Among the 257 raw poultry samples, 93 (36%) isolates were identified as E. coli. Based on serological and microbiological tests, 36 (38.7%), 7 (7.5%), and 12 (12.9%) of the E. coli isolates were characterized as STEC (stx1 and/or stx2 and eae+/eae_), EPEC (positive for eae), and AEEC strains (stx1 and/or stx2 and eae+ or negative for stx1 and stx and positive for eae), respectively. All of the STEC isolates showed colorless colonies on the MacConkey sorbitol media culture.
The results of the antimicrobial susceptibility test conducted on 93 E. coli isolates are shown in Figure 1. Based on the results of the antimicrobial susceptibility test, all of the isolates were susceptible to cefotaxime, cefoxitin, ceftazidime, and aztreonam. A high-level of resistance to nalidixic acid (91.4%), tetracycline (89.2%), ampicillin (82.8%), and sulfamethoxazole-trimethoprim (71%) was detected among the E. coli isolates. The PCR results showed that the distribution of the virulence genes stx1, stx2, and eae among the 93 E. coli isolates was 15 (16.1%), 31 (33.3%), and 12 (12.9%), respectively. All of E. coli O157 strains showed stx1/stx2/eae (1 isolate), stx1/eae (2 isolates), and sx2/eae (2 isolates) patterns. The hlyA gene was not detected in any of the E. coli isolates (Figure 2). The analysis of the ERIC-PCR results showed genetic diversity among E. coli O157 strains because five different ERIC patterns were observed among these strains (Figure 3).
The results of our study have shown that chicken meat can be contaminated with E. coli. This organism was isolated from 93 (36%) raw chicken meat samples and 36 (38.7%), 7 (7.5%), and 12 (12.9%) of the E. coli isolates were characterized as STEC, EPEC, and AEEC strains. The stx2 gene was the most frequent virulence factor among the STEC isolates. The major animal source of STEC is primarily cattle, followed by sheep, goats, pigs, and poultry. Poultry meat is known as the potential source of STEC contamination compared to other sources of meat. In Korea, STEC was isolated in 22.6% of beef, 7.3% of poultry, and 2.0% of pork meat samples . In the current study, O157 E. coli isolates having stx1 and/or stx2 and eae were detected in 5.3% of the poultry meat samples and recognized as STEC strains. Although the prevalence of this isolate was not significant, this rate of infection is considerable from the public health point of view.
The prevalence of STEC and AEEC in the current study is different from that of some studies in Iran and other countries. In the current study, higher STEC and lower AEEC isolates were detected compared to the study of Momtaz el al. They reported that the prevalence of STEC and AEEC were 21% and 34%, respectively, among 422 raw poultry meat samples in different cities of Iran . They also reported that stx1 was the most frequent (96%) virulence factor among the isolates. In contrast, in the current study, stx1 was found only in 16% of the isolates. One of the reasons for this difference in frequency can be the difference in the number of samples studied. However, Guran et al. showed that the overall prevalence of E. coli O157 in poultry meat samples collected from supermarkets in Diyarbakir, Turkey was 1.3% . One of the significant results of the current study is that 12.9% of the E. coli isolates were identified as AEEC. Intimin genes are present in EPEC and in some STEC. Atypical enteropathogenic E. coli (EPEC) or AEEC appears to be more closely related to STEC [13-15]. Based on the results of the current study, the role of AEEC strains in gastrointestinal infection needs further investigations .
In this study, the E. coli O157 strains were positive for stx1, stx2, and eae genes. In India, Dutta et al. reported that 14 (33.33%) isolates carried at least 1 virulence gene. 10 (23.81%) of these isolates (collected from poultry samples) were recorded as STEC and 4 (9.52%) of them were recorded as EPEC [11, 17].
Some gastrointestinal infections caused by E. coli have a bacterial origin and need to be treated by antibiotics. In a review study by Roth et al., the resistance rates of E. coli strains (isolated from broiler samples) to tetracycline, sulfamethoxazole, streptomycin, and ampicillin were more than 40% in all the studied countries. Increasing antibiotic resistance is a major concern for animal and human health because of the high consumption of antibiotics in veterinary medicine. Resistant bacteria can spread from food-producing animals to humans. The information from the evaluated countries indicates that such antibiotics as tetracycline, aminoglycoside, sulfonamide, and penicillin are usually used in poultry industry .
In this study, the resistance levels of STEC to some antimicrobial agents such as nalidixic acid, ampicillin, tetracycline, and trimethoprim-sulfamethoxazole ranged from 71 to 91%. According to these results, the poultry meat contaminated with STEC strains can be a potential source of antimicrobial resistance. In a study conducted in Thailand, 100% of the E. coli isolates showed resistance to tetracycline, ampicillin, and erythromycin.
Momtaz et al. reported the high resistance of STEC strains to tetracycline, chloramphenicol, and nitrofurantoin (63 to 77%). According to our findings and studies by others, the prescription of tetracycline is recommended neither in cases of E. coli infection nor in veterinary medicine with respect to poultry products . There are few reports about the molecular typing of STECs from poultry sources in Iran and other countries. In the current study, ERIC-PCR genotyping demonstrated 5 different ERIC-genotypes from 5 E. coli O157 isolates. Therefore, the results of the current study showed genetic diversity among E. coli O157 isolates as well as the different potential sources of E. coli O157 contamination. The results of the present research also indicated the usefulness of the PCR-based genotyping method in the epidemiological investigations of virulent E. coli strains and of STEC strains. There is some limitation in this study due to the lack of access to human samples because finding relevant human samples is very difficult except during infection outbreaks. Consistent with our results, in a study by Shekar et al. in India, the ERIC-PCR results discriminated 12 STEC isolates from poultry samples into 11 ERIC-PCR genotypes .
In conclusion, the results of the current study revealed that poultry meat can be considered as a source of pathogenic E. coli strains. Pathogenic E. coli strains in poultry meat samples were detected by such accurate and quick techniques as PCR assay. The detection of STEC (38%) was a significant finding. The stx2 was identified as the most frequent virulence factor among the STEC isolates. Our results highlight the need to pay more attention to controlling poultry meat and also antibiotic prescription in veterinaries. The results indicated that the E. coli virulence genes especially stx1, stx2, and eae existed to a large extent in pathogenic E. coli strains isolated from poultry meat.
One of the most important limitations of this study was the rather few number of raw poultry meat samples. More samples are required for such molecular studies. We also had some limitations in financial support for obtaining information about poultry raising systems and slaughter systems to discuss the sources of contamination by robust typing methods.