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ORIGINAL ARTICLE Molecular typing of Staphylococcus aureus isolated from food samples in Iran Masoud Haghkhah & Zahra Esmailnezhad & Narjes Ghaffari Received: 6 August 2012 / Accepted: 15 May 2013 # Springer-Verlag London 2013 Abstract Staphylococcus aureus is the third most common cause of confirmed food poisoning in the world and is the predominant species involved in staphylococcal food poi- soning outbreaks. Considerable genetic heterogeneity has been shown in natural populations of S. aureus isolates. Coagulase gene typing is one of the numerous molecular techniques to identify and compare S. aureus genotypes. The present study was conducted to type the coagulase gene in 25 S. aureus isolates isolated from food samples. All isolates were identified by routine biochemical tests and then confirmed by species-specific PCR and yielded prod- ucts with the expected molecular size of 1.3 kb. PCR am- plification of DNA with the primers COAG2 and COAG3 yielded single-banded PCR products in 24 isolates with the molecular size of approximately 500 bp (n =2, 8 %), 750 bp (n = 1, 4 %), 850 bp (n =12, 48 %), and 950 bp (n = 9, 36 %), while one isolate produced no band in PCR amplification of coagulase gene. Since human and bovine reservoirs of S. aureus represent two subpopulations that rarely cross-infect, detection of single bands by coagulase PCR in S. aureus isolates suggests that these isolates may be of bovine origin not human one, and contamination of food samples may initiate from the animal source not the food handlers. Digestion of coagulase PCR products with restriction endo- nuclease enzymes AluI and Hin6I yielded four different restriction profiles that indicate presence of heterogeneity in the coagulase gene of the isolates. This work showed that restriction analysis of the coagulase gene can be considered as a reliable and fast method for determining the origin of S. aureus in food samples. Keywords Food . Iran . Molecular typing . Staphylococcus aureus Introduction The predominant species involved in staphylococcal food poisoning outbreaks is Staphylococcus aureus (Aragon- Alegro et al. 2007) which is considered the third most common cause of confirmed food poisoning in the world (Acco et al. 2003). Considerable genetic heterogeneity has been shown in natural populations of S. aureus isolates (Tenover et al. 1994; Kapur et al. 1995). Coagulase gene typing is one of the numerous molecular techniques to identify and compare S. aureus genotypes. This technique has been considered as a simple, accurate, sufficiently reproducible, specific, easy to interpret, and discriminatory method for typing S. aureus isolates (Goh et al. 1992; Raimundo et al. 1999; Shopsin et al. 2000; Schlegelova et al. 2003; Dastmalchi Saei et al. 2009; Momtaz et al. 2011). Molecular studies of S. aureus isolated from human or bovine mastitic milk have indicated that human and bovine reservoirs of S. aureus represent two subpopulations that rarely cross-infect (Lange et al. 1999; Larsen et al. 2000; Karahan and Cetinkaya 2007). Lack of proper hygienic measures during food preparation is one of the major sources of contamination as the food handlers themselves can harbor the pathogenic bacterium (Stewart et al. 2002; Huong et al. 2010). On the other hand, the bacterium can be frequently isolated in raw milk of cattle suffering from mastitis and various dairy products and milk can harbor toxigenic strains (Le Loir et al. 2003; Huong et al. 2010). Proper knowledge about the source of contamination of M. Haghkhah (*) : Z. Esmailnezhad : N. Ghaffari Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz 71345-1731, Iran e-mail: [email protected] Z. Esmailnezhad e-mail: [email protected] N. Ghaffari e-mail: [email protected] Comp Clin Pathol DOI 10.1007/s00580-013-1764-y

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Page 1: Molecular typing of Staphylococcus aureus isolated from food samples in Iran

ORIGINAL ARTICLE

Molecular typing of Staphylococcus aureus isolated from foodsamples in Iran

Masoud Haghkhah & Zahra Esmailnezhad & Narjes Ghaffari

Received: 6 August 2012 /Accepted: 15 May 2013# Springer-Verlag London 2013

Abstract Staphylococcus aureus is the third most commoncause of confirmed food poisoning in the world and is thepredominant species involved in staphylococcal food poi-soning outbreaks. Considerable genetic heterogeneity hasbeen shown in natural populations of S. aureus isolates.Coagulase gene typing is one of the numerous moleculartechniques to identify and compare S. aureus genotypes.The present study was conducted to type the coagulase genein 25 S. aureus isolates isolated from food samples. Allisolates were identified by routine biochemical tests andthen confirmed by species-specific PCR and yielded prod-ucts with the expected molecular size of 1.3 kb. PCR am-plification of DNA with the primers COAG2 and COAG3yielded single-banded PCR products in 24 isolates with themolecular size of approximately 500 bp (n=2, 8 %), 750 bp(n=1, 4 %), 850 bp (n=12, 48 %), and 950 bp (n=9, 36 %),while one isolate produced no band in PCR amplification ofcoagulase gene. Since human and bovine reservoirs of S.aureus represent two subpopulations that rarely cross-infect,detection of single bands by coagulase PCR in S. aureusisolates suggests that these isolates may be of bovine originnot human one, and contamination of food samples mayinitiate from the animal source not the food handlers.Digestion of coagulase PCR products with restriction endo-nuclease enzymes AluI and Hin6I yielded four differentrestriction profiles that indicate presence of heterogeneityin the coagulase gene of the isolates. This work showed thatrestriction analysis of the coagulase gene can be considered

as a reliable and fast method for determining the origin of S.aureus in food samples.

Keywords Food . Iran . Molecular typing . Staphylococcusaureus

Introduction

The predominant species involved in staphylococcal foodpoisoning outbreaks is Staphylococcus aureus (Aragon-Alegro et al. 2007) which is considered the third mostcommon cause of confirmed food poisoning in the world(Acco et al. 2003).

Considerable genetic heterogeneity has been shown innatural populations of S. aureus isolates (Tenover et al.1994; Kapur et al. 1995). Coagulase gene typing is one ofthe numerous molecular techniques to identify and compareS. aureus genotypes. This technique has been considered asa simple, accurate, sufficiently reproducible, specific, easyto interpret, and discriminatory method for typing S. aureusisolates (Goh et al. 1992; Raimundo et al. 1999; Shopsin etal. 2000; Schlegelova et al. 2003; Dastmalchi Saei et al.2009; Momtaz et al. 2011).

Molecular studies of S. aureus isolated from human orbovine mastitic milk have indicated that human and bovinereservoirs of S. aureus represent two subpopulations thatrarely cross-infect (Lange et al. 1999; Larsen et al. 2000;Karahan and Cetinkaya 2007). Lack of proper hygienicmeasures during food preparation is one of the majorsources of contamination as the food handlers themselvescan harbor the pathogenic bacterium (Stewart et al. 2002;Huong et al. 2010). On the other hand, the bacterium can befrequently isolated in raw milk of cattle suffering frommastitis and various dairy products and milk can harbortoxigenic strains (Le Loir et al. 2003; Huong et al. 2010).Proper knowledge about the source of contamination of

M. Haghkhah (*) : Z. Esmailnezhad :N. GhaffariDepartment of Pathobiology, School of Veterinary Medicine,Shiraz University, Shiraz 71345-1731, Irane-mail: [email protected]

Z. Esmailnezhade-mail: [email protected]

N. Ghaffarie-mail: [email protected]

Comp Clin PatholDOI 10.1007/s00580-013-1764-y

Page 2: Molecular typing of Staphylococcus aureus isolated from food samples in Iran

food samples by S. aureus is important for the effectivecontrol of staphylococcal food poisoning outbreaks andimprovement of hygienic measures; therefore, the presentstudy was conducted. In this study, coagulase gene restric-tion fragment length polymorphism (RFLP) patterns wereused to investigate the heterogeneity in the genes of theisolates of S. aureus from food samples in Iran.

Materials and methods

Bacterial isolates

Twenty-five S. aureus isolates isolated from food sampleswerekindly provided by Dr. T. Zahraei Salehi, College of VeterinaryMedicine, Tehran University, Iran. All isolates were identifiedbiochemically as S. aureus and stored in Trypticase Soy Broth(Merck) containing 15 % glycerol at −20 °C. S. aureus ATTC1556 and Staphylococcus epidermidis were served as positiveand negative controls, respectively.

DNA extraction

Each isolate was subcultured overnight on sheep blood Agar(Merck) before DNA extraction. Bacterial DNA wasextracted from overnight cultures using Genomic DNAPurification Kit (Fermentas, EU).

Amplification of S. aureus-specific gene

PCRs for confirmation of S. aureus were performed in athermocycler (Eppendorf, Germany) in a reaction volume of50 μl, containing 5 μl 10× PCR buffer (500 mM KCl andTris HCl, pH 8.4), 5 μl 25 mM MgCl2, 200 mM of eachdeoxynucleotide triphosphate, 1 U Taq DNA Polymerase(CinnaGen, Iran), 50 pmol of each primer, and 25 ng oftemplate DNA. Primers Sau327 (5′ GGACGACATTAGACGAATCA 3′) and Sau1645 (5′ CGGGCACCTATTTTCTATCT 3′), derived from the 23S rRNA gene of S. aureus,were used for species-specific PCR (Karahan and Cetinkaya2007). After initial denaturation at 94 °C for 3 min, PCRproducts were amplified by 35 cycles of denaturation at94 °C for 45 s, annealing at 58.5 °C for 45 s, and extensionat 72 °C for 2 min, with a final extension step at 72 °C for10 min. The amplified products were detected by stainingwith ethidium bromide (CinnaGen, Iran) in 1 % agarose gel(Fermentas, EU) and photographed under UV light.

PCR amplification of coagulase gene

DNA samples were amplified by PCR for the coagulasegene using the primers COAG2 (5′ CGAGACCAAGATTCAACAAG 3′) and COAG3 (5′ AAAGAAAACCAC

TCACATCA 3′) (Karahan and Cetinkaya 2007). The PCRmixture was prepared as described above and amplified asfollowing: initial denaturation at 95 °C for 3 min; 30 cyclesof 95 °C for 45 s, 58 °C for 45 s, 72 °C for 1 min; and finalextension at 72 °C for 10 min. Presence of coagulase genewas confirmed by gel electrophoresis.

Restriction enzyme digestion

The PCR products were digested with AluI and Hin6I(Fermentas, EU) for RFLP analysis, a technique that exploitsvariations in homologous DNA sequences. For this purpose,12.5 μl of each PCR product was mixed with 2 U enzyme and1.5 μl 10× restriction buffer and incubated at 37 °C overnight(Karahan and Cetinkaya 2007). The digested products wereseparated in a 2 % agarose gel (Fermentas, EU), stained withethidium bromide (CinnaGen, Iran), and photographed underUV illumination.

Results

S. aureus species-specific PCR

All 25 S. aureus isolates isolated from food samples as wellas positive control were confirmed as S. aureus by species-specific PCR with the primers Sau327 and Sau1645 andyielded products with the expected molecular size of 1.3 kb.S. epidermidis which served as negative control producedno DNA products following PCR amplification.

Coagulase gene PCR

PCR amplification of DNA from food S. aureus isolates withthe primers COAG2 and COAG3 yielded approximately500 bp (n=2, 8 %), 750 bp (n=1, 4 %), 850 bp (n=12,48 %), and 950 bp (n=9, 36 %) PCR products, one isolateproduced no band in PCR amplification of coagulase gene.One isolate produced no band in PCR amplification of coag-ulase gene. A single band in coagulase gene PCR was ob-served in 24 isolates that yielded PCR amplification of coag-ulase gene. The primers used to amplify species-specific genefor S. aureus and coagulase gene are shown in Table 1.

Table 1 Primers used to amplify species-specific gene for S. aureusand coagulase gene

Name Sequence

Sau327 5′ GGACGACATTAGACGAATCA 3′

Sau1645 5′ CGGGCACCTATTTTCTATCT 3′

COAG2 5′ CGAGACCAAGATTCAACAAG 3′

COAG3 5′ AAAGAAAACCACTCACATCA 3′

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Page 3: Molecular typing of Staphylococcus aureus isolated from food samples in Iran

Restriction analysis

Digestion with AluI

Digestion of coagulase PCR products with AluI yielded fourdifferent restriction profiles. Following AluI digestion, 10strains contained three bands of approximately 420, 250,and <100 bp, 5 strains contained three bands of approxi-mately 650, 160, and <100 bp, 2 strains contained twobands of approximately 420 and <100 bp, and 1 straincontained two bands of approximately 420 and 340 bp(Fig. 1). Six isolates were not digested following AluI di-gestion. The results are shown in Table 2.

Digestion with Hin6I

Digestion of coagulase PCR products with Hin6I yielded fourdifferent restriction profiles. Digestion of coagulase gene usingHin6I resulted in 10 strains that contained three bands ofapproximately 600, 160, and <100 bp, 5 strains that containedthree bands of approximately 420, 250, and <100 bp, 5 strainsthat contained three bands of approximately 500 and <100 bp,and 2 strains that contained three bands of approximately 420,160, and <100 bp (Fig. 2). Two isolates were not digestedfollowing Hin6I digestion. The results are shown in Table 2.

Discussion

S. aureus is a common pathogen associated with seriouscommunity- and hospital-acquired diseases and has for longbeen considered as a major problem of public health(Pesavento et al. 2007; Pereira et al. 2009). Although someauthors have reported that other coagulase-positive staphy-lococci, such as Staphylococcus intermedius and

Staphylococcus hyicus, may be enterotoxigenic (Vernozy-Rozand et al. 1996), S. aureus is the predominant speciesinvolved in staphylococcal food poisoning outbreaks(Aragon-Alegro et al. 2007). This organism is the third mostcommon cause of confirmed food poisoning in the world(Acco et al. 2003; Aragon-Alegro et al. 2007). Due to therelatively quick progression of staphylococcal food poison-ing and an equally quick return to normal health status (formost individuals), it is speculated that most cases areunreported (Pinto et al. 2005; Aragon-Alegro et al. 2007).

Staphylococci grow in the temperature range of 7–48 °Cand produce enterotoxin from 10 to 48 °C, with optimumenterotoxin production at 40–45 °C. Although growth usu-ally is constrained by the presence of competing organisms,staphylococci thrive in environments relatively free ofcompetition from other bacteria, such as foods with highconcentrations of salt and sugar that impede the growth ofother organisms (Aycicek et al. 2005). Foods that aresubjected to post-processing contamination by S. aureus

Fig. 1 Agarose gel of coagulase gene of food isolates digested withAluI. Lines 1–6 genotype codes I–VI, respectively.M 100 bp molecularweight marker

Table 2 The sizes of coagulase gene and RFLP patterns in food S.aureus isolates

Genotypecode

Coagulasegene

Hin6Ipatterns

AluI patterns No. ofisolates

(bp) (bp) (bp)

I 850 600, 160, <100 420, 250, <100 10

II 950 420, 250, <100 650, 160, <100 5

III 950 500, <100 – 4

IV 850 420, 160, <100 – 2

V 500 – 420, <100 2

VI 750 500, <100 420, 340 1

– – – –

Fig. 2 Agarose gel electrophoresis of coagulase gene of food isolatesdigested with Hin6I. Lines 1 and 2 genotype codes I and II, respec-tively. Line 3 genotype codes III and VI. Lines 4 and 5 genotype codesIV and V, respectively. M 100 bp molecular weight marker

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Page 4: Molecular typing of Staphylococcus aureus isolated from food samples in Iran

represent a significant health hazard because microbes thatwould normally outcompete S. aureus have been eliminat-ed (Sandel and McKillip 2004).

Enterotoxins involved in food poisoning are produced byapproximately one third of strains of S. aureus (Halpin-Dohnalek and Marth 1989; Aragon-Alegro et al. 2007) andare the cause of the gastrointestinal symptoms observed dur-ing intoxications (Tamarapu et al. 2001; Normannoa et al.2005). The enterotoxins produced during cell growth gener-ally do not affect the sensory characteristics of the contami-nated food and may therefore go unnoticed (Sandel andMcKillip 2004). Even though food-borne S. aureus poisoningis a mild, generally self-limiting disease, with symptoms thatinclude vomiting with or without diarrhea (Dinges et al.2000), hospitalization is required in approximately 10 % ofthe cases (Holmberg and Blake 1984; Normannoa et al. 2005).Nevertheless, staphylococcal food poisoning represents a con-siderable social burden in terms of hospital expenses, loss ofpatients’ working days, and productivity, coupled with theproblems and the cost of disposing the contaminated food(Normannoa et al. 2005).

The staphylococci are the causative agents of many op-portunistic human and animal infections (Ghafari et al.2012). S. aureus is a habitant bacterium in some sites inhumans; however, its carriage can play a key role in theepidemiology and pathogenesis of staphylococcal infections(Motamedifar et al. 2010). Although this bacterium primar-ily inhabits the nasopharyngeal region, the skin and hair ofwarm-blooded animals (Huong et al. 2010) and environ-mental surfaces, humans are thought to be the primarysource of strains associated with food matrix staphylococcalintoxication (Rosec et al. 1997; Aycicek et al. 2005). Lackof proper hygienic measures during food preparation is oneof the major sources of contamination as the food handlersthemselves can harbor the pathogenic bacterium. Besides, S.aureus can tolerate a wide range of temperature, pH, andsalinity (Stewart et al. 2002; Huong et al. 2010). On theother hand, the bacterium can be frequently isolated in rawmilk of cattle suffering from mastitis and various dairyproducts including cheese, meat, and milk can harbor toxi-genic strains (Le Loir et al. 2003; Huong et al. 2010).Molecular and other studies of S. aureus isolated fromhuman or bovine mastitic milk have indicated that humanand bovine reservoirs of S. aureus represent two subpopu-lations that rarely cross-infect (Lange et al. 1999; Larsen etal. 2000; Karahan and Cetinkaya 2007). Single-banded co-agulase PCR products have been reported in S. aureus iso-lates originating from animals (Lange et al. 1999; Raimundoet al. 1999), whereas double-banded amplification productshave been reported from some isolates of human origin(Goh et al. 1992; Schwarzkopf and Karch 1994). In thepresent study, the detection of single bands by coagulasePCR in S. aureus isolates suggests that these isolates may be

of bovine origin not human one, and contamination of foodsamples may initiate from the animal source not the foodhandlers.

DNA sequencing of the 3′-end of coagulase gene hasdisclosed heterogeneity in the region containing the 81 bptandem repeats of the coagulase C-terminal region. PCRamplification of this region yields different sizes of DNAfragments and can be further differed by enzyme digestion.This fact can be used to identify and differ the genotype ofstrains (Schlegelova et al. 2003). Coagulase gene typing hasbeen considered as a simple, accurate, sufficiently reproduc-ible, specific, easy to interpret, and discriminatory methodfor typing S. aureus isolates (Schlegelova et al. 2003;Dastmalchi Saei et al. 2009). In the present study, digestionof coagulase PCR products with AluI and Hin6I yielded fourdifferent restriction profiles that indicate the presence ofheterogeneity in the coagulase gene of the isolates.

Studies carried out by other researchers (Su et al. 2000;Goh et al. 1992; Van belkum et al. 1997; Schlegelova etal. 2003; Karahan and Cetinkaya 2007; Reinoso et al.2008; Dastmalchi Saei et al. 2009; Momtaz et al. 2011;El-Jakee et al. 2010; Esmailnezhad et al. 2012) showeddifferent length and restriction analysis profiles of coagu-lase gene. The reason for this polymorphism among S.aureus isolates is unclear, but it seems to be because ofdeletion or insertion mutations by which a portion of the3′ end region of the coagulase gene is deleted or severalnucleotides are inserted and as a consequence change thecoagulase gene size and probably antigenic properties ofthe coagulase enzyme (Dastmalchi Saei et al. 2009; El-Jakee et al. 2010).

In conclusion, restriction analysis of the coagulase genecan be considered a reliable method for determining theorigin of S. aureus isolates in food samples. The results ofthe present study might help better control and managefood-associated places especially during the initial steps ofproduction. However, further studies using a large collectionof strains should be conducted to determine the commoncharacteristics of the predominant strains.

Acknowledgments The authors would like to appreciate ShirazUniversity for financial support.

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