Molecular analysis of Shiga toxin-producing Escherichia coli O157:H7 and non-O157 strains isolated from calves

Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 are food-borne pathogens and contaminants of foods of animal origin. This study was conducted to investigate the presence of virulence and integrase genes in STEC isolates from diarrhoeic calves in Fars Province, Iran. Five hundred and forty diarrheic neonatal calves were randomly selected for sampling. Rectal swabs were collected and cultured for isolation and identification of E. coli following standard methods. The isolates were analysed for the presence of class 1 integrons and bacterial virulence factors using polymerase chain reaction (PCR). Antimicrobial susceptibility testing was performed using the Kirby–Bauer disc diffusion method. Out of 540 diarrhoeic faecal samples, 312 (57.7%) harboured E. coli and 71 (22.7%) of them were identified as STEC: 41(69.5%) carried the stx2 gene, 21 (35.6%) carried the stx1 gene and 3 (5%) carried both. Twenty-six (44%) of the isolates showed the eae gene. Among the STEC isolates examined for susceptibility to eight antimicrobial agents, erythromycin and penicillin (96.8%) resistance were most commonly observed, followed by resistances to ampicillin (71.8%), tetracycline (62.5%) and trimethoprim/sulfamethoxazole (39%). Integrons were detected by PCR in 36% of the STEC tested isolates, 57 (89%) of which showed resistance to at least three antimicrobial agents. Our findings should raise awareness about antibiotic resistance in diarrhoeic calves in Fars Province, Iran. Class 1 integrons facilitate the emergence and dissemination of multidrug-resistance (MDR) among STEC strains recovered from food animals.


Introduction
New research provides the strongest evidence that Shiga toxin-producing Escherichia coli (STEC) non-O157:H7 and in particular serogroup O157 are linked to severe gastrointestinal diseases . The clinical manifestations of STEC infection can vary, from asymptomatic carriage to very serious illnesses such as haemolytic uremic syndrome (HUS), thrombocytopenic purpura (TTP) and haemorrhagic colitis (HC) (Thomas et al. 2012). Estimates vary, but experts suggest that gastrointestinal infections are responsible for approximately 1.5 million deaths per year, over 90% of which are in developing countries (Montenegro et al. 2011). For instance, non-O157:H7 serogroups are found in more than 36 000 cases of infections annually and at least 73 000 are infected with O157:H7 serogroup in the United States (US) (Zhao et al. 2001).
Studies have revealed that O157 and non-O157 strains of cattle origin can cause the disease in humans via consumption of raw milk and undercooked meat. In fact, cattle, especially young animals, are known to be the primary reservoirs of both non-O157 and O157 STEC (Moura et al. 2012). The pathogenicity of STEC is associated with Shiga toxin (stx) encoded by Shiga toxinogenic (stx) genes 1, 2 (stx1 and stx2) and an outer membrane protein which is encoded by the chromosomal eae gene (Pradel et al. 2008).
The problems with some new STEC strains isolated from neonatal calf diarrhoea (NCD) (Rigobelo et al. 2008), which is recognised as a disease complex characterised by acute, undifferentiated diarrhoea in newborn calves, are that antibiotic multiresistance (De Verdier et. al. 2012) and STEC strains can be transmitted to humans by contact occupational exposure and the food chain (Schroeder et al. 2002). Epidemiological observations show high levels of antimicrobial resistance in bacterial pathogens from veterinary and human medicine (Zhao et al. 2001). This has led to the discovery that these bacteria are able to acquire antibiotic resistance by resistance-conferring genes, many of which are carried on transposons, plasmids or integrons (Bakhshi, Najibi & Sepehri-Seresht 2014). An integron is mainly composed of an integrase gene that encodes a sitespecific recombinase, by which an insertion site of integron is recognised. Moreover, an integron Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 are food-borne pathogens and contaminants of foods of animal origin. This study was conducted to investigate the presence of virulence and integrase genes in STEC isolates from diarrhoeic calves in Fars Province, Iran. Five hundred and forty diarrheic neonatal calves were randomly selected for sampling. Rectal swabs were collected and cultured for isolation and identification of E. coli following standard methods. The isolates were analysed for the presence of class 1 integrons and bacterial virulence factors using polymerase chain reaction (PCR). Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method. Out of 540 diarrhoeic faecal samples, 312 (57.7%) harboured E. coli and 71 (22.7%) of them were identified as STEC: 41(69.5%) carried the stx2 gene, 21 (35.6%) carried the stx1 gene and 3 (5%) carried both. Twenty-six (44%) of the isolates showed the eae gene. Among the STEC isolates examined for susceptibility to eight antimicrobial agents, erythromycin and penicillin (96.8%) resistance were most commonly observed, followed by resistances to ampicillin (71.8%), tetracycline (62.5%) and trimethoprim/sulfamethoxazole (39%). Integrons were detected by PCR in 36% of the STEC tested isolates, 57 (89%) of which showed resistance to at least three antimicrobial agents. Our findings should raise awareness about antibiotic resistance in diarrhoeic calves in Fars Province, Iran. Class 1 integrons facilitate the emergence and dissemination of multidrugresistance (MDR) among STEC strains recovered from food animals. contains a variable region which is the place for gene cassettes to be inserted . Depending on the sequence of the encoded integrases (intI) catalysing excision and integration of deoxyribonucleic acid (DNA) units, eight distinct integron classes have been identified up to now, and class 1 integrons have shown to be the major contributors to multidrug-resistant (MDR) infections in the Enterobacteriaceae family (Singh et al. 2005).
Many studies in various countries including Iran have shown that the distribution of integrons among enteric bacteria has increased over time (Eftekhari et al. 2013;Gonzalez et al. 1998;Hamada, Oshima & Tsuji 2003;Martinez-Freijo et al. 1998, 1999Najibi et al. 2012). In Iran, only a few studies have reported antimicrobial resistance properties and virulence genes in the pathogenic E. coli (Bakhshi et al. 2014;Shahrani et al. 2014). Unfortunately, there is no conclusive data on the distribution of virulence genes and the antimicrobial resistance properties of STEC strains isolated from Iran, particularly from Fars, which is one of the major agricultural and animal husbandry areas in Iran, with nearly 400 000 cattle and 8 000 000 sheep and goats (Shams et al. 2012).

Study design and study areas Sampling and Escherichia coli identification
A total of 540 recto-anal mucosal swabs from diarrhoeic calves (< 30 days of age) were collected over 1 year from November 2015 to November 2016.
These calves were raised on 33 farms from eight geographic areas in Fars Province, including industrial, semi-industrial and traditional farms, with a herd size of 25-500 cows. These farms had a recognised scouring problem in neonatal calves. Sick calves which showed abnormal faecal consistency and/ or signs of dehydration and weakness were selected. None of them had been vaccinated. All samples were immediately placed in cooled boxes and transported to the laboratory. The swab samples were incubated overnight at 37 °C in trypticase soy broth (TSB) (Merck KgaA, Darmstadt, Germany). Each sample was then streaked onto MacConkey's agar (MC, Merck, Germany) (24 hours at 37 °C). Lactose positive colonies were cultured on eosin methylene blue agars (EMB, Merck, Germany) (24 h at 37 °C). Green colonies with a metallic lustre were considered typical E. coli colonies. Such colonies were confirmed as E. coli using standard biochemical tests (citrate utilisation, indole production, glucose, lactose fermentation, urease negative and hydrogen sulphate production). The biochemically confirmed E. coli colonies were subjected to DNA analysis.

DNA extraction
A single colony of overnight TSB culture was suspended in 100 μL of distilled water and exposed to boiling for 10 min at 100 °C. After a 13 min freeze, the frozen cell pellets were centrifuged at 14 000 rpm for 10 min  and the supernatant, containing bacterial DNA, was subjected to PCR analysis.

Polymerase chain reaction detection of virulence factors and class 1 integron in Shiga toxin-producing Escherichia coli strains
Polymerase chain reaction assays were used to detect the presence of the following virulence genes coding regions including stx1, stx2 and eae. To detect class 1 integron in confirmed STEC isolates, a PCR protocol was employed. Preparation of the DNA samples was done as described in previously published paper . Primer sequences, sizes of PCR products and PCR conditions are shown in Table 1. DNA from E. coli O157:H7 EDL933 strain and ATCC 25922 strains were used as positive and negative controls, respectively. The amplified DNA products were separated by 1.5% agarose gel electrophoresis (Sigma-Aldrich, St. Louis, MO, United States). The gels were stained with ethidium bromide (Merek, Germany). Visualisation of amplified products was done by ultraviolet (UV) illumination and photographed using a Kodak camera system (Gel Logic 200).

Statistical analysis
The chi-square (χ 2 ) test and Fisher's exact test were used to assess whether integron-positive strains were significantly more resistant than integron-negative strains for each of the tested antibiotics. A p value < 0.05 was considered statistically significant. Statistical calculations were made using GraphPad Prism for Windows version 5 (GraphPad Software, San Diego, CA).

Characterisation of virulence genes
Of 312 E. coli strains, 71 isolates (22.7%) were identified as STEC. The virulence genes stx2, stx1 and eae were detected at 76%, 46.4% and 53.5% in STEC isolates, respectively. Of isolates that were not characterised as STEC, 101 (32.3%) were positive for eae gene (Figures 1 and 2). These findings are summarised in Table 2. Out of 59 non-O157 strains (PNU6, PNU11, PNU12 and PNU16) in the diarrhoeic calves, four were positive for both the stx1 and stx2 genes and three non-O157 harboured all of the stx1, stx2 and eae genes.

Integrons
Class 1 integrons were detected among 23 (36%) of the STEC isolates ( Figure 4 and Table 4). Integron-positive strains were significantly more resistant to enrofloxacin, trimethoprim/ sulfamethoxazole and tetracycline than integron-negative strains (p < 0.05). Nevertheless, resistance to ampicillin, erythromycin, penicillin, cefixime and chloramphenicol could not be directly related to the presence of integrons ( Table 5). All of the integron-positive strains displayed multidrug resistance. The most prevalent MDR phenotypes in integron-positive strains were AM-CFM-E-P-TET (26% of the 52 non-O157 strains).

Discussion
Antibiotic resistance developed in STEC isolates from humans and animals (Van Meervenne et al. 2013). Integrons, which are known to be associated with many antimicrobial resistance genes, were suspected to serve as pools of antimicrobial resistance genes worldwide (El-Sokkary & Abdelmegeed 2015). Class 1 integrons are commonly found in gram-negative pathogens (Maguire et al. 2001).
In this study, the presence of major virulence factors and resistance to antimicrobials belonging to classes generally   http://www.ojvr.org Open Access utilised in Iran was investigated in zoonotic STEC isolates from calves with diarrhoea. Owing to the close contact of humans with animals, the presence of virulence and antimicrobial resistance genes in E. coli strains harboured by animals leads to public health concerns (Torkan et al. 2016). Escherichia coli, which has been implicated as an aetiological factor of calf diarrhoea, harbours many virulence genes that enable it to cause disease in a particular host (Nagarjuna et al. 2015). In the present study, among 312 E. coli strains from diarrhoeic calves, 71 (22.7%) were STEC. The results are in agreement with those of Dastmalchi et al. (2012), who screened 51 E. coli isolates from diarrhoeic calves in the Urmia region, which is located in west Azerbaijan Province, Iran, and illustrated that 19.6% of isolates were stx positive. Most epidemiological studies in diarrhoeic calves in Iran have disclosed that the prevalence of STEC infection ranges between 6.4% and 34.5% (Pourtaghi, Dahpahlavan & Momtaz 2013; Shahrani et al. 2014). These discrepancies can be attributed to the small sample size and geographical differences. In other words, STEC prevalence in calves may be influenced by environmental factors (Dastmalchi et al. 2012). Higher prevalence of the stx2 gene (54 isolates) compared to the stx1 gene (33 isolates) in this study corroborates the findings of previous reports in Iran (Dastmalchi et al. 2012;Tahamtan, Hayati & Namavari 2010). However, these results contrast with other reports that have shown that most STEC from diarrhoeic calves only produce stx1, whereas stx2-positive strains are the dominant types in healthy calves (Nguyen, Vo & Vu-Khac 2011). The differences in these findings suggest that stx2 may be associated with a majority of E. coli isolates from diarrhoeic calves in Iran. Shiga toxin producing E. coli infection, which is associated with diarrhoea in calves, may result in severe diseases in humans such as HUS and HC (Bastos et al. 2006). The diarrhoeal phase of diseases associated Note: Antibiotic resistance profile was determined for eight antibiotics: ampicillin (AM), tetracycline (TET), erythromycin (E), enrofloxacin (ENR), trimethoprim/sulfamethoxazole (SXT), chloramphenicol (C), penicillin (P) and cefixime (CFM). stx, isolates carrying stx1 and/or stx2 genes; MDR, multidrug-resistant isolates; S, antibiotic-susceptible isolates; R, antibiotic-resistant isolates. +, positive for int gene or MDR. with STEC is usually self-limiting, and the role of early antimicrobial treatment in the prevention of HUS is still regarded as controversial (Shahrani et al. 2014). Current recommendations and the available data suggest that not only do antibiotic exposure increase the risk of HUS in children via inducing expression of stx through replication of temperate bacteriophages carrying stx-encoding genes (Ochoa et al. 2007), it turns out to have another perilous effect on the frequency of STEC antimicrobial resistance (Shahrani et al. 2014), which could result in an increase of frequency of STEC and perhaps greater shedding. Resistance could contribute to greater contamination of animal food products with STEC (Torkan et al. 2016). Several reports have documented that a significant increase of antimicrobial resistance in STEC strains isolated from animals and humans has acquired antibiotic resistance genes almost 20 years ago (Zhao et al. 2001). In STEC strains, class 1 integrons are strongly associated with multidrug resistance (Colello et al. 2015 (Singh et al. 2005;Zhao et al. 2001). Class 1 integrons appear to be common in the endemic STEC strains. In the present study, class 1 integron was identified in 23 (36%) out of 71 STEC isolates. Our data revealed low distribution of class 1 integrons among STEC isolates from calves with diarrhoea in the south of Iran compared with a similar study in northern Iran in 2014 for which the authors found a higher percentage (53%) of the strains containing integron class 1 (Bakhshi et al. 2014). The various percentages of class 1 integrons in different parts of the world could be attributed to the characteristics of the analysed collection and differences in the prevalence of antibiotic consumption in each country (Kargar et al. 2014). In general, exposure to antibiotics, heavy metals or biocides and a high multiplicity of other different environmental factors are among the main reasons for an increase of cells containing integrons (Baquero, Martínez & Cantón 2008).

Conclusion
We report the presence of class 1 integrons in the most familiar STEC strains from diarrhoeic calves. Results imply that stx2, stx1 and eae putative virulence gene, the IntI integrase gene and resistance to erythromycin, penicillin, ampicillin, tetracycline and trimethoprim/sulfamethoxazole were the most commonly detected characteristics of the STEC strains isolated from diarrhoeic calves in southern Iran. Our investigation demonstrated that calves are possible reservoirs of STEC strains and developed resistance to multiple classes of antimicrobials. Emerging data suggest an association between MDR and integrons which may play a significant role in the dissemination of resistance genes. Therefore, it is advised to stop routine antimicrobial treatment and conduct further molecular studies to detect other antimicrobial resistance and virulent genes in STEC isolates obtained in this study.