مقاومت آنتی‌بیوتیکی سویه‌های وروتوکسیژن باکتری اشریشیا کلی جدا شده از سبزیجات

Introduction

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 37^oC for 24 h. Then, 0.1 ml of EC broth was transferred to MC agar and incubated at 37^oC 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).

Table 2.The E. coli isolates possess virulence genes from vegetables.

Results

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.