Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
349
MOLECULAR DETECTION OF TEM GENE IN ESCHERICHIA COLI
O157:H7 ISOLATED FROM CHILDREN AND BUFFALOES IN
BASRAH PROVINCE
Raneem A. Kareem , Bassam Y. Khudaier*
Department of Microbiology, College of Veterinary Medicine, University of Basrah,
Basrah, Iraq
Key words: Escherichia coli O157:H7, tem gene, PCR.
ABSTRACT
During a period of five months (August 2015 to December 2015), a total of 250 samples were
collected 125 from hospitalized children suffering from diarrhea, and 125 from buffalo feces samples
collected from different regions in Basra Province, (Basra City Center, Abo alkaseeb, alqurna,
karmat Ali, A lzobeer). All specimens were screened for the presence of E. coli O157H7. A total of
104 (41.6%) of suspected E. coli isolates were obtained : 62 from children stool and 42 from buffalo
feces, All suspected isolates were tested biochemically. 6 out of 62 from children stool 9.7% and 4
out of 42 from buffalo feces 9.5% were Non-Sorbitol Fermented E. coli (NSFEC). All the isolates
were found to be resistant to at least 7 antibiotics to which they were subjected. Therefore, all these
four isolates were considered to be multidrug resistant. PCR assay for amplification of tem gene
revealed that 6 of the E. coli O157:H7 isolates that isolated from children and buffalo were positive
for tem gene.
INTRODUCTION
B-lactam are a large group of important antibiotics, because their effectiveness and
generally low toxicity [1]. There are four main groups of B-lactam antibiotics including
penicillins, cephalosprins, carbapenems and monobactams which are arranged according to
structure [2]. Bacteria expressing Extended-Spectrum β-Lactamases (ESBLs) enzymes
hydrolysing penicillins and cephalosporins, may not respond to therapy using some of these
antibiotics. ESBLs producing Gram-negative rods have a higher rates of antimicrobial
resistance than other areas of hospital [3]. The ESBLs producing organisms are often resistant
to several other type of antibiotics, as the plasmids with the gene encoding ESBLs often have
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
350
other resistance determinants. The ESBLs are commonly produced by many member of
enterobacteriaceae, especially Escherichia coli efficiently hydrolyze oxyimino cephalosporins
conferring resistance to third generation cephalosporins such as cefotaxime, ceftazidime,
ceftriaxone and to monobactums such aztreonam. ESBL-producing enterobacteriaceae might
become a major threat to children health as the infection cause by these bacteria have
increasingly been reported worldwide in last decade [4].
Escherichia coli is a major cause of food and water-borne illnesses characterized by
bloody diarrhea, hemorrhagic colitis (HC) and life-threatening hemolytic uremic syndrome
(HUS) in developed nations across the globe [5]. Ruminants are a major source of E. coli
O157:H7, and transmission principally occurs through consumption of contaminated food but
also through direct or indirect contacts with contaminated buffalos or persons [6; 7; 8 and 9].
The aim of the present study is to investigate the antimicrobial susceptibility for E. coli
O157:H7 isolated from buffalo and children and determine the occurrence of tem betalactamases
genes (extended-spectrum β-lactamase ESBL).
MATERIAL AND METHODS
Collection of Samples
In the present study, isolation of E. coli was carried out in stool and faecal samples
collected during the period October 2015 through January 2016. Two hundred fifty stool and
faecal samples (125 of each group) were collected from children and buffalos, respectively,
from outpatients wards at Aben-Kzwan hospital, Basra, Iraq.
Culturing of E. coli
Loopful of each samples were inoculated in 5 ml of trypticase Soy broth (TSB-V)
supplemented with (1.5 g) bile salt and incubated at 37 °C for 18-24h [10]. A loopfull of
bacterial growth was streaked on EMB and MacConkey agar then incubated overnight for E.
coli. Typical colonies on MacConkey agar and Eosin-Methylene Blue agar (EMB) were
streaked on sorbitol MacConkey agar supplemented with cefixime (0.05mg/L) and potassium
tellurite (2.5mg/L ) and incubated for additional overnight to identify on NSFEC [10].
Identification of E. coli
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
351
The identification of E. coli isolates was performed according to standard method
described by [11]. Tentative identification for all the isolates was done by traditional
culture characteristics, gram staining, indole test, simmon's citrate test and triple sugar iron
test (Hi-Media, India). Definitive identification up to species level was made with the Api
20E System Identification (Pioneer, Germany).
Antimicrobial Sensitivity Testing
The antimicrobial test of all positive isolates were performed according to [12] by
modified disc-diffusion method using 10 different antibiotic discs in the following
concentrations: amoxicillin (25μg), amikacin (30μg), cephalothin (30μg), cefotaxime
(30μg), ciprofloxacin (30μg), gentamicin (10μg), caphalothin (300μg), ceftazidime (5μg),
imipenem (20μg) and tetracycline (30 μg), using [12 and 13]. Mueller-Hinton (MH) agar
plates (Difco Laboratories, Detroit, USA) were overlaid with each of the E. coli strains
inoculum (turbidity equivalent to that of a 0.5 McFarland Standard). Inhibition zone
diameters were measured after 24 and then 48 hrs incubation [12].
Molecular detection tem gene by PCR assay
The DNA was purified and extracted according to the instructions of the company
(Kiagen, Germany). PCR amplifications were carried out on a DNA thermal cycler instrument
(Tc-312Techne (UK)). The primer design were performed as previously described Table (1).
The composition of the reaction mixture was as follows: 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 0.1% Triton X-100, 1.5 mM MgCl2, each of the four deoxynucleoside triphosphates at a
concentration of 0.2 mM, and 1.2 U of Taq DNA polymerase in a total volume of 25 μl. A total
of 2.5 μl of template DNA was added to the reaction mixture, and the mixture was centrifuged
briefly. The PCR program consisted of an initial denaturation step at 95°C for 15 min, followed
by 30 cycles of DNA denaturation at 94°C for 30 s, primer annealing at 50°C for 1min, primer
extension at 72°C for 2 min, and final extension at 72°C for 10 min. After the last cycle the
products were stored at 4°C.
Table (1): Oligonucleotide primer sequences used for PCR amplification of tem gene.
Gene Primer Sequence Size* Reference
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
352
(bp)
tem
F
5'-TCG CCG CAT ACA CTA TTC TCA GAA
TGA-3'
445
[14]
R
5'-ACG CTC ACC GGC TCC AGA TTT AT-3'
*: Size: PCR product (bp).
The PCR products (1/10 volume) were analyzed by electrophoresis with 1% agarose
gels in TBE buffer (0.04 M Tris-OH, 0.002 M EDTA [pH 8.5]). The gels were stained with
ethidium bromide, and the PCR products were visualized with UV light. A single band was
observed for tem amplified products with a single primer set.
RESULTS
Out of 250 samples 104 isolates were suspected E. coli (62 and 42) from children and
buffaloes respectively. On the other hand, the prevalence of E. coli isolated from children and
buffaloes were 33.6% and 49.6% respectively (Table. 2).
The percentage of frequency of NSFEC was 9.6% based on the fermentation of the
sorbitol in Sorbitol MacConkey agar (Table 3).
All E. coli O157:H7 isolates (100%) were resistance to cephalothin, gentamycin,
amoxicillin and ceftazidime. (90%) were resistance to amikacin and cefotaxime and (75%)
were resistance to tetracycline, in addition to (50%) were resistant to ceftriaxone. While (100%
and 75%) E. coli O157:H7 isolates were sensitive to imipenem and ciprofloxacin, respectively
(Table 4; Figure 1).
The PCR analysis was applied to DNA extracted from pre-conventional microbiological
confirmed of E. coli O157:H7 isolates figure (2). Six isolates were produced PCR products
corresponding to tem gene (445 bp) corresponding for tem gene analyzed by PCR assay
(Figure 3).
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Table (2): Prevalence of E. coli isolated from children and buffalos.
Sample
source
Total No. of
sample
No. of E. coli
isolate
(%)
children 125 42 (33.6)
buffalo 125 62 (49.6)
Total 250 104 (41.6)
Table (3): Percentage of non-sorbitol fermenting E coli (NSFEC).
Sample
source
Total No. of
sample
Non-sorbitol
fermenter (NSF)
(%)
children 62 6 (9.7)
buffalo 42 4 (9.5)
Total 104 10 (9.6)
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Table (4): Antimicrobial susceptibility of E. coli O157:H7 isolated from children and
buffalos.
Type of antibiotic Sensitive (%) Intermediate (%) Resistance (%)
Gentamycin 100 0 0
Cephalothin 0 0 100
Imipenem 100 0 0
Ciprofloxacin 75 25 0
Amoxicillin 0 0 100
Amikacin 10 0 90
Tetracyclin 0 25 75
Ceftazidime 0 0 100
Ceftriaxone 50 0 50
Cefotaxime 0 10 90
AK
IPM
AMC
CAZ
CRO
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Figure (1): Antimicrobial susceptibility test of E. coli O157:H7 isolated from children and
buffalos.
IPM: Imipenem; AK: Amikacin; CAZ: Ceftazidime; AMC: Amoxicillin; CRO: Ciprofloxacin.
Total genomic DNA
Figure (2): Total genomic DNA extracted from E. coli O157:H7 isolates using 1% agarose
gel electrophoresis.
1 2 3 4 5 6
500bp
200bp
445bp
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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Figure (3): PCR amplification of tem gene (445 bp) in E. coli O157:H7. Lane 1: molecular size
marker; Lanes 4, 5 and 6: E. coli O157:H7 tem gene isolates.
DISCUSSION
Trypticase Soy broth (TSB) was used as enrichment medium while MacConkey agar
and EMB agar were used for selection and isolation purposes. In the present study, 104 E. coli
isolates out of 250 collected samples were result 62 from children and 42 from buffalo. These
results agrees with the results reported by [15 and 16] who found the rates of E. coli isolates in
stool 38.3% and 38%, respectively. [17] found that the percentage of prevalence of E. coli was
59.4%, while [18] found lower rates of E. coli isolates in stool 29%.
The occurrence of NSFEC in stool samples which detected by conventional
microbiological methods were 9.5% in children. The present occurrence of NSFEC in children
stool sample was lower than the result of obtained by [19] who reported 15.6%. It is also lower
than the finding of [20 and 21] who detected 57.5% and 73.9% , respectively.
The overall rates of prevalence of NSFEC in all tested samples were found to be 19.2%
a factor which accounts for the increased prevalence could be the methodology of isolation
employed such as variation in media used for isolation, sampling procedure. In addition, to
sows, hooks are not properly cleaned and disinfected.
Multiple antibiotic resistant strains can be transferred from buffalo to children through
contaminated food. [22] reported that multiple resistant bacterial strains were transmitted to
childrens by raw meat and milk. Cattle feces are a potential source of antibiotic resistant
bacteria. If released into the environment, resistant strains may contaminate water and food
sources and can be a potential threat to children health [23]. Antibiotic resistance has become a
major clinical and public health problem during the lifetime of most people [24]. There are
many reasons for this problem, one of which is an over use of antibiotics [25].
The disc diffusion test was used to screening the antimicrobial phenotypes of the four
isolates of EHEC O157 : H7. The finding of 100% E. coli O157:H7 were resistant to tested
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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antibiotics cephalothin, amoxicillin and ceftazidime, among the important findings of
antimicrobial testing was that 100% of E. coli O157:H7 isolates were sensitive to imipenem,
gentamycin and 50% was sensitive to ceftriaxone in addition 25% prevalence of intermediate
other antimicrobials including ciprofloxacin and tetracycline. The level of resistance was
significantly higher than sensitivity. The present result in line with [26] who found E. coli
O157:H7 that was found to be sensitive to amikacin and ciprofloxacin. On the other hand, it
was found resistant to all other antibiotics, ranging from 8.5%-90%. [27] in (1997) study
reported multidrug-resistance in E. coli with persistent diarrhea in Kenyan children where the
isolates were resistant to tetracycline. Another study [28] recorded the highest rates of
resistance against amoxicillin, gentamycin, tetracycline and ciprofloxacin (100%) and the most
common resistance pattern was for amoxicillin, gentamycin, ciprofloxacin, tetracycline (38.7%)
and the least common resistance patterns was for F (3.2%).
The tem genes are by far the most widespread with unknown origin [29]. tem-1, which is
responsible for most of the ampicillin resistance in; 94% of E. coli strains isolated in Spain, 89% of
E. coli strains isolated in Hong Kong, and in 78% of E. coli strains isolated in London [30].
However, [31] reported that up to 90% of ampicillin resistance in E. coli is due to the production of
TEM-1. This enzyme has the ability to hydrolyze penicillins and early cephalosporins such as
cephalothin and cephaloridine, TEM -2 β-lactamase is widespread in E. coli, although they are
much rare than TEM-1.The classical TEM-1 and TEM-2 enzymes have minimal activity against
newer cephalosporins [32]. In the past 30 years, however, there have been an increasing emergency
of ESBLs, which attack many newer cephems and monobactams as well as third generation
cephalosporins and anti-Gram-negative bacterial penicillins (31). Most of these enzymes are mutant
of TEM-1 and TEM-2 such as TEM-3, TEM-4, TEM-10, TEM-27, TEM-92 [31]. Although strains
that produce ESBL are characteristically resistant to new cephalosporins and/or aztreonam, many
strains producing these enzymes appear susceptible or intermediate to some or all of these agents in
vitro, while expressing clinically significant resistance in infected patients [3].
It was recently reported by [33] in (2011) whom agree with present results which
reported presence of tem gene in all the 23strains. [34 and 35] founded tem β-lactamases genes
presence in 50.5% and 57.1 %, respectively.
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O157:H المعزولة من جراثیم الاشرشیا القولونیة 7 tem الکشف الجزیئی لجینات
المعزولة من الانسان والحیوان فی محافظة البصره
رنیم عبد الکریم لفتھ ، بسام یاسین خضیر
فرع الاحیاء المجھریة ، کلیة الطب البیطری ، جامعة البصرة ،البصره ،العراق
الخلاصة
خلال فترة خمسة اشھر ( اب 2015 - دیسمبر 2015 ) تم جمع 250 عینة ، 125 عینة من براز الاطفال الذین یعانون من الاسھال
و 125 عینة من براز الجاموس من مناطق مختلفة من محافظة البصرة (مرکز المحافظة، کرمة علی ، الزبیر ، القرنھ ، ابو
تم الحصول على .O157:H الخصیب). فحصت جمیع العینات عن وجود الاشرشیا القولونیة و الاشرشیا القولونیة النمط المصلی 7
104 عزلة ( 41,6 %) من الاشرشیا القولونیة المشکوک بھا : 42 عزلة من براز الجاموس و 62 عزلھ من عینات غائط الاطفال.
وجد ان 6 من اصل 62 عزلة من براز الجاموس بنسبة 9.5 %. و 4 عزلة من غائط الاطفال من اصل 42 عزلة بنسبة 9.7 % غیر
مخمرة للسوربیتول. أظھرت جمیع العزلات نمط المقاومة المتعددة للمضادات الحیویة. عرضت عزلات الاشرشیا القولونیة النمط
اظھرت النتائج إن 6 عزلات .tem المنتجة لإنزیم البیتالاکتامیز لاختبار تفاعل السلسلة المتبلمرة للمورث O157:H المصلی 7
. tem الاشریکیا القولونیة معزولھ من غائط الاطفال وبراز الجاموس تحتوی على الجین
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