1دانشیار بهداشت و کنترل کیفی مواد غذایی، دانشکده دامپزشکی، پژوهشکده بیماریهای مشترک، دانشگاه شهرکرد، ایران
2دانش آموخته کارشناسی ارشد بهداشت مواد غذایی، دانشکده دامپزشکی، دانشگاه شهرکرد
مقدمه: عفونتهای گوارشی ناشی از سویههای وروتوکسینزای باکتری اشریشیا کلی در دهههای اخیر شناسایی شده است. اشریشیا کلیO157:H7 مهمترین سروتیپ از این گروه است که موجب اسهال خونی و سندرم همولیتک اورمیک در انسان میشود. این مطالعه به منظور شناسایی سویههای وروتوسینزای باکتری اشریشیا کلی جداشده از سبزیجات و مقاومت آنتی بیوتیکی آنها طراحی و اجرا شد. مواد و روشها: 500 نمونه سبزیجات عرضه شده در شهرکرد به طور تصادفی از مغازههای خرده فروشی سطح شهر جمع آوری شد. آزمونهای میکروبیولوژی و بیوشیمیایی به منظور جداسازی باکتری اشریشیا کلی روی نمونهها انجام شد. آزمون زنجیرهای پلیمراز برای شناسایی ژنهای stx1, stx2, eaeو rbfE روی جدایهها انجام و همچنین مقاومت آنتیبیوتیکی جدایهها نسبت به آنتیبیوتیکها به روش دیسک انتشاری ارزیابی شد. نتایج: نتایج نشان داد که از میان 25 باکتری اشرشیا کلی جدا شده 40 درصد واجد ژن eaeA ، 12 درصد واجد ژن STx2 و 4 درصد واجد هر دو ژن بودند. هیچیک از جدایهها واجد ژنهای rfbE، H7 و Stx1نبودند. آزمونهای آنتیبیوگرام نشانگر مقاومت بالای جدایهها در برابر جنتامایسین و سفوتوکسیم بود. بحث و نتیجهگیری: حضور سویههای وروتوکسینزای باکتری اشریشیا کلی در سبزیجاتو مقاومت بالای جدایهها در برابر آنتیبیوتیکها میتواند یک خطر بالقوه برای بهداشت انسان باشد.
Antibiotic resistance of Verotoxigenic Escherichia coli isolated from vegetables
Introduction: Human gastrointestinal disease caused by verotoxigenic Escherichia coli has been diagnosed for recent decades. Escherichia coli O157:H7 is the most important serotype of verotoxigenic Escherichia coli that cause hemolytic uremic syndrome and hemorrhagic colitis in humans. This study was conducted to determine the occurrence of verotoxigenic E. coli and antibiotic resistance of the isolates from vegetables. Materials and methods: A total of 500 fresh vegetable samples were collected randomly from retail shops in Shahrekord, Iran. E. coli was isolated and identified using bacteriological and biochemical tests. PCR method was used to identify the rbfE, stx1, stx2 and eae genes. Also, antibiotic resistance of the isolates was determined by disk diffusion method. Results: The results represented that among 25 isolates possess virulence genes, 40, 12 and 4% of the isolates contained eaeA, STx2, and both genes, respectively. But none of them contained H7, STx1, and rfbE genes. The antibiotic resistance pattern demonstrated that the isolates were highly resistant to Gentamycin and cefotoxime. Discussion and conclusion: The results of this study showed that the presence of verotoxigenic E.coli in vegetables; and high resistance of the isolates to antibiotics could be hazardous for public health.
Shiga toxin-producing Escherichia coli (STEC) is currently considered as an important food-borne pathogen. The serotype O157: H7 is a widespread Enterohemorrhagic Escherichia coli (EHEC) serotype that causes several diseases such as hemorrhagic colitis, hemolytic uremic syndrome and thrombocytopenic purpura. The pathogenic capacity of STEC is due to a number of virulence factors such as Shiga toxins (Stx1 and Stx2) and intimin (1). In fact, the ability of EHEC to colonize in human and animal intestinal mucosa and cause disease is associated with a number of virulence factors, including expression of Shiga toxins (Stx) (2) and the capacity to induce attaching/effacing (A/E) lesions (3). A/E lesions are characterized by intimate bacterial attachment to the host cell membrane and destruction of microvilli at the site of bacterial adhesion. Fresh vegetables can become contaminated in different points in the food processing chain. Thus, knowledge of survival and persistence of enteric pathogens on edible crops and during production provides information for agricultural practices. Leaves are inhabited by diverse highly-adapted microbes and are colonized frequently by transient microorganisems, including human pathogens (4).
A wide range of animal species are known to carry STEC and EHEC strains, but ruminants are the most important natural reservoir and excrete these bacteria through their feces (5). The main routes of infection transmission are person-to-person and consumption of contaminated meat and milk. Moreover, ingestion of contaminated vegetables or water and direct contact with animals or soil are associated with EHEC-associated outbreaks. It has been reported that human infections can result from ingestion of fewer than 100 viable EHEC cells (5). Moreover, EHEC and STEC can persist and remain infectious for several weeks in slurries, farmyard manure and sewage sludge as well as on pasture land (6- 8).
The aim of this study was to identify the virulence genes and antibiotic resistance of the verotoxigenic E. coli isolated from vegetables.
Materials and methods
Sample collection: A total of 500 fresh vegetable samples including Mentha, Ocimumbasilicum, Lepidiumsativum, Allium ampeloprasumpersicum, and Allium fistulosum were collected randomly from retail shops. Detection of E. coli: A portion of vegetable sample (100 g) was washed with 200 ml of sterile distilled water. 100 ml of raising water was centrifuged, 1ml of sediment transferred to EC broth medium and incubated at 37oC for 24 h. Then, 0.1 ml of EC broth was transferred to MC agar and incubated at 37oC for 24 h. Suspected colonies were selected and differential biochemical tests such as IMViC and culture on Eosin methylen blue medium were conducted to confirm E.coli. (9).
Identification of virulence genes by PCR: In order to identify the virulence genes, PCR assay was performed. E. coli colonies were subcultured in trypton soy broth. The bacterial cells were suspended in 250 µl of sterile water and boiled at 100°C for 5 min to release the DNA, and the aliquot was centrifuged in 1200 rpm for 3 min. The supernatant was used in the PCRas template (10).
The strain of E.coli O157:H7 was prepared from microbiology laboratory of veterinary medicine of Tehran University as a positive control and sterile distilled water was used as a negative control. Sequences and predicted sizes of amplified products for the specific oligonucleotide primers are shown in Table 1. PCR was used for detection of stx1, stx2, eae A, H7 and rfbE genes. Amplification of bacterial DNA was performed using 30µl volumes containing 50 ng of the prepared sample supernatant, 150 ng of the oligonucleotide primers, 0.2 mM (each) dATP, dGTP, dCTP, dTTP, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 50 mMKCl and 1 U of Taq DNA polymerase (Bioline, London, United Kingdom).
The conditions performed in a thermal cycler (Hybaid, Southhampton, United Kingdom) were 94°C for 2 min followed by 35 cycles of 94°C for 1 min, 55–64°C for 1 min and 72°C for 1 min. The amplified products were visualized by standard submarine gel electrophoresis using 10 µl of the final PCR product on a 2% agarose gel in 0.5% TBE buffer. The samples were electrophoresed for 40 min at 100 V. Amplified DNA fragments of specific sizes were located by UV fluorescence after staining with Ethidium bromide (10).
Antibiogram tests: The antibiotic susceptibility of the isolates contained virulence genes were performed by disc diffusion method. The isolates were inoculated in TSB medium and incubated for 24 h at 37˚C. After observing turbidity in TSB medium, a swab of bacteria was plated onto Mueller Hinton agar medium and antibiotic disks containing Ampicillin 10, Ciprofloxacin 5, Tetracycline 30, Trimethoprim-sulfamethoxazol 20, Cefotaxime 30 and Gentamicin 10, (Padtan Teb, Co) were placed in the plates. Plates were incubated for 24h at 37˚C. The zone of growth inhibition for each antibiotic disc was measured. The zone diameters around all disks were interpreted using the recommendation of the Clinical and Laboratory Standards Institute (CLSI).
Table 1- Characterization of primers for detection of virulence genes of isolated E. coli (Rey 2006), (21).
Oligonucleotide Sequence (5'-3')
Table 2.The E. coli isolates possess virulence genes from vegetables.
eaeA and STx2
No of Sample
Type of sample
Overall, Escherichia coli was detected in 25 vegetable samples. E. coli O157:H7 was not found in samples, and 4 numbers (16%) of the isolates were non-O157 STEC. Among the isolates possess virulence genes, 40, 12 and 4% of them contained eaeA, STx2 and both genes, respectively. But none of the isolates contained STx1gene (Table 2) (Figs 1- 4).
A total of 14 E. coli isolates possess virulence genes, were examined for antimicrobial resistance. The results showed that resistance against Tetracycline, Ciprofloxacin, Ampicillin, Cefotaxime, Trimetoprim-Sulfamethoxazol and Gentamicin were 57.14, 35.71, 57.14, 85.71, 57.14 and 100% respectively. (Table 3).
Table 3- Antimicrobial resistance of verotoxigenic E.coli isolated from vegetables.
Discussion and conclusion
In recent years, verotoxigenic Escherichia coli caused epidemics in some parts of the world and for this reason it has considered by researchers. Blanco reported that E. coli O157: H7in level of 1 to10 CFU/g may cause illnesses. It can survive for 15 days in lettuce (10). Also Bell et al. demonstrated that E. coli O157: H7 can be present in fruits such as apples (11). E.coli is not only regarded as an indicator of fecal contamination but more likely as an indicator of poor hygiene and sanitary practices during processing and further handling of food products. High prevalence of E.coli in milk was reported by Ali (63%), (12) and Lingathurai (70%), (13). The incidence of E. coli in food products is not significant because E. coli is normally an ubiquitous organism (14), but the pathogenic strains, if present could be harmful for consumers. In this study, from a total of 500 vegetable samples examined, 25 isolates of E. coli were identified; and none of the isolates were O157: H7. It could be due to the fact that in fall and winter the prevalence of serotype O157: H7 is lower than in spring and summer. So absence of serotype O157: H7 strains in vegetables examined in this study is acceptable.
Fig 1- PCR for identifing eaeA gene (expected band: 775 bp) La: Ladder, C+: Positive control, C- : Negative control 1, 2 and 3: positive samples
Fig 2- PCR for identifing O157 gene (expected band: 259 bp) La: Ladder, C+: Positive control, C-: Negative control, No sample was positive
Fig 3- PCR for identifing STx1 gene (expected band: 302 bp) La: Ladder, C+: Positive control, C-: Negative control. No sample was positive
Unlike our results, in a study in Canada, 26 samples of unpasteurized Gouda cheese were tested and two samples were positive for E. coli O157: H7. This study was conducted due to incidence of HUS syndrome in Canada, in which 13 cases of HUS was observed. So consumption of cottage unpasteurized Gouda cheese was considered as the source of infection (15). In a study in Pennsylvania, from 248 milk samples tested for the presence of food-borne Pathogens, 4.2% of samples were positive for STEC (16).
In the present study, 5% of samples were contaminated with E.coli. This could be due to using human and animal manure as fertilizer which is a major source of this bacteria. Several studies indicated that E. coli can persist in manure-fertilized soil for several months (17 & 18). It is recommended to use compost manure for fertilizing organic Vegetables instead of human and animal manure (19). Practices of manure application have been shown to vary considerably among organic vegetable producers (20).
The results of the present study showed that none of the strains of E.coli possess genes STx1, H7 and rfbE, but 40% of the isolates contain eaeA genes. Similarly, in a study in Spain on Shiga toxin-producing Escherichia coli, serotype O157: H7 strains isolated from tanks of fresh milk and cheese contained eaeA genes. In addition, it was found that 12% of isolates possess STx2 gene and 4% contain both eaeA and STx2 genes (21). Similarly, in a study by Garcia et al. in Mexico, from 22 STEC strains isolated from patients with diarrhea, most strains harbored Stx2 gene or combination of eaeA and STx1 genes (22).
Based on the results of the present study, 85.71, 35.71, 57.14 and 100% of samples were resistant to cefotaxime, ciprofloxacin, trimetoprim-sulfamethoxazol and gentamycin respectively. In a study in Kermanshah-Iran, Mohajeri et al. found that from a total of 200 strains of E. coli 27, 5.22 and 26% of samples were resistant to cefotaxime, ceftaxidim, and ciprofloxacin, respectively (23). Many researchers reported the highest levels of resistance to those antimicrobials. About antimicrobial resistance of isolated strain in vegetables, highly variable resistance results were obtained and the resistance rate was quite high in some cases and low in others (24- 27). However, in most cases, a direct comparison of studies is difficult because of different types of samples involved, different scopes of bacterial species targeted, different methods of strain isolation and different antimicrobials tested (28- 30).
The presence of verotoxigenic E.coli in vegetables and high resistance of the isolates to antibiotics may be deduced from the results of this study. It could be a public health problem at the moment.
(2) O’Brien AD., Tesh VL., Donohue-Rolfe A. Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis. Current Topics in Microbiology and Immunology 1992; 180: 65- 944.
(3) Phillips AD., Navabpor S., Hicks S., Dougan A. Enterohaemorrhagic Escherichia coli O157:H7 targets Peyer’s patches in man and causes attaching-effacing lesions in both human and bovine intestine. Gut 2000; 47: 377- 811.
(4) Lindow SE., Brandl MT. Microbiology of the phyllosphere. Applied and Environmental Microbiology 2003; 69: 1875- 833.
(5) Caprioli A., Morabito S., Brugere H. Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission. Veterinary Research 2005; 36: 289- 3111.
(6) Duffy G. Verocytoxigenic Escherichia coli in animal feces, manures and slurries. Journal of Applied Microbiology 2003; 94 (Suppl.): 94- 103.
(7) Hepburn N F., MacRae M., Ogden ID. Survival of Escherichia coli O157 in abattoir waste products. Letter in Applied Microbiology 2002; 35: 233- 66.
(8) Maule A. Survival of verocytotoxigenic Escherichia coli O157 in soil, water and on surfaces. Applied Microbiology 2000; 29: 71- 88.
(10) Blanco M., Blanco JE., Mora A. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from healthy sheep in Spain. Journal of Clinical Microbiology 2003; 41: 1351- 655.
(11) Bell C., Kyriakides A. Pathogenic Escherichia coli. in: Blackburn C., McClure P., eds, Foodborne pathogens. Cambridge 2000; 294- 55.
(12) Ali AA., Abdelgadir WS. Incidence of quality of milk and milk products with special Escherichia coli in raw cow milk in Khartoum reference to Salmonella and its public health State British. Journal of Dairy Sciences 2011; 2 (1): 23- 6.
(13) Lingathurai S., Vellathurai P. Bacteriological Quality and Safety of Raw Cow Milk in Madurai, South India. Available at: http://www.webmedcentral.com/article_view/1029, 2010.
(14) Hahn G. Pathogenic bacteria in raw milk situation and significance. In: Bacteriological quality of raw milk. Brussels (Belgium). Internation Dairy Federation 1996; 67- 833.
(15) Alves D., Kamali MA. 20-year population –based study of haemolytic syndrome: evidence for the importance of shiga- like toxin producing E.coli. Recent Advance In VTEC Infractions. Infection and Immunity 1999; 655 (7): 221- 34.
(16) Jayarao BM., Donaldson SC., Straley BA. A survey of foodborn pathogens in bulk tank milk and raw milk consumption among farm families in Pennsylvania. Journal of Dairy Sciences2006; 89: 2451- 8.
(17) Ingham SC., Losinski JA., Andrews MP. Escherichia coli contamination of vegetables grown in soils fertilized with noncomposted bovine manure: garden-scale studies. Applied and Environmental Microbiology 2004; 70:: 6420- 7.
(18) Islam M., Doyle MP., Phatak SC. Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or irrigation water. Journal of Food Protectin 2004; 67: 1365- 70.
(19) Anonymous. Soil fertility and crop nutrient management practice standard. National Organic Program. United States Department of Agriculture, Washington DC 2000; 205- 3.
(20) Landau B., Koller M., Mader P. Pathogenic identification of critical control points for organic vegetable crops. Report Online Organic. Available at: http://orgprints org/20389/2013.
(21) Rey J., Sanchez S., Blanco JE. Prevalence, serotypes and virulence genes of shiga toxin-producing E.coli isolated from ovine and caprine milk and other dairy products in spain. International Journal of Food Microbiology2006; 107 (2): 207- 12.
(22) Garcia T., Cerna JF., Paheco- Gil L. Drug resistant diarrheogenic Escherichia coli, Mexico. Emerging Infectious Diseases 2005; 11: 1306- 88.
(23) Mohajeri P., Izadi B., Rezai M., Falahi B. Assessment of the frequency of extended spectrum Beta Lactamases producing Escherichia coli isolated from urinary tract infections and its antibiotic resistance pattern inn Kermanshah. Journal of Ardabil University of Medical Sciences2011; 11 (1): 86- 94. [in Persian]
(24) Osterblad M., Pensala O., Peterzens M. Antimicrobial susceptibility of Enterobacteriaceae isolated from vegetables. Journal of Antimicrobial Chemotherapy 1999; 43: 503- 99.
(25) Schwaiger K., Helmke K., Holzel CS. Antibiotic resistance in bacteria isolated from vegetables with regards (sic) to the marketing stage (farm vs. supermarket). International Journal of Food Microbiology 2011; 148: 191- 6.
(26) Hassan SA., Altalhi AD., Gherbawy YA. Bacterial load of fresh vegetables and their resistance to the currently used antibiotics in Saudi Arabia. Foodborne Pathogen Diseases 2011; 8: 1011- 18.
(27) Holvoet K., Sampers I., Callens B. Moderate prevalence of antimicrobial resistance in Escherichia coli isolates from Lettuce, irrigation water, and soil. Applied and Environmental Microbiology 2013; 79 (21): 6677- 82.
(28) McGowan LL., Jackson CR., Barrett JB. Prevalence and antimicrobial resistance of enterococci isolated from retail fruits, vegetables, and meats. Journal of Food Protection 2006; 69: 2976- 82.
(29) Boehme S., Werner G., Klare I. Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs. Molecular Nutrition and Food Research 2004; 48: 522- 31.
(30) Johnston LM., Jaykus LA. Antimicrobial resistance of Enterococcusspecies isolated from produce. Applied and Environmental Microbiology 2004; 70: 3133- 77.