn 000 b 1668 plusmn 120 a(5) FLC 1048 2560 plusmn 153 d 2373 plusmn 208 b 2369 plusmn 200 b 1283 plusmn 173 a(6) FLC 1167 2371 plusmn 100 b 2161 plusmn 058 a 2127 plusmn 000 a 1118 plusmn 089 a(7) FLC 1249 2965 plusmn 153 b 2861 plusmn 100 a 2877 plusmn 133 a 1851 plusmn 208 a(8) FLC 1055 3451 plusmn 115 c 2970 plusmn 100 c 2438 plusmn 133 a 1486 plusmn 100 a(9) FLC 1054 3151 plusmn 115 d 2970 plusmn 100 c 25 78 plusmn 133 a 1486 plusmn 100 a(10) FLC 1085 3990 plusmn 100 c 2879 plusmn 100 b 2810 plusmn 000 b 1558 plusmn 120 a(11) FLC 1244 3570 plusmn 153 d 2873 plusmn 208 b 2859 plusmn 200 b 1263 plusmn 173 a(12) FLC 1165 3971 plusmn 100 c 2961 plusmn 058 c 2917 plusmn 000 b 1648 plusmn 089 a(13) FLC 1086 3955 plusmn 153 c 2861 plusmn 100 c 2787 plusmn 133 b 1221 plusmn 208 a(14) FLC 1053 3451 plusmn 115 c 8970 plusmn 100 c 2758 plusmn 133 b 1486 plusmn 100 a(15) FLC 1095 3151 plusmn 115 c 2970 plusmn 100 b 2518 plusmn 133 a 1486 plusmn 100 a(16) FLC 1219 3895 plusmn 100 b 2879 plusmn 100 b 2810 plusmn 000 b 1668 plusmn 120 a(17) FLC 1235 3670 plusmn 153 d 2773 plusmn 208 c 2779 plusmn 200 b 1283 plusmn 173 a(18) DSM8696 3971 plusmn 100 c 2961 plusmn 058 c 2967 plusmn 000 b 1758 plusmn 089 a(19) DSM9025 3965 plusmn 153 c 2861 plusmn 100 c 2617 plusmn 133 b 1810 plusmn 208 a(20) DSM10973 3451 plusmn 115 c 2970 plusmn 100 c 2328 plusmn 133 b 1486 plusmn 100 a(21) ATCC33559 3151 plusmn 115 d 2970 plusmn 100 c 2158 plusmn 133 b 1486 plusmn 100 a(22) CVVI E210 8890 plusmn 100 c 8879 plusmn 100 c 6920 plusmn 000 b 1478 plusmn 120 a(23) CVVI E173 3650 plusmn 153 d 2773 plusmn 208 c 2799 plusmn 200 b 2283 plusmn 173 a(24) CVVI E126 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 4458 plusmn 089 a(25) CVVI E16 3855 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 1151 plusmn 208 a(26) CVVI E320 3851 plusmn 115 c 2970 plusmn 100 c 2718 plusmn 133 b 1486 plusmn 100 a(27) CVVI E130 3071 plusmn 115 c 2970 plusmn 100 c 2768 plusmn 133 a 1446 plusmn 100 a(28) CVVI E48 3860 plusmn 100 c 2879 plusmn 100 c 2920 plusmn 000 a 1668 plusmn 120 a(29) CVVI KH10 9960 plusmn 153 d 8173 plusmn 208 c 6839 plusmn 200 b 6233 plusmn 173 a(30) CVVI K10B 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 6058 plusmn 089 a(31) CVVI E298 3975 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 2011 plusmn 208 a(32) CVVI E273 3451 plusmn 115 c 2970 plusmn 100 c 2658 plusmn 133 b 2286 plusmn 100 a(33) CVVI K436 3185 plusmn 115 d 2970 plusmn 100 c 2718 plusmn 133 b 1586 plusmn 100 a(34) CVVI E98 3793 plusmn 100 c 2879 plusmn 100 c 2950 plusmn 000 b 1668 plusmn 120 a(35) CVVI E77 3369 plusmn 153 d 2173 plusmn 208 c 2729 plusmn 200 b 1983 plusmn 173 a(36) CVVI E148 3971 plusmn 100 c 2961 plusmn 058 c 2957 plusmn 000 b 2258 plusmn 089 a(37) CVVI E10 8965 plusmn 153 c 8861 plusmn 100 c 6167 plusmn 133 b 5079 plusmn 208 a(38) CVVI E215 3451 plusmn 115 c 2970 plusmn 100 c 1758 plusmn 133 b 2186 plusmn 100 a(39) NLK99F5 9151 plusmn 115 d 8970 plusmn 100 c 6768 plusmn 133 b 6186 plusmn 100 a(40) NL12BF5 8890 plusmn 100 c 8879 plusmn 100 c 6960 plusmn 000 b 6368 plusmn 120 a(41) NLK99-1lowast 9770 plusmn 153 d 8173 plusmn 208 c 6869 plusmn 200 b 6283 plusmn 173 a(42) NLK99-3lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 a(43) NLK99-5lowast 8965 plusmn 153 c 7861 plusmn 100 c 6767 plusmn 133 b 5081 plusmn 208 a(44) NLK99-7lowast 8451 plusmn 115 c 7970 plusmn 100 c 6758 plusmn 133 b 4486 plusmn 100 a(45) NLK99-9lowast 9151 plusmn 115 c 7970 plusmn 100 c 6728 plusmn 113 b 6486 plusmn 100 a(46) NLK99-11lowast 8890 plusmn 100 c 7879 plusmn 100 c 6960 plusmn 000 b 6668 plusmn 120 a(47) NLK99-15lowast 9970 plusmn 153 d 9163 plusmn 028 c 6869 plusmn 200 b 6283 plusmn 173 a(48) NLK99-19lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 aThree plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Values in a row followed by differentlowercased letters are significantly different at 119875 le 005

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

CVVIK10B (from calve feces) and NLK99-3lowast (from calvefeces) both revealed resistance to CFX No GIZ was detectedin plates treated with negative controls (TWN andWTR)

As shown in Table 5 the percent bacterial GR revealed at100 120583L 10 120583L 1 120583L and 01 120583L NO concentrations was in therange 2371plusmn 100ndash9970plusmn 153 2161plusmn 056ndash9163plusmn 008 1758plusmn 133ndash6957 plusmn 000 and 1118 plusmn 089ndash6758 plusmn 089

There is a significant difference of antibacterial activ-ity among the isolates and the NO concentrations tested(Table 5) The highest percent bacterial GRs were detected at100 120583L NO and they concerned mainly enteropathogenic Ecoli isolates Amplicons of the expected sizes from virulencegenes of enteropathogenic E coli isolates were not detectedwhen bacterial viable cells were checked in samples treatedwith 100 120583L and 10 120583L NO concentrations On the contraryamplicons of the expected size were revealed in the samesamples using primer pair numbers 16 and 17 as reported inTable 1 that specifically amplify unique sequences of E coli16S rRNA

The antibiotic activity of ciprofloxacin is to bind andinhibit bacterial topoisomerase types II and IV thus beingable to interfere with the bacterial processes of replicationtranscription and DNA repair

An increasing ciprofloxacin resistance of E coli isolateswas reported [20] according several epidemiological studiesE coli the most commonly isolated bacterium in clini-cal samples from patients affected by different severity ofdiarrheal symptoms shows high antibiotic resistance [2122] Diarrheagenic E coli considered in the experimentshowed a resistance or less susceptibility to ciprofloxacinin comparison with the other nonenteropathogenic isolatestested

The viable cells of the fourteen diarrheagenic E coliwere checked after NO treatment with primer pairs listedin Table 1 and PMA dye The dye propidium monoazide(PMA Biotium Inc Hayward CA USA) is a photoreactivedye with high affinity for DNA The dye intercalates intoDNA and forms a covalent linkage upon exposure to intensevisible light It is cellmembrane impermeableWhen a samplecomprising both live and dead bacteria is treated with PMAonly dead cells are susceptible to DNA modification dueto their compromised cell membranes [23 24] Thereforeselective detection of the sole live cells is achieved

The fourteen ciprofloxacin resistantless susceptible diar-rheagenic E coli seem to lose their virulence after NO treat-ment because amplicons were obtained only with the primerpairs numbers 16 and 17 (Table 1) This let us suppose thatantibacterial activity acts on adhesion factor and membraneand its permeability with possible loss of extrachromosomalDNA

4 Conclusions and Future Implications

Studies of new antimicrobials from plant derived extractsand agroindustrial byproducts as antimicrobials andpreservatives are important issues in applied microbiologyand biotechnology for both implementing and improvingeffective alternative technologies to tackle antimicrobial

resistance The potential use of plant natural antimicrobialswould require amendments of several different legal textsinvolving areas such as food additives food packaging andhygiene Anyway the applications could concern either thenatural preservation in the food industries or an accessibleand safe alternative to synthetic antimicrobial drugs

Abbreviations

ATCC American Type Culture CollectionCFX Antibiotic hydrochloride monohydrateCRA Agricultural Research Council ItalyCVVI Central Vietnam Veterinary InstituteDAEC Diffusely adherent E coliDSMZ Leibniz-Institut DSMZmdashDeutsche

Sammlung von Mikroorganismen undZellkulturen GmbH

EAEC Enteroaggregative E coliEHEC Enterohemorrhagic E coliEIEC Enteroinvasive E coliELISA Enzyme-linked immunosorbent assayEPEC Enteropathogenic E coliETEC Enterotoxigenic E coliGIZ Growth inhibition zoneHPTLC High performance thin layer

chromatographyHUS Hemolytic uremic syndromeLSD Least significant differenceNCE Neem cake extractNL Department of Bacteriology and Animal

Science University of WageningenNetherlands

NO Neem oilOD Optical densityPMA Propidium monoazideRAPD PCR Random amplified polymorphic DNA

polymerase chain reactionRf Ratio between the migration distance of

substance and the migration distance ofsolvent front

SAS Statistical Analysis System (SAS InstituteInc Cary NC USA)

SD Standard deviationTWN Tween 80WTR Sterile distilled water

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Paola Del Serrone carried out research concept and designcollection andor assembly of biological assay data collectionandor assembly of molecular biology assay data data analy-sis and interpretation statistical analysis writing the papercritical revision of the paper and the final approval of the

8 BioMed Research International

Table 5 Bacterial growth reduction () at 24 h in liquidmediumwith different concentrations ofNO using as reference the control treatment(without NO)

E coli isolates Percent growth reduction zone ()NO (100120583L) NO (10120583L) NO (1 120583L) NO (01 120583L)

(1) FLC 1056 3925 plusmn 143 c 3561 plusmn 100 b 2167 plusmn 133 a 1131 plusmn 208 a(2) FLC 1247 2551 plusmn 115 c 2470 plusmn 100 b 2188 plusmn 133 a 1186 plusmn 100 a(3) FLC 1059 3151 plusmn 115 c 2570 plusmn 100 b 2458 plusmn 133 a 1486 plusmn 100 a(4) FLC 1243 3890 plusmn 100 d 2879 plusmn 100 c 2940 plusmn 000 b 1668 plusmn 120 a(5) FLC 1048 2560 plusmn 153 d 2373 plusmn 208 b 2369 plusmn 200 b 1283 plusmn 173 a(6) FLC 1167 2371 plusmn 100 b 2161 plusmn 058 a 2127 plusmn 000 a 1118 plusmn 089 a(7) FLC 1249 2965 plusmn 153 b 2861 plusmn 100 a 2877 plusmn 133 a 1851 plusmn 208 a(8) FLC 1055 3451 plusmn 115 c 2970 plusmn 100 c 2438 plusmn 133 a 1486 plusmn 100 a(9) FLC 1054 3151 plusmn 115 d 2970 plusmn 100 c 25 78 plusmn 133 a 1486 plusmn 100 a(10) FLC 1085 3990 plusmn 100 c 2879 plusmn 100 b 2810 plusmn 000 b 1558 plusmn 120 a(11) FLC 1244 3570 plusmn 153 d 2873 plusmn 208 b 2859 plusmn 200 b 1263 plusmn 173 a(12) FLC 1165 3971 plusmn 100 c 2961 plusmn 058 c 2917 plusmn 000 b 1648 plusmn 089 a(13) FLC 1086 3955 plusmn 153 c 2861 plusmn 100 c 2787 plusmn 133 b 1221 plusmn 208 a(14) FLC 1053 3451 plusmn 115 c 8970 plusmn 100 c 2758 plusmn 133 b 1486 plusmn 100 a(15) FLC 1095 3151 plusmn 115 c 2970 plusmn 100 b 2518 plusmn 133 a 1486 plusmn 100 a(16) FLC 1219 3895 plusmn 100 b 2879 plusmn 100 b 2810 plusmn 000 b 1668 plusmn 120 a(17) FLC 1235 3670 plusmn 153 d 2773 plusmn 208 c 2779 plusmn 200 b 1283 plusmn 173 a(18) DSM8696 3971 plusmn 100 c 2961 plusmn 058 c 2967 plusmn 000 b 1758 plusmn 089 a(19) DSM9025 3965 plusmn 153 c 2861 plusmn 100 c 2617 plusmn 133 b 1810 plusmn 208 a(20) DSM10973 3451 plusmn 115 c 2970 plusmn 100 c 2328 plusmn 133 b 1486 plusmn 100 a(21) ATCC33559 3151 plusmn 115 d 2970 plusmn 100 c 2158 plusmn 133 b 1486 plusmn 100 a(22) CVVI E210 8890 plusmn 100 c 8879 plusmn 100 c 6920 plusmn 000 b 1478 plusmn 120 a(23) CVVI E173 3650 plusmn 153 d 2773 plusmn 208 c 2799 plusmn 200 b 2283 plusmn 173 a(24) CVVI E126 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 4458 plusmn 089 a(25) CVVI E16 3855 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 1151 plusmn 208 a(26) CVVI E320 3851 plusmn 115 c 2970 plusmn 100 c 2718 plusmn 133 b 1486 plusmn 100 a(27) CVVI E130 3071 plusmn 115 c 2970 plusmn 100 c 2768 plusmn 133 a 1446 plusmn 100 a(28) CVVI E48 3860 plusmn 100 c 2879 plusmn 100 c 2920 plusmn 000 a 1668 plusmn 120 a(29) CVVI KH10 9960 plusmn 153 d 8173 plusmn 208 c 6839 plusmn 200 b 6233 plusmn 173 a(30) CVVI K10B 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 6058 plusmn 089 a(31) CVVI E298 3975 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 2011 plusmn 208 a(32) CVVI E273 3451 plusmn 115 c 2970 plusmn 100 c 2658 plusmn 133 b 2286 plusmn 100 a(33) CVVI K436 3185 plusmn 115 d 2970 plusmn 100 c 2718 plusmn 133 b 1586 plusmn 100 a(34) CVVI E98 3793 plusmn 100 c 2879 plusmn 100 c 2950 plusmn 000 b 1668 plusmn 120 a(35) CVVI E77 3369 plusmn 153 d 2173 plusmn 208 c 2729 plusmn 200 b 1983 plusmn 173 a(36) CVVI E148 3971 plusmn 100 c 2961 plusmn 058 c 2957 plusmn 000 b 2258 plusmn 089 a(37) CVVI E10 8965 plusmn 153 c 8861 plusmn 100 c 6167 plusmn 133 b 5079 plusmn 208 a(38) CVVI E215 3451 plusmn 115 c 2970 plusmn 100 c 1758 plusmn 133 b 2186 plusmn 100 a(39) NLK99F5 9151 plusmn 115 d 8970 plusmn 100 c 6768 plusmn 133 b 6186 plusmn 100 a(40) NL12BF5 8890 plusmn 100 c 8879 plusmn 100 c 6960 plusmn 000 b 6368 plusmn 120 a(41) NLK99-1lowast 9770 plusmn 153 d 8173 plusmn 208 c 6869 plusmn 200 b 6283 plusmn 173 a(42) NLK99-3lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 a(43) NLK99-5lowast 8965 plusmn 153 c 7861 plusmn 100 c 6767 plusmn 133 b 5081 plusmn 208 a(44) NLK99-7lowast 8451 plusmn 115 c 7970 plusmn 100 c 6758 plusmn 133 b 4486 plusmn 100 a(45) NLK99-9lowast 9151 plusmn 115 c 7970 plusmn 100 c 6728 plusmn 113 b 6486 plusmn 100 a(46) NLK99-11lowast 8890 plusmn 100 c 7879 plusmn 100 c 6960 plusmn 000 b 6668 plusmn 120 a(47) NLK99-15lowast 9970 plusmn 153 d 9163 plusmn 028 c 6869 plusmn 200 b 6283 plusmn 173 a(48) NLK99-19lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 aThree plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Values in a row followed by differentlowercased letters are significantly different at 119875 le 005

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

Table 5 Bacterial growth reduction () at 24 h in liquidmediumwith different concentrations ofNO using as reference the control treatment(without NO)

E coli isolates Percent growth reduction zone ()NO (100120583L) NO (10120583L) NO (1 120583L) NO (01 120583L)

(1) FLC 1056 3925 plusmn 143 c 3561 plusmn 100 b 2167 plusmn 133 a 1131 plusmn 208 a(2) FLC 1247 2551 plusmn 115 c 2470 plusmn 100 b 2188 plusmn 133 a 1186 plusmn 100 a(3) FLC 1059 3151 plusmn 115 c 2570 plusmn 100 b 2458 plusmn 133 a 1486 plusmn 100 a(4) FLC 1243 3890 plusmn 100 d 2879 plusmn 100 c 2940 plusmn 000 b 1668 plusmn 120 a(5) FLC 1048 2560 plusmn 153 d 2373 plusmn 208 b 2369 plusmn 200 b 1283 plusmn 173 a(6) FLC 1167 2371 plusmn 100 b 2161 plusmn 058 a 2127 plusmn 000 a 1118 plusmn 089 a(7) FLC 1249 2965 plusmn 153 b 2861 plusmn 100 a 2877 plusmn 133 a 1851 plusmn 208 a(8) FLC 1055 3451 plusmn 115 c 2970 plusmn 100 c 2438 plusmn 133 a 1486 plusmn 100 a(9) FLC 1054 3151 plusmn 115 d 2970 plusmn 100 c 25 78 plusmn 133 a 1486 plusmn 100 a(10) FLC 1085 3990 plusmn 100 c 2879 plusmn 100 b 2810 plusmn 000 b 1558 plusmn 120 a(11) FLC 1244 3570 plusmn 153 d 2873 plusmn 208 b 2859 plusmn 200 b 1263 plusmn 173 a(12) FLC 1165 3971 plusmn 100 c 2961 plusmn 058 c 2917 plusmn 000 b 1648 plusmn 089 a(13) FLC 1086 3955 plusmn 153 c 2861 plusmn 100 c 2787 plusmn 133 b 1221 plusmn 208 a(14) FLC 1053 3451 plusmn 115 c 8970 plusmn 100 c 2758 plusmn 133 b 1486 plusmn 100 a(15) FLC 1095 3151 plusmn 115 c 2970 plusmn 100 b 2518 plusmn 133 a 1486 plusmn 100 a(16) FLC 1219 3895 plusmn 100 b 2879 plusmn 100 b 2810 plusmn 000 b 1668 plusmn 120 a(17) FLC 1235 3670 plusmn 153 d 2773 plusmn 208 c 2779 plusmn 200 b 1283 plusmn 173 a(18) DSM8696 3971 plusmn 100 c 2961 plusmn 058 c 2967 plusmn 000 b 1758 plusmn 089 a(19) DSM9025 3965 plusmn 153 c 2861 plusmn 100 c 2617 plusmn 133 b 1810 plusmn 208 a(20) DSM10973 3451 plusmn 115 c 2970 plusmn 100 c 2328 plusmn 133 b 1486 plusmn 100 a(21) ATCC33559 3151 plusmn 115 d 2970 plusmn 100 c 2158 plusmn 133 b 1486 plusmn 100 a(22) CVVI E210 8890 plusmn 100 c 8879 plusmn 100 c 6920 plusmn 000 b 1478 plusmn 120 a(23) CVVI E173 3650 plusmn 153 d 2773 plusmn 208 c 2799 plusmn 200 b 2283 plusmn 173 a(24) CVVI E126 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 4458 plusmn 089 a(25) CVVI E16 3855 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 1151 plusmn 208 a(26) CVVI E320 3851 plusmn 115 c 2970 plusmn 100 c 2718 plusmn 133 b 1486 plusmn 100 a(27) CVVI E130 3071 plusmn 115 c 2970 plusmn 100 c 2768 plusmn 133 a 1446 plusmn 100 a(28) CVVI E48 3860 plusmn 100 c 2879 plusmn 100 c 2920 plusmn 000 a 1668 plusmn 120 a(29) CVVI KH10 9960 plusmn 153 d 8173 plusmn 208 c 6839 plusmn 200 b 6233 plusmn 173 a(30) CVVI K10B 8981 plusmn 100 c 8961 plusmn 058 c 6937 plusmn 000 b 6058 plusmn 089 a(31) CVVI E298 3975 plusmn 153 c 2861 plusmn 100 c 2757 plusmn 133 b 2011 plusmn 208 a(32) CVVI E273 3451 plusmn 115 c 2970 plusmn 100 c 2658 plusmn 133 b 2286 plusmn 100 a(33) CVVI K436 3185 plusmn 115 d 2970 plusmn 100 c 2718 plusmn 133 b 1586 plusmn 100 a(34) CVVI E98 3793 plusmn 100 c 2879 plusmn 100 c 2950 plusmn 000 b 1668 plusmn 120 a(35) CVVI E77 3369 plusmn 153 d 2173 plusmn 208 c 2729 plusmn 200 b 1983 plusmn 173 a(36) CVVI E148 3971 plusmn 100 c 2961 plusmn 058 c 2957 plusmn 000 b 2258 plusmn 089 a(37) CVVI E10 8965 plusmn 153 c 8861 plusmn 100 c 6167 plusmn 133 b 5079 plusmn 208 a(38) CVVI E215 3451 plusmn 115 c 2970 plusmn 100 c 1758 plusmn 133 b 2186 plusmn 100 a(39) NLK99F5 9151 plusmn 115 d 8970 plusmn 100 c 6768 plusmn 133 b 6186 plusmn 100 a(40) NL12BF5 8890 plusmn 100 c 8879 plusmn 100 c 6960 plusmn 000 b 6368 plusmn 120 a(41) NLK99-1lowast 9770 plusmn 153 d 8173 plusmn 208 c 6869 plusmn 200 b 6283 plusmn 173 a(42) NLK99-3lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 a(43) NLK99-5lowast 8965 plusmn 153 c 7861 plusmn 100 c 6767 plusmn 133 b 5081 plusmn 208 a(44) NLK99-7lowast 8451 plusmn 115 c 7970 plusmn 100 c 6758 plusmn 133 b 4486 plusmn 100 a(45) NLK99-9lowast 9151 plusmn 115 c 7970 plusmn 100 c 6728 plusmn 113 b 6486 plusmn 100 a(46) NLK99-11lowast 8890 plusmn 100 c 7879 plusmn 100 c 6960 plusmn 000 b 6668 plusmn 120 a(47) NLK99-15lowast 9970 plusmn 153 d 9163 plusmn 028 c 6869 plusmn 200 b 6283 plusmn 173 a(48) NLK99-19lowast 8971 plusmn 100 c 7961 plusmn 058 c 6957 plusmn 000 b 6758 plusmn 089 aThree plates for each bacterium were considered The experiment was repeated twice Values are given as mean plusmn SD Values in a row followed by differentlowercased letters are significantly different at 119875 le 005

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

paper Chiara Toniolo was responsible for collection andorassembly of chemical data data analysis and interpretationcritical revision of the paper and final approval of the paperMarcello Nicoletti carried out collection andor assembly ofchemical data data analysis and interpretation writing thepaper critical revision of the paper and final approval of thepaper

Acknowledgments

The authors thank Dr D Carminati of the Fodder andDairy Productions Research Centre (CRA FLC) of LodiAgricultural Research Council (CRA) Italy Dr vu Kackof Central Vietnam Veterinary Institute and Dr F G VanZijderveld of the University of Wageningen The Netherlandfor kindly providing the E coli isolates

References

[1] J Raloff ldquoDrugged watersrdquo Science News vol 153 pp 187ndash1891998

[2] DMMcKeon J P Calabrese andGK Bissonnette ldquoAntibioticresistant gram-negative bacteria in rural groundwater suppliesrdquoWater Research vol 29 no 8 pp 1902ndash1908 1995

[3] M Droge A Puhler and W Selbitschka ldquoPhenotypic andmolecular characterization of conjugative antibiotic resistanceplasmids isolated from bacterial communities of activatedsludgerdquoMolecular and General Genetics vol 263 no 3 pp 471ndash482 2000

[4] A R Manges S P Smith B J Lau et al ldquoRetail meatconsumption and the acquisition of antimicrobial resistantEscherichia coli causing urinary tract infections a case-controlstudyrdquo Foodborne Pathogens and Disease vol 4 no 4 pp 419ndash431 2007

[5] P Del Serrone R Contillo S Failla et al ldquoAssessment of themicrobiological quality of retail fresh porkmeat in central ItalyrdquoItalian Journal of Food Science vol 18 no 4 pp 397ndash408 2006

[6] K Palaniappan and R A Holley ldquoUse of natural antimicrobialsto increase antibiotic susceptibility of drug resistant bacteriardquoInternational Journal of Food Microbiology vol 140 no 2-3 pp164ndash168 2010

[7] A Rawani S Pal and G Chandra ldquoEvaluation of antimicrobialproperties of four plant extracts against human pathogensrdquoAsian Pacific Journal of Tropical Biomedicine vol 1 no 1 ppS71ndashS75 2011

[8] P Del Serrone and M Nicoletti ldquoAntimicrobial activity of aneem cake extract in a broth model meat systemrdquo InternationalJournal of Environmental Research and Public Health vol 10 no8 pp 3282ndash3295 2013

[9] EFSA ldquoTheCommunity summary report on trends and sourcesof zoonoses zoonotic agents and foodborne outbreaks inEuropean Union in 2008rdquoThe EFSA Journal vol 8 no 1 2010

[10] Comunicazione della Commissione al Parlamento Europeo eal Consiglio ldquoPiano drsquoazione di lotta ai crescenti rischi diresistenza antimicrobica (AMR)rdquo Tech Rep COM 2011-7482011

[11] ldquoConclusioni del Consiglio del 22 giugno 2012 sullrsquoimpattodella resistenza antimicrobica nel settore della salute umana enel settore veterinariomdashuna prospettiva di tipo lsquoOne HealthrsquordquoGazzetta Ufficiale 2012C 21102 2012

[12] National Research Council ldquoNeem a tree for solving globalproblemsrdquo Report of an Ad Hoc Panel of the Board on Sci-ence and Technology for International Development NationalAcademy Press Washington DC USA 1992

[13] M Baswa C C Rath S K Dash and R K Mishra ldquoAntibac-terial activity of Karanj (Pongamia pinnata) and Neem (Azadir-achta indica) seed oil a preliminary reportrdquoMicrobios vol 105no 412 pp 183ndash189 2001

[14] M SaiRam G Ilavazhagan S K Sharma et al ldquoAnti-microbialactivity of a new vaginal contraceptive NIM-76 from neem oil(Azadirachta indica)rdquo Journal of Ethnopharmacology vol 71 no3 pp 377ndash382 2000

[15] M Zago B Bonvini A M M Platero G Mucchetti DCarminati and G Giraffa ldquoCharacterisation of Escherichia coliisolated from rawmilk cheesesrdquo Annals of Microbiology vol 57no 1 pp 49ndash54 2007

[16] F G van Zijderveld and E Overdijk ldquoExperiences with theELISA for detection of the E coli K99 antigen in calf faecesrdquoAnnales de Recherches Veterinaires vol 14 no 4 pp 395ndash3991983

[17] M Nicoletti S Mariani O MacCioni T Coccioletti and KMurugan ldquoNeem cake chemical composition and larvicidalactivity on Asian tiger mosquitordquo Parasitology Research vol 111no 1 pp 205ndash213 2012

[18] M Nicoletti ldquoHPTLC fingerprint a modern approach for theanalytical determination of botanicalsrdquo Brazilian Journal ofPharmacognosy vol 21 no 5 pp 818ndash823 2011

[19] AW BauerWMKirby J C Sherris andM Turck ldquoAntibioticsusceptibility testing by a standardized single disk methodrdquoAmerican Journal of Clinical Pathology vol 36 pp 493ndash4961966

[20] F E Abdullah A A Memon M Y Bandukda and M JamilldquoIncreasing ciprofloxacin resistance of isolates from infectedurines of a cross-section of patients in Karachirdquo BMC ResearchNotes vol 5 article 696 2012

[21] H Momtaz F S Dehkordi M J Hosseini M Sarshar and MHeidari ldquoSerogroups virulence genes and antibiotic resistancein Shiga toxin-producingEscherichia coliisolated fromdiarrheicand non-diarrheic pediatric patients in Iranrdquo Gut Pathogensvol 5 no 1 article 39 2013

[22] V N Trung V L Phung H L Chinh and A WeintraubldquoAntibiotic resistance in diarrheagenic Escherichia coli andShigella strains isolated from children in Hanoi VietnamrdquoAntimicrobial Agents and Chemotherapy vol 49 no 2 pp 816ndash819 2005

[23] A Nocker C-Y Cheung and A K Camper ldquoComparison ofpropidiummonoazide with ethidiummonoazide for differenti-ation of live vs dead bacteria by selective removal of DNA fromdead cellsrdquo Journal of Microbiological Methods vol 67 no 2 pp310ndash320 2006

[24] S Chen F Wang J C Beaulieu R E Stein and B Ge ldquoRapiddetection of viable Salmonellae in produce by coupling propid-ium monoazide with loop-mediated isothermal amplificationrdquoApplied and Environmental Microbiology vol 77 no 12 pp4008ndash4016 2011

[25] J Osek ldquoMultiplex polymerase chain reaction assay for iden-tification of enterotoxigenic Escherichia coli strainsrdquo Journal ofVeterinary Diagnostic Investigation vol 13 no 4 pp 308ndash3112001

[26] B Ojeniyi P Ahrens and A Meyling ldquoDetection of fimbrialand toxin genes in Escherichia coli and their prevalence in

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 BioMed Research International

piglets with diarrhea The application of colony hybridizationassay polymerase chain reaction and phenotypic assaysrdquo Jour-nal of Veterinary Medicine vol 41 no 1 pp 49ndash59 1994

[27] M Blanco N L Padola A Kruger et al ldquoVirulence genes andintimin types of Shiga-toxin-producing Escherichia coli isolatedfrom cattle and beef products in Argentinardquo InternationalMicrobiology vol 7 no 4 pp 269ndash276 2004

[28] T Yamamoto and M Nakazawa ldquoDetection and sequences ofthe enteroaggregative Escherichia coli heat- stable enterotoxin 1gene in enterotoxigenic E coli strains isolated from piglets andcalves with diarrheardquo Journal of Clinical Microbiology vol 35no 1 pp 223ndash227 1997

[29] G Wang C G Clark and F G Rodgerst ldquoDetection inEscherichia coli of the genes encoding the major virulence fac-tors the genes defining the O157H7 serotype and componentsof the type 2 Shiga toxin family by multiplex PCRrdquo Journal ofClinical Microbiology vol 40 no 10 pp 3613ndash3619 2002

[30] H V Vu Khac E Holoda E Pilipcinec et al ldquoSerotypesvirulence genes and PFGE profiles of Escherichia coli isolatedfrom pigs with postweaning diarrhoea in Slovakiardquo BMCVeterinary Research vol 2 article 10 2006

[31] H Imberechts N van Pelt H de Greve and P LintermansldquoSequences related to the major subunit gene fedA of F107fimbriae in porcine Escherichia coli strains that express adhesivefimbriaerdquoFEMSMicrobiology Letters vol 119 no 3 pp 309ndash3141994

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology