The First International Scientific Conference of Health ...kti.mtu.edu.iq/conf/researches/3.pdf · Urinary pathogens vary depending upon age, sex, catheterization, hospitalization

The First International Scientific Conference of Health and Medical Specialties……15-16 April 2019

Kut Technical Institute/ Middle Technical Universty

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Biochemical and molecular diagnosis of Escherichia coli and Pseudomonas aeruginosa

isolated from UTI patients

Suzan Saadi Hussain1, Ali HusseinAlwan2, MaysoonKhaleefa Abbas

3 Nihad Khalawe Tektook

4

1;2 &3 Al-Mustansiriya University ∕ College of science;*4 Middle Technical University/Collage of Medical & Health Technology,

Medical laboratory techniques dep. Iraq

Gmail: [email protected]

Background/Aims: A total of (75) clinical isolates of gram negative bacteria primary identified as Escherichia coli and

Pseudomonas aeruginosa were collected from Samples from nine hospitals in Baghdad included: AL-Kindy General Teaching

Hospital, Ibn-Al Balady Hospital, Teaching Laboratories in Medical City, ,Education Baghdad Hospital, Al-Kadhymia teaching

Hospital in Medical Cityfor six month.From March to August, 2018 in Baghdad city ,They were obtained from mid stream urine

from patients suffering from urinary tract infections. The isolates were initially identified by culturing on MacConkey agar and

blood agar then diagnosed by performing some morphological and biochemical tests. A confirmatory test as a final diagnoses was

done using api20E system. According to this, the 75 isolates were distributed as 45 isolates of Escherichia coli while the remaining

35 isolates were Pseudomonas aeruginosa.

Antibiotics sensitivity test was done by used 24antibiotics, the isolates were drug resistance.TheEscherichia coliisolates showed

resistance as follow Amoxicillin and Tobramycin high resistance about 78.6% but high sensitive Ticarcillin/Clavulanic acid and

Pipracllin 93.3%,whilewerePseudomonas aeruginosaisolatesshowed high resistanceNitrofurantoin and Amikacin

78.6%butNorfloxacinshowed high sensitive 93.3% and Levofloxacin 92%.

Key ward: MTII, MDA, Histopathology and liver homogenate.

I. INTRODUCTION

The urethra. Urinating after sexual intercourse also helps to prevent an overgrowth of bacteria that may be in the urethra (1).

In summary, urinary tract infections are very common, especially in women and in healthcare settings. Bacteria, namely E. coli, is the

main causative agent of these infections. (2).

Urinary tract infections caused by bacteria associated with an increased antimicrobial resistant species are common in all age

groups, Urinary tract infections are responsible for a large amount of medical expenses and research into vaccination has started due

to the problem of antibiotic resistance. Until successful vaccinations are created, prevention is of utmost importance.(3,39)

Urinary pathogens vary depending upon age, sex, catheterization, hospitalization and previous exposure of antimicrobials

(38).The microorganism Escherichia coli, known as E. coli, is responsible for about 80% of urinary tract infections that have not

passed the urinary bladder (4). This is a very common microbe in the human body and is normally harmless in the intestinal tract

however when in the urinary tract, certain strains can cause infection. E. coli can enter into your urinary tract through the urethra. This can happen from sexual intercourse or from improper hygiene techniques.(5)

Women are 8 times more likely to get urinary tract infections, due to the closeness of female’s urethra to the anal opening,

where E. coliare found in fecal matter (6).

Pseudomonas aeruginosa(P. aeruginosa) is a Gram-negative bacterium, which has an ability to be alive and adapt in different environments like water, soil, municipal wastewater and hospital (7). The bacterium can grow aerobicly and anaerobicly(8).

P.aeruginosais an opportunistic pathogen that infects human, animals and plants. It causes various infections in patients with Acquired

Immune Deficiency Syndrome (AIDS), cancer or immunocompromised patients by burn, wounds, otitis externa, surgery, keratitis,

and urinary tract infections (8) as well as it characterizes one of the causative pathogens which cause severe damage of tissue in

diabetic foot ulcers (9).

P.aeruginosahas a large genome when it is compared with other bacteria, its size varies between strains, ranging from (5.5 to 7)

Mbp (10).

There are numerous methodsthat can be used in the identification of pathogens include cultural tests, biochemical tests (API

20E test) and molecular tests.

mailto:[email protected]



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P. aeruginosa is aerobic, non-spore forming, non-fermentingfor glucose, gram negative rods which is slightly curved or straight

and measuring about 0.5–1.5μm wide and about 1–5μm long (11). It is motile by one or more polar flagella and it is capsulated or it

has slime layers which they play as a barrier against antibiotics, phagocytes and lymphocytes (12).

This bacteria is an obligate aerobic respiratory metabolism by oxygen, but in some cases, it uses nitrate as an alternative source (as respiratory electron accepter) that allows growth anaerobically (7), due to these traits, P. aeruginosais a ubiquitous organism, and

its isolation has been documented from soil, fresh and salt water, plants, animals, and inanimate objects (13).P.aeruginosacauses many

infections which have blue-green appearance of the pus due to the mixing of pyoverdin (yellow-green and fluorescent)and

pyocyanin(blue), so the production of this pigment is referred to this organism, some strains of this bacteria secrete another pigments

like pyorubin(red) and pyomelanin(brown) (14).

A mainly dangerous and dreaded pathogen has a natural tendency for the expansion of resistance to antibiotics (15). This

bounds future therapeutic usages of antibiotics against this bacterium and increases rates of mortality. The bacterium is naturally

resistant to various antibiotics including tetracyclines and benzyl penicillin by reason of the permeability barrier afforded by its outer membrane lipopolysaccharide (LPS) (15).

E.coli and Pseudomonas aeruginosaidentification was conducted using API 20E system depending on the procedure from the

manufacturing company .

A specific and rapid identification of the pathogen leads to successful treatment of the bacterial infection, the identification of causative agents by commercial methods can represent the conclusive element of diagnostic process, different methods such as

culturing the organisms on differentiation media and biochemical tests like oxidase and)API 20E test( (16), especially culturing

clinical samples before antibiotic treatment and determining antimicrobials susceptibility for this pathogen, so using nucleic acid-

based tests (NATs) to identify pathogens reliably and rapidly by using PCR technique (17). PCR techniques are accepted as standard

methods for pathogens detection, improving time to result, sensitivity and specificity (18).there are some genes have been used as

markers for the identification of P. aeruginosa, one of this genes are 16SrDNA and other genes, using specific primers for each gene

to amplify the target sequence(19).

Aims of the study

Isolate and diagnosis Escherichia coli and Pseudomonas aeruginosafrom UTI patients by Biochemical and molecular methods.

II. MATERIALS AND METHODS

Clinical isolates (75) of gram negative bacteria primary identified as 45 clinical isolates of Escherichia coli and 35 isolates of Pseudomonas aeruginosa were collected from Samples from fife hospitals in Baghdad included: AL-Kindy General Teaching

Hospital, Ibn-Al Balady Hospital, Teaching Laboratories in Medical City, ,Education Baghdad Hospital, Al-Kadhymia teaching and

Al- HareryHospital in Medical Cityfor six month.

From March to August, 2018 in Baghdad city , They were obtained from mid stream urine from patients suffering from

urinary tract infections, and their diagnosis was confirmed on the basis of microscopic and genetic methods, biochemical tests and

final diagnosis using( Bio Merieux France).

Sterilization of materials and tools was done by various methods of sterilization, isolating and cultured the bacteria on the

appropriate pure culture media

Bacterial DNA was extracted and bacterial DNA was Migrate by gel electrophoresis.

Laboratory Diagnosis by Morphological Examination and Microscopic Examination.

Bacterial DNA isolation

The DNA isolates were isolated using several processed DNA extracts from Promega (USA). The results were extracted using

a gel concentration of 1%.

Molecular Diagnosis :

DNA Extraction from Gram Negative bacteria and Bacterial DNA extraction was carried by using (Genomic DNA purification KitWizard®Promega. USA).Isolates inoculated on nutrient broth for 24hr at 37 ˚C, then suspended in 1 ml of inoculated nutrient

broth in eppendorf tube and mixed gently.

Polymerase Chain Reaction (PCR) Amplification Technique



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A PCR technique was used with primers (22).Detection genes by PCR product by Agarose Gel Electrophoresis.

Antimicrobial Agents Disc :

The following antimicrobial agents were used (24) types and the results of intibition zone of P. aeruginosain appendix

1compared with those indicated in (20).

Table (1)Antibiotic Discs

No. Antibiotics Codes Mg /disk Manufacturing Company (origin)

1 Amoxicillin- clavulanic acid AMC (20 μg) Bioanalyse (Turkey)

2 Azithromycin AZM (30μg) Bioanalyse

3 Tobramycin TOB (10 μg) Bioanalyse

4 Aztreonam ATM (30 μg) Bioanalyse

5 Ticarcillin/Clavulanic acid TIM (10, 75 μg) Bioanalyse

6 Cefotaxime CTX (30 μg) Bioanalyse

7 Ceftazidime CAZ (30μg) Bioanalyse

8 Augmentin AUG (30μg) Bioanalyse

9 Ciprofloxacin CIP (10 μg) Bioanalyse

10 Cephalexin CL (10 μg) Bioanalyse

11 Ceftazidime CDZ (30μg) Bioanalyse

12 Gentamicin GEN (10 μg) Bioanalyse

13 Imipenem IPM (10 μg) Bioanalyse

14 Levofloxacin LEV (5 μg) Bioanalyse

15 Meropenem MEM (10 μg) Bioanalyse

16 Norfloxacin NOR (10 μg) Bioanalyse

17 Nitrofurantoin NIT (300μg) Bioanalyse

18 Piperacillin PI (100 μg) Bioanalyse

19 Mefoxin FOX (20 μg) Bioanalyse

20 Dextromethorphan DXT (30μg) Bioanalyse

21 Samaphenecol SAM (20μg) Bioanalyse

22 Ticarcillin-clavulanic acid TCC (75/10μg) Bioanalyse

23 Amikacin AK (30μg) Bioanalyse

24 Trimethoprim- sulfamethoxazole TS (25 μg) Bioanalyse

The test was performed using the Mueller-Hinton agar medium and the computed diameters were calculated according to (11),

The diameter of the inhibition zone of each antibiotic disc was measured by ruler and the results were recorded into resistant (R),

susceptible (S) or intermediary (I)



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III. RESULTS AND DISCUSSION

A total of (75) clinical isolates of gram negative bacteria primary identified as Escherichia coli and Pseudomonas

aeruginosa were collected from Samples from nine hospitals in Baghdad included: AL-Kindy General Teaching Hospital, Ibn-Al

Balady Hospital, Teaching Laboratories in Medical City, ,Education Baghdad Hospital, Al-Kadhymia teaching Hospital in Medical

Cityfor six month.. This Gram-negative bacteria considered opportunistic pathogen with most problematic bacterial challenges in the

infectious disease community as recognized by the Infectious Disease Society of America(20 ,21).the most bacteria isolated from

UTIs were Escherichia coli ( 30%); Klebsiella pneumoniae (10%); Pseudomonas aeruginosa (8%), this results conferred with

tektook, 2005 who identified bacteria in UTI patients as Escherichia coli 16( 32%);Klebsiella pneumoniae 9 (18%); Proteus mirabilis 12 (24%) ,Streptococcus agalactiae 2 (4%) ;Pseudomonas aeruginosa 7 (14%) ;Staphylococcus aureus

4 (8%)

Escherichia coligrowth on EMB Agar the colonies seem as a green metalic color and it is producing a diffusing green pigment

in the agar, Figure (1) , but it growth on MaCconky Agar the colonies seem as a pink color and it is producing a pale color of the

agarFigure (2). Sensitivity Test using Antimicrobial Discs against Escherichia coli cultured on Mueller Hinton agar Figure (3)

WhileP. aeruginosacolonies on MacConkey agar appeared as a pale color because this pathogen is not fermenting

lactoseFigure (4) and it produces a diffusing green pigment in the agar (13)Figure (5). These isolates cultured on pseudomonas agar,

all colonies exhibit growth with blue-green or brown pigment that represents an evidence for these bacteria (21), then it was used

pseudomonas agar in the identification of P. aeruginosa, depending on the manufacture company information that determine the color

of P.aeruginosacolonies, it is easily distinguished because of the green colony color and the color of the medium that change from

pale or colorless to pale green, the rest of bacteria are inhibited and in case of growing, they grow as dark green colonies Figure(6).

P.aeruginosaisolates were cultured on blood agar and gave a positive results , that represented by producing large flat spreading, β-

hemolytic, mucoid, pigmented colonies with distinguished metallic sheen(22).The colonies appeared bright greenish especially under

UV light.

All isolates were cultured on nutrient agar and all of them gave a distinguished blue-green pigment .

Figure (1): Escherichia coligrowth on EMB Agar the colonies seem as a green metalic color and it is producing a diffusing green pigment in the agar.



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Figure (2): Escherichia coligrowth on MaCconky Agar the colonies seem as a pink color and it is producing a pale color

of the agar.

Figure (3): Sensitivity Test using Antimicrobial Discs againstEscherichia coli cultured on Mueller Hinton agar

Figure (4): Pseudomonas aeruginosagrowth MaCconky Agar the colonies seem as a Pink in the agar.



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Figure (5): Pseudomonas aeruginosagrowth on Mueller Hinton agar the colonies seem as a green color and it is producing a

diffusing green pigment in the agar.

Figure (6): ): Sensitivity Test using Antimicrobial Discs against Pseudomonas aeruginosacultured on Mueller Hinton agar.

Microscopic Examination

Gram stain showed that gram negative (red) rods seem as a single, short chain or groups, which they showed under

compound light microscope at 40X and 100X (12).

Biochemical Tests Results

P. aeruginosaisolates were identified by traditional biochemical tests including Oxidase test, Catalase test and by API 20E

biochemical kit, the results of the biochemical tests for Escherichia coli and P.aeruginosaisolates

Oxidase reaction gave a positive results in 2-3seconds, but the mucoid strains (from some of wound and burn infection) of

this bacterium gave a positive after ~20 seconds, the positive result refers to oxidase production that means the bacteria contain

cytochrome c oxidase, so they can use oxygen to produce energy by turning O2 to H2O or H2O2 by electron transfer chain (21).

All isolates showed a positive results for catalase test, producing catalase enzyme, that represented by the creation of

gaseous bubbles, which are caused by catalyzing the breaking down of hydrogen peroxide to oxygen and water (22). There are no difference in the catalase test between isolates of P.aeruginosafrom wound,burn infection and other infections.

Table (2): Results of susceptibility tests of Escherichia coli against 24 antimicrobial agents according to (20)



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symbol Antibiotics

Number of bacterial isolates and their percentege

R I S

AMC Amoxicillin- clavulanic acid 59(78,6%) - 16(21.3%)

AZM Azithromycin 53 (70.6%) - 22(29.3%)

TOB Tobramycin 59(78,6%) 7(9.3%) 9(12%)

ATM Aztreonam 50(66.6%) - 25(33.3)

TIM Ticarcillin/Clavulanic acid 5(6.6%) - 70(93,3%)

CTX Cefotaxime 15(20% ) - 60(80%)

CAZ Ceftazidime 40(53,3%) - 35(46.6%)

AUG Augmentin 57(76%) - 18(24%)

CIP Ciprofloxacin 24(32%) - 51(68%)

CL Cephalexin 36(48%) - 39(52%)

CDZ Ceftazidime 53(70.6%) 22(29.3%)

GEN Gentamicin 40(30%) 3(4 ) 35(60%)

IPM Imipenem 7(9.3%) - 69(92%)

LEV Levofloxacin 62(82%) - 13(18%)

MEM Meropenem 55(73,3%) 5(6%) 20(26,6%)

NOR Norfloxacin 16(21.3%) - 59(78,6%)

NIT Nitrofurantoin 55(73,3%) - 20(26.6%)

PRL Piperacillin 5(6.6%) 70(93,3%)

FOX Mefoxin 51(68%) - 24(32%)

DXT Dextromethorphan 9(12%) - 66(88%)

SAM Samaphenecol 46(61.3%) 29(38.6%)

TCC Ticarcillin-clavulanic acid 40(53,3%) - 35(46.4%)

AK Amikacin 17(22.6%) - 58(77.3% )

TS Trimethoprim- sulfamethoxazole 47(62.6%) 28(37.3%)

R=resistance I=intermediate S=sensitive

Table (3): Results of susceptibility tests of P.aeruginosa against 24 antimicrobial agents according to (20).

symbol Antibiotics

Number of bacterial isolates and their percentege

R I S

AMC Amoxicillin- clavulanic acid 47(62.6%) 28(37.3%)

AZM Azithromycin 53 (70.6%) - 22(29.3%)



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TOB Tobramycin 35(70%) 40(30%)

ATM Aztreonam 15(20% ) - 60(80%)

TIM Ticarcillin/Clavulanic acid 41(54.6%) - 24(45.3%)

CTX Cefotaxime 55(73,3%) - 20(26.6%)

CAZ Ceftazidime 57(76%) - 18(24%)

AUG Augmentin 55(73,3%) - 20(26.6%)

CIP Ciprofloxacin 24(32%) - 51(68%)

CL Cephalexin 57(76%) - 18(24%)

CDZ Ceftazidime 43(57.3%) 32(42.6%)

GEN Gentamicin 24(32%) - 51(68%)

IPM Imipenem 15(20% ) - 60(80%)

LEV Levofloxacin 7(9.3%) - 69(92%)

MEM Meropenem 50(66.6%) - 25(33.3)

NOR Norfloxacin 5(6.6%) 70(93,3%)

NIT Nitrofurantoin 59(78,6%) - 16(21.3%)

PI Piperacillin 17(22.6%) - 58(77.3% )

FOX Mefoxin 62(82%) - 13(18%)

DXT Dextromethorphan 40(30%) 35(60%)

SAM Samaphenecol 24(32%) - 51(68%)

TCC Ticarcillin-clavulanic acid 50(66.6%) - 25(33.3)

AK Amikacin 59(78,6%) - 16(21.3%)

TS Trimethoprim- sulfamethoxazole 24(32%) - 51(68%)

R=resistance I=intermediate S=sensitive

The range of sensitivity ,intermediate and resistant are shown in (Table (3) according to (20), the present study indicated that

P. is becoming resistance to commonly used of antibiotic due to excessing consumption of antibiotics exerting selected present

bacteria .

The result confirmed the occurrence of MDR strains of ,which agree with(21) "who found that the P. aeruginosamulti-resistance to most antibiotic.

In this study Escherichia coliisolates were resistant Amoxicillin- clavulanic acid (6,87)% , Azithromycin (6.07)% ,

Tobramycin (6,87)% , Aztreonam (7707)% ,

,Ceftazidime (3585)% ,Augmentin (67)% ,Ciprofloxacin (53)% ,Cephalexin (4,)% ,Ceftazidime (6.07)% ,Gentamicin (5.)% ,Levofloxacin (,3)% ,Meropenem (6585)% , Nitrofurantoin (6585)% ,Piperacillin

(707)% ,Mefoxin (7,)% ,, Samaphenecol (7.05)% ,

Ticarcillin-clavulanicacid (3585)% , Trimethoprim- sulfamethoxazole (7307)% , isolates were sensitive are Amikacin

(3307)% ,Ticarcillin/Clavulanic acid (707)% ,Cefotaxime (3.) % , Imipenem (305%) , Norfloxacin

(3.05)% , Dextromethorphan (.3)% .



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Results of susceptibility tests of P.aeruginosa against 24 antimicrobial agents according to (20) ,and were resistant

Amoxicillin- clavulanic acid (62.6%),Azithromycin (70.6%) ,Tobramycin (70%),Ticarcillin/Clavulanic acid(54.6%)

,Cefotaxime (73,3%),Ceftazidime(76%),Augmentin (73,3%) ,Ciprofloxacin (32%), Cephalexin (76%)

,Ceftazidime (57.3%) ,Gentamicin (32%), Meropenem (66.6%), Nitrofurantoin (78,6%),Mefoxin (82%), Ticarcillin-

clavulanic acid(66.6%) ,Amikacin (78,6%) , isolates were sensitive are Trimethoprim- sulfamethoxazole(32%),Aztreonam

(20%),Dextromethorphan (30%),Samaphenecol (32%) , Norfloxacin (6.6%), Piperacillin (22.6%), Imipenem (20%

),Levofloxacin (9.3%) .

The present results agree with another study that showed low resistance to LEV is 4% and CIP is 28%(23), and low level of

resistance to CIP 20.6% (25), While the present study shows low level of bacterial resistance to LEV that disagree with the other study

(25), in which it illustrated the best antimicrobial against this bacteria is quinolones like LEV and CIP, and disagree with (26), study

about the sensitivity to LEV that showed the resistance of P.aeruginosa against LEV is 69.23% . Levofloxacin and (CIP) are member

of fluoroquinolones which inhibit bacterial growth via binding to DNA gyrase. Mutations in the DNA gyrase subunits may play a

major role in forming a high rate of resistance to fluoroquinolone in P. aeruginosaand other Gram-negative bacteria (10).

P.aeruginosa high resistant against AMC that agreed with other study in Pakistan (25) reported a very high resistant 97.6% of

P.aeruginosa against AMC, while the same study showed a lower resistant 10.4% to Imipenem that is closed to our study as well as

the present study agrees with (27,39, 40) in the low resistant to Imipenem which is 30%.

P.aeruginosa showed low resistance to PRL, this antibiotic is a β-lactam group of class ureidopenicillin. The present study

result is agree with to (28,37) result who found that around 24% of P.aeruginosa isolates were resistant to these antimicrobial agents.

In contrast the present study results 62% that is agreed with the other study in India that showed high resistant to PRL (29, 37, 38,40).

Gentamicin is a type of aminoglycoside. In the present data, P.aeruginosa shows high resistance to GEN 86%. This result is

closed to the result for other study (30) who found that the resistance of P.aeruginosa against GEN is 100%, but disagreed with (31)

which recorded its sensitivity as 35.9%. TOB 78% that is agree with (31).Amikacin 42% this study agree with (32,36).

Ticarcillin and Clavulanic acid is a combination of a β-lactam antibiotic and a β-lactamase inhibitor respectively. It is tested against P.aeruginosa. P.aeruginosais showed high resistance against it 50% in the present study, this result agreed with other result

showed that P.aeruginosagave moderate resistance 57.1% against this antimicrobial agent (33,34 and 35).

Molecular Diagnosis

The method of DNA extraction by kit was perfect and successful, the extracted DNA was pure and intact The presence of the16S rRNA gene was identified by850bp, as shown in figure 7.

leader 100Bp 680 Bp



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Figure (,): Agarose Gel Electrophoresis of Bacterial Genomic DNA using 0.7% agarose, TBE (1x) and imagined by Ethidium

Bromide Stain.

The results in this figure showed table 2 demonstrated that the percentage of the16S rRNA gene detection was 71.4%, whilst

the percentage of the negative results was 24.6%,,This result agreement with (36).

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The First International Scientific Conference of Health ...kti.mtu.edu.iq/conf/researches/3.pdf · Urinary pathogens vary depending upon age, sex, catheterization, hospitalization