Enteropathogenic E. Coli Isolates Research Articles

Serotypes, Pathotypes, Shiga Toxin Variants and Antimicrobial Resistance in Diarrheagenic Escherichia coli Isolated from Rectal Swabs and Sheep Carcasses in an Abattoir in Mexico

Sheep represent one of the main reservoirs of diarrheagenic Escherichia coli; this microorganism is an etiological agent of food-borne diseases; therefore, this work aimed to identify and characterize the principal pathotypes of diarrheagenic E. coli (DEC) obtained through rectal swabs and carcasses samples from sheep slaughtered in an abattoir at the central region of Mexico. The isolates were subjected to bacteriological identification, serotyping; phylogenetic classification; detection for virulence factors, and antimicrobial sensibility. A total of 90 E. coli isolates were obtained. It was observed through 49 E. coli isolates (54%), 8 of them from carcasses, and 43 from feces was DEC. DEC serotypes with health public relevance were found: O76:H19 (n = 5), O146:H21 (n = 3), O91:H10 (n = 1), O6:NM (n = 1), and O8:NM (n = 1). Regarding the presence of Shiga toxin-producing E.coli (STEC), 43/90 (47.7%) isolates have the stx1 w/o stx2 genes, and therefore were assigned as STEC non-O157; only one isolate expressed stx1 and eae genes and was classified as t-STEC (typical STEC). Additionally, 3/90 (3.3%) harbored only the eae gene and were classified as enteropathogenic E. coli (EPEC), the stp gene was found in 2/90 isolates (2.2%) and were classified as enterotoxigenic E. coli (ETEC); 1/90 (1.1%) isolates harboring the ipaH were classified as enteroinvasive E. coli EIEC. Regarding stx1 genes subtypes, stx1c only was found in 60.5% (26/43), followed by stx1a-stx1c 20.9% (9/43) and stx1a-stx1d 2.3% (1/43). The presence of both, stx1 and stx2 genes was found in 7/43 isolates (16.3%) from rectal swabs; the combination stx1c-stx2g was detected in 3/43 isolates (6.9%), while 4 (9.4%) isolates showed different patterns (stx1a-stx1c-stx2g; stx1c-stx2b-stx2g; stx1c-stx2b and stx1a-stx1c-stx2b-stx2g). STEC isolates showed the major diversity of phylogenetic groups, although phylogroup B1 was predominant in 90.6% (39/43) while there was only one isolate (2.3%) in each remaining phylogroup (A, B2, C, and F). All EPEC, ETEC, and EIEC isolates were clustered in phylogroup B1. We observed that 27.9% (12/43) of STEC isolates carried at least one antibiotic resistance: nine isolates expressed the tetB gene, one isolate the tetA gene, two isolates the sul2 gene, one isolate the sul1 and one isolate the sul1-tetB genes. These results highlight the importance of diarrheagenic E. coli as a potential risk for public health during the slaughtering process.

Shiga Toxin-Producing Escherichia coli in Diarrheic Neonatal Dairy Calves: Matching Small Intestinal and Fecal Isolates in Terms of Isolation Rates and Antimicrobial Resistance

Background: Shiga toxin-producing strains have been considered remarkable diarrheagenic agents and foodborne pathogens. Several studies have mentioned the role of some Shiga toxin-producing Escherichia coli (STEC) strains in diarrhea and dysentery in calves. Enteropathogenic E. coli (EPEC) have also been isolated from diarrheic calves. Generally, the culture and antibiogram results obtained from fecal samples are used to select antibiotics to treat calf diarrhea. However, the value of such a sampling method has not been evaluated yet. Objectives: This study aimed to evaluate the clinical utility of fecal sample cultures for isolating STEC and EPEC in calf diarrhea by comparing them with small intestine samples. Materials and Methods: The small intestine and fecal samples were simultaneously collected from 35 diarrheic calves. Small intestine samples were collected under the ultrasonographic guide. A total of 70 confirmed E. coli isolates were screened by the multiplex polymerase chain reaction to detect genes encoding Shiga toxin1 (stx1), Shiga toxin2 (stx2), intimin (eae), and hemolysin (E-hly). We also compared the presence of class 1 and 2 integrons and antimicrobial resistance properties in the STEC and EPEC isolates recovered from the small intestine and fecal samples. Finally, the presence of important STEC/EPEC serogroups, including O26, O103, O111, O113, O145, and O157 in isolates from both samples, was determined as well. Results: STEC strains were detected in 25.7%, and 20% of E. coli isolates obtained from the small intestine and fecal samples, respectively. The stx1 was the sole Shiga toxin subtype detected among STEC in intestinal and fecal isolates. EPEC was detected only in one and two E. coli isolates from the small intestine and fecal samples, respectively. Conclusion: A numerically higher prevalence of STEC was observed in the small intestine compared to fecal samples; there was no significant difference in the frequencies of STEC and EPEC isolates between the small intestine and fecal samples. The results indicated that the fecal sample, as a non-invasive and practical method, could be used for isolating STEC and EPEC in calf diarrhea. The antibiogram showed the presence of a high degree of multi-drug resistance among the isolates.

Antibacterial Activity of Traditionally Used Plants against the Resistant Enteropathogenic Escherichia coli

Background: It is well known that the herbs have therapeutic efficacy against infectious agents including bacterial ones. In the current study, the antibacterial activities of methanolic extracts of nine herbs were evaluated against the resistant enteropathogenic E. coli (EPEC) using disc diffusion method. Methods: Isolation and identification of E. coli and EPEC was done by standard bacteriological methods and PCR assays. Sensitivity of the EPEC isolates to antibiotics was determined by disc diffusion method. The methanolic extracts of the herbs were prepared and growth inhibition test was done by disc diffusion method. Minimum inhibitory concentration and minimum bactericidal concentration were determined by agar microdilution method. Result: Out of 150 faecal samples, a total of 135 E. coli isolates were isolated and among them, a total of 36 isolates were identified as EPEC. EPEC isolates showed resistance to multiple antibiotics. The maximum mean inhibition zone of antibacterial effect against the resistant clinical isolates of EPEC was demonstrated by the methanolic extract of Holarrhaena antidysenterica (19.16±0.76 mm) followed by Acacia nilotica (18.00±1.00 mm) and Punica granatum (16.67±1.53 mm). This study validates that the said plants can be used as therapy against colibacillosis in neonatal goat kids.

Evaluation of the frequency of Escherichia coli pathogroups in Brassica oleracea cultivars.

Background and Objectives:Pathogenic diseases resulting from microbial contamination of food have been widely distributed in many parts of the world. Among these, Escherichia coli is one of the most important foodborne pathogenic bacteria. Diarrhea is one of the major causes of children’s death in developing countries, with approximately 2 million deaths annually. The current study aimed to determine the frequency of diarrheagenic E. coli pathotypes such as Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), Enteroaggregative E. coli (EAEC), and Shiga toxin-producing E. coli (STEC) in Brassica oleracea cultivars in order to provide information on the assessment of diarrheagenic E. coli pathogenesis risk.Materials and Methods:100 samples of vegetables were collected in Tehran, including cabbage, cauliflower, broccoli and Brussels sprouts. After homogenizing samples, enrichment was done in the EC broth medium. Five colonies of pure culture were used for DNA extraction. Pathotypes were identified by PCR using virulence genes.Results:The results showed that the prevalence of diarrheagenic E. coli strains was 7%. The EPEC prevalence was 3%, All EPEC isolates were atypical. The ETEC frequency was 3%, And the EAEC prevalence was 1%.Conclusion:These findings indicated that Brassica oleracea cultivars could be consideredas a source of contamination with diarrhea-causing E. coli strains.

Characterization of shiga-toxin producing E.coli (STEC) and Enteropathogenic E. coli (EPEC) from domestic animals for virulence and colonisation factors and their antimicrobial resistance

Aim: To determine the occurrence and molecular characterization of Shiga toxin-producing E. coli (STEC) and Enteropathogenic E. coli (EPEC) from different domestic animals for serogrouping, virulence factors and their antibiotic resistance from Jammu region. Methodology: Faecal samples were collected from 200 different animals including calves (39), rabbits (24), dogs (38), slaughtered pigs (22), poultry (41), sheep and goat (36). All the 200 strains were screened for the presence of stx1, stx2 and eae genes by m-PCR and screening of confirmed EPEC isolates with respect to their serogroup, virulence factors (bfpA, astA and ecpA genes) by PCR and antibiotic resistance. Results: Of total 200 E. coli isolates, 38 (19.00%) were detected as EPEC and 28 (14.00%) as STEC. 5 (12.82%), 17 (43.58%) isolates from calves and 12 (33.33%), 11 (30.55%) isolates from sheep were found as EPEC and STEC, respectively. In rabbits, pigs and dogs, 6 (25.0%), 10 (45.45%) and 5 (13.15%) isolates were detected as EPEC, respectively. The most predominant EPEC serogroups were O88 (36.84%), O118 (18.42%). All 38 EPEC (100%) isolates carried ecpA gene and 18 (47.36%) isolates carried astA gene. Of 38 EPEC, only 5 (13.15%) isolates from dog carried bfpA gene, therefore considered as typical EPEC and 33 (86.84%) isolates were designated as atypical EPEC. Antimicrobial sensitivity test (AST) of 38 EPEC isolates showed resistance to nalidixic acid (55.26%), kanamycin (42.10%) followed by streptomycin (42.10%), doxycycline hydrochloride (28.94%), ciprofloxacin (13.15%), tetracycline (13.15%). Surprisingly, all the 38 isolates were sensitive to ampicillin.

Raccoons (Procyon lotor) in the Madrid region of Spain are carriers of antimicrobial-resistant Escherichia coli and enteropathogenic E.coli.

The role of wildlife in the epidemiology of antimicrobial resistance is unclear. Raccoons in North America can carry a variety of enteric bacteria, with associated antimicrobial resistance, that could infect humans and livestock. The potential for raccoons to carry these bacteria in Europe, where they are an invasive species, has not been explored. Our objectives were to determine the prevalence of Escherichiacoli with associated antimicrobial resistance in raccoons from the Madrid region of Spain and to determine whether they are carriers of potential human pathogens, including verotoxin-producing E.coli (VTEC) and enteropathogenic E.coli (EPEC). In total, we tested 237 E.coli isolates from the faeces of 83 euthanized raccoons for susceptibility to 14 antimicrobial agents and the presence of VTEC and EPEC. Antimicrobial resistance to at least one antimicrobial was detected in the faeces of 51% (42/83; 95% CI, 40.1-61.1) of the raccoons tested. A high percentage of raccoons carried, in their faeces, E.coli isolates resistant to ampicillin (33%), streptomycin (33%), tetracycline (30%), sulphafurazole (31%) and trimethoprim-sulphamethoxazole (23%). We detected one isolate of extended-spectrum β-lactamase-producing E.coli from the faeces of one raccoon. We detected VTEC in the faeces of one raccoon, and EPEC in the faeces of 12% (10/83) of the raccoons. Of the raccoons that carried EPEC in their faeces, 60% (6/10) carried EPEC isolates that exhibited characteristics associated with pathogenicity in humans. Raccoons in Madrid can carry pathogenic and antimicrobial-resistant E.coli in their faeces and may be a risk to public health because of their potential to contaminate food and the environment with their faeces.

Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves.

Escherichia coli field isolates from calves were characterized and categorized into the most significant diarrheagenic pathotypes using polymerase chain reaction (PCR) assays with different specific primers. The used PCR systems were designed to detect sequences representing the group-specific virulence genes encoding fimbriae f5 (K99), Shiga toxins (stx1 and stx2), heat-stable enterotoxins (st), heat-labile enterotoxins (lt), intimin (eae), hemolysin (hylA), and EAEC heat-stable enterotoxin (astA). In the present work, a total of 150 E. coli field isolates were recovered from 150 fecal swabs collected from 100 diarrheic and 50 apparently healthy in-contact cattle and buffalo calves under 3 months old. Out of these 150 isolated E. coli, 106 isolates from 77 diarrheic and 29 in-contact calves harbored one or more of the investigated virulence genes. The pathotyping of the isolates could classify them into shigatoxigenic E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC) with a 30.7, 2.7, 12.7, and 7.3% distribution, respectively. Meanwhile, the detection rates of f5, stx1, stx2, st, lt, eae, hylA, and astA genes were 17.3, 27.3, 6.7, 10, 37.3, 17.7, 9.3, and 20.7%, respectively. These virulence genes were found either single or in different combinations, such as stx/eae, stx/st/f5, eae/st/f5, or st/lt/f5. Four attaching-effacing shigatoxigenic E. coli isolates (AE-STEC) harboring stx/eae were retrieved from diarrheic calves. Although none of the stx-or eae-positive isolates was verified as O157:H7, STEC isolates detected in apparently healthy calves have potential pathogenicity to humans highlighting their zoonotic importance as reservoirs. Atypical combinations of ETEC/STEC and ETEC/EPEC were also detected in percentages of 14.7 and 2.7%, respectively. Most of these atypical combinations were found more in buffalo calves than in cattle calves. While STEC and EPEC isolates were detected more in cattle calves than in buffalo calves, ETEC isolates were the same in the two species. The pathogenic E. coli infection in calves was recorded to be higher in the first weeks of life with the largest numbers of virulence factor-positive isolates detected at the age of 4 weeks. Histopathological examination of five intestinal samples collected from four dead buffalo calves revealed typical attaching and effacing (AE) lesion which was correlated with the presence of intimin encoding virulence gene (eae). Other lesions characterized by hemorrhagic enteritis, shortening and fusion of intestinal villi and desquamation of the lining epithelium of intestinal mucosa had also been detected.

Typical and atypical enteropathogenic Escherichia coli in children with acute diarrhoea: Changing trend in East Delhi

BackgroundWorldwide around 2 million deaths occur every year due to diarrhoeal illnesses among children less than 5 years of age. Among diarrhoeagenic Escherichia coli, Enteropathogenic E. coli (EPEC) is highly prevalent in both community and hospital settings and is one of the main causes of persistent diarrhea in children in developing countries. EPEC remains underdiagnosed in India due to lack of conventional tools for identification. MethodsWe in this study investigated the prevalence and regional variation of EPEC in paediatric population suffering from diarrhoea in East Delhi, India. Two hundred stool samples were collected from children, aged between 0.5 and 5 years, with acute diarrhoea. E. coli were identified by conventional tests and PCR. ResultsWe observed 7% atypical EPEC (aEPEC) and 2.5% typical EPEC (tEPEC), with an overall 9.5% EPEC prevalence amongst total samples. E. coli phylogenetic group A was the predominant. The most common age group affected was 6–23 months with common symptoms being vomiting, watery diarrhoea and severe dehydration. High drug resistance pattern was observed in EPEC isolates. ConclusionThe study depicts a changing trend of aEPEC over tEPEC in children less than 5 years with diarrhoea, an emerging drug resistant enteropathogen and a public health concern demanding monitoring and surveillance.

First report of enteropathogenic and enteroinvasive <i>Escherichia coli</i> with multiple antibiotic resistance indices from African catfish (<i>Clarias glariepinus</i>) in Nigeria

Background: There are increasing reports of food safety issues associated with intensive production of fish which increase the chances of disease outbreaks from stressful growth conditions accompanying mass production and presence of bacterial pathogens. Methodology: Two hundred gastrointestinal tract (GIT) samples from two hundred African Cat Fish ( Clarias glariepinus ) were assessed for the presence of enteric Escherichia coli species including E. coli 0157, Enteropathogenic E. coli (EPEC) and Enteroinvasive E. coli (EIEC) which are traditionally associated with infantile gastroenteritis. The antibiotic resistance profile and Multiple Antibiotic Resistance Index (MARI) for these isolates were determined. The serogrouping of the E. coli isolates was done using E. coli agglutinating sera (Oxoid) and E. coli 0157 latex reagent (Oxoid). Antibiotic susceptibility was determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: A total of 35 (17.5%) E. coli isolates were recovered from the fish intestines among which 9 (25.7%) were EPEC and 2 (5.7%) were EIEC. No E. coli 0157 strain was recovered. Thirty-three (94.0%) isolates had a MARI greater than 0.2. Antibiotic resistance to cefoxitin and amoxicillin-clavulanic acid were 77.1% and 74.3% respectively. All isolates were susceptible to meropenem and amikacin but all EPEC and EIEC isolates were AmpC (resistance to all penicillins, cephalosporins and beta lactamase inhibitors) positive. Conclusion: The isolation of EPEC and EIEC which can cause fatal gastroenteritis coupled with high MARI among isolates in this study represents a public health concern. Strict monitoring of administration of antibiotics in aquaculture is recommended. Keywords: EPEC; EIEC; Multiple antibiotic resistance; Aquaculture

Occurrence and characterization of stx and/or eae-positive Escherichia coli isolated from wildlife, including a typical EPEC strain from a wild boar

Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains are food-borne pathogens associated with acute diarrhea. Haemolytic-uremic syndrome (HUS) is often a complication of STEC infection. In order to examine the occurrence, serotypes, virulence and antimicrobial-resistance profiles of STEC and EPEC in wildlife, 326 faecal E. coli strains from 304 clinically healthy animals were analyzed. For this approach stx1, stx2 and eae genes, as well as accessory virulence determinants (ehx, hlyA, saa, tia, bfp, subAB) were PCR-screened and sequenced. Serotyping was performed employing all available O (O1–O185) and H (H1-H56) antisera. Genetic diversity was analyzed by XbaI-PFGE and phylotyping. Thirteen STEC (4.3%) and 10 EPEC (3.3%) were identified among 12 deer, 3 mouflon, 6 wild boars and 2 birds. Nine STEC showed seropathotypes B (O145:[H28]) and C (O22:H8, O128:[H2]) associated with HUS, and D (O110:H28, O146:H21, O146:[H28], ONT:H8) associated with human diarrhea. Although most isolates harbored stx2b and stx1c variants, stx2a and stx1a (related with severe disease) were also detected. Additionally, the eae gene was present in one stx2a–positive O145:[H28] STEC from a deer and 11 STEC harbored subAB genes (mainly the subAB2 variant). EPEC isolates showed 7 different intimin variants (β1, β2, γ1, ε1, ζ1, ι1-A, κ). Interestingly, the O49:[H10] eae-κ EPEC isolated from a wild boar was bfpA-positive showing a combination of serotype/virulence profile previously detected among human clinical tEPEC. Based on present results, wild ruminants, wild boars and to a lesser extent birds would be carriers of potentially pathogenic STEC and EPEC strains.

Distribution of Integrons and Phylogenetic Groups among Enteropathogenic Escherichia coli Isolates from Children

Integrons by means of horizontal gene transfer carry multidrug resistance genes (MDR) among bacteria, including E. coli. The aim of this study was to determine the antibiotic resistance profiles and the genes associated with them, to gain insights in the distribution of phylogroups, prevalence, and characterization of class 1, 2 and 3 integrons among Enteropathogenic E. coli (EPEC) isolates, from children upto 5 years of age from Delhi and National Capital Region (NCR), India. A total of 120 E. coli isolates, including 80 from diarrheagenic E. coli (cases) and 40 from healthy isolates (controls) were recruited in this study. After isolation of E. coli, screening for EPEC was done by conventional multiplex PCR. Antibiotic suseptibility test was performed using disk diffusion method and further confirmed by minimum inhibitory concentration (MICs) by E-test. The presence and characterization of integrons and antimicrobial resistance genes were performed by PCR and DNA sequencing. Phylogeny determination was carried out by quadruplex PCR. EPEC strains were found in 64 of the 80 diarrheagenic cases, out of which 38 were MDR. In the 40 healthy controls, 23 were found to be EPEC strain, out of which only 2 were MDR. Amongst 80 diarrheagenic cases, class 1 integron were observed in 43 isolates, class 2 integron in 12 isolates and 9 isolates were found with co-existence of both. Similarly, in healthy controls; class 1 integron in 9 and class 2 integron in 7 isolates were observed with co-existence in 3 isolates. None of the isolates included class 3 integron. The dfr was the most commonly identified gene cassette within the integron-positive isolates. Phylogenetic studies showed considerable representation of phylogroup B2 in both diarrheagenic cases and healthy controls. This study reiterates the importance of class 1 integron predominantly for acquisition of antibiotic resistance genes among EPEC isolates. Furthermore, it also ascertains the possible association between multidrug resistance and presence of integrons. Approximately 91% of isolates were easily assigned to their respective phylogroups. Assessment of the relationship between antibiotic resistance and dominant phylogroups detected was also attempted. This study also highlights the increased burden of antimicrobial resistance in healthy controls.

Characterization of Enteropathogenic Escherichia coli Associated with Diarrhea Among Iranian Infants

Background: Diarrhea is a cause of concern due to high morbidity and mortality in children. Enteropathogenic E. coli (EPEC) strains contribute to outbreaks of infantile diarrhea, especially in developing countries. Objectives: This study was performed to investigate the contribution of EPEC as a cause of infectious diarrhea among Iranian infants. Methods: A total of 140 E. coli isolates from children with diarrhea and 110 from children without diarrhea were evaluated for the presence of EPEC. The E. coli isolates were examined for uidA, eae, and stx genes by polymerase chain reaction (PCR). The eae positive isolates were tested for the bfpA gene to differentiate typical and atypical EPEC. The EPEC isolates were typed by random amplified polymorphic DNA (RAPD-PCR). Results: The E. coli isolates were confirmed by the presence of the uidA gene. The EPEC was diagnosed in 6 cases (4.2%) with diarrhea, which were positive for the eae gene, compared with negative results in the asymptomatic group. The bfpA were positive in 5 isolates (3.6%) that were categorized as tEPEC and 1 (0.09%) as aEPEC. All isolates showed genetically different patterns in RAPD-PCR. Conclusions: This study suggests that EPEC strains are important contributors to diarrhea in Iranian children. Because of the weakness of routine microbiological tests and poor specifity of serological tests, it is recommended that the EPEC strains are better detected by molecular methods.

Diarrheagenic Escherichia coli pathotypes investigation revealed atypical enteropathogenic E. coli as putative emerging diarrheal agents in children living in Botucatu, São Paulo State, Brazil.

The aim of the present study was to investigate the prevalence of Diarrheagenic Escherichia coli (DEC) pathotypes, a leading cause of diarrhea worldwide, among diarrheal and healthy children, up to 5 years of age, living in the city of Botucatu, São Paulo, Brazil. DEC, investigated by PCR detection of virulence factor-encoding genes associated with the distinct pathotypes, was isolated from 18.0% of the patients, and 19.0% of the controls, with enteroaggregative E. coli (EAEC), the most frequent pathotype, being detected in equal proportion between patients and controls (10.0%). Among the enteropathogenic E. coli (EPEC) isolates, only one isolate was able to produce the localized adherence pattern to HeLa cells, being thus the only typical EPEC identified. All the remaining EPEC were classified as atypical (aEPEC), and detected in 8.0% and 8.5% of the patients and controls, respectively. Regarding the serotypes, 26.5% of the analyzed EPEC isolates belonged to classical EPEC-serogroups, and the only two STEC found were serotyped as O26:H11 (patient) and O119:H7 (control). Antimicrobial susceptibility tests revealed that 43.6%, 29.5% and 2.6% of the DEC isolates were resistant to ampicillin, cotrimoxazole and gentamicin, respectively. Our data indicate that EAEC remains prevalent among children living in Botucatu, and revealed atypical EPEC as emerging putative diarrheal agents in this geographical region.

Isolation and Molecular Characterization of Shigatoxigenic and EnteropathogenicEscherichia colifrom diverse sources

Total 45 E. coli isolates were recovered from faecal samples of 77 diarrhoeic and 85 healthy animals and birds, 51 milk samples and 48 diarrhoeic human stool samples. Multiplex PCR based molecular characterization targeting the virulence genes (stx1, stx2, eae and bfpA) could reveal presence of 24 Shiga toxin-producing E. coli (STEC) and 21 as enteropathogenic E. coli (EPEC). Among the STEC 19 isolates belonged to 13 different serogroups while four were untypable and one rough. Majority of STEC isolates carried stx2 gene. Out of 21 EPEC isolates, 15 were serogrouped into 9 different serogroups and 6 were either untypable or rough. All the four EPEC isolates of milk origin belonged to serogroup O2. Only two isolates from dierrhoeic buffaloes were found to be typical.

Occurrence of SHV, TEM and CTX-M β-Lactamase Genes Among Enteropathogenic Escherichia coli Strains Isolated From Children With Diarrhea

Background:Antibiotic resistance is widespread among diarrheagenic Escherichia coli in developing countries, where the overuse of antibiotics is common. Information regarding β-lactamases, especially Extended-Spectrum β-Lactamases (ESBLs) in diarrheagenic pathogens should be considered in clinical management when an optimal treatment is needed.Objectives:The main objective of this study was to investigate the prevalence of blaCTX-M, blaSHV and blaTEM β-lactamase genes among enteropathogenic E. coli (EPEC) isolates in Tehran, Iran.Materials and Methods:Stool specimens were collected from children with diarrhea during a 17-month period from 2011 to 2013. Routine biochemical tests were performed for identification of E. coli isolates. The isolates were further examined by PCR for the presence of eae, stx1, stx2 and bfp genes. EPEC isolates have been screened for different β-lactamase genes. Genotyping EPEC isolates harboring blaCTX-M15 gene was performed through Multi-Locus VNTR Analysis (MLVA).Results:Of 42 EPEC, eight isolates carried the blaCTX-M1. None of the isolates carried blaCTX-M2 and blaCTX-M9. The blaCTX-M15 variant was identified in all of blaCTX-M1-positive isolates. Furthermore, blaSHV and blaTEM genes were detected in 40.5% (n = 17) and 19% (n = 8) of all EPEC isolates, respectively. No significant association was observed between the existence of bfp gene and presence of those β-lactamase genes (P > 0.05). MLVA analysis revealed high genetic diversity among blaCTX-M15-positive isolates.Conclusions:Our study emphasized the increasing role of ESBL genes, especially blaCTX-M15 in EPEC isolates.

Potentially pathogenic Escherichia coli in healthy, pasture-raised sheep on farms and at the abattoir in Brazil

Sheep harbor pathogenic Escherichia coli, which may cause severe disease in humans. In this study, the prevalence of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) was examined in sheep feces and carcasses on three farms and at an abattoir in Brazil. The isolates were further characterized for the presence of markers recently associated with disease in humans, to investigate their possible origin and role as food-borne pathogens. At the abattoir, 99 carcass samples yielded two STEC and 10 EPEC isolates while 101 fecal samples yielded five EPEC and eight STEC isolates. On the other hand, on the farms, 202 samples yielded 44 STEC and eight EPEC isolates. The 77 isolates were typed by PFGE. Isolates with the same PFGE pattern and also those that were not restricted with XbaI were termed as “clones” (n=49). The isolates of any one clone mostly originated from the same sampling site. In addition, seven isolates encoded for novel Stx2 variants and five for Stx2e, the subtype related to porcine edema disease, which was for the first time isolated from sheep feces and carcasses. Also, three stx2-only isolates harbored genes of predicted Stx2 variants that were formed by A and B subunits of different types including Stx2a and Stx2d. The EPEC isolates were heterogeneous, 21 (91.3%) of them possessing efa1, ehxA, lpfAO113 or paa genes associated with diarrhea in humans. Thus, using markers recently associated with disease, we have demonstrated that E. coli similar to those pathogenic for humans are present in the sheep intestinal microflora, particularly at the abattoir, underlining the potential for food-borne transmission.

Shiga Toxigenic and Atypical EnteropathogenicEscherichia coliin the Feces and Carcasses of Slaughtered Pigs

Escherichia coli is a pathogen of major importance in swine and public health. To determine the prevalence of Shiga toxigenic E. coli (STEC) and enteropathogenic E. coli (EPEC), samples were collected from the feces and carcasses of swines. In total, 441 samples were collected in four samplings, of which 141 samples tested positive for either the stx1, stx2, and/or eae genes. From the positive samples, one STEC and 15 atypical EPEC (aEPEC) isolates were obtained, and all originated from the same sampling. In addition to eae, lpfA(O157/OI-141), ehxA, toxB, and lpfA(O113) were present in the aEPEC isolates. The only stx2-containing isolate carried stx2e and belonged to serotype O103:HNT. Resistance to four or more antimicrobials was found in almost half of the isolates, and some isolates shared the same fingerprint patterns by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The presence of certain virulence genes and the high level of resistance to antimicrobials, as well as the possible fecal contamination of carcasses showed that some of the isolates are of public health concern.

Molecular characterization of diarrheagenic Escherichia coli isolated from meat products sold at Mansoura city, Egypt

This study was carried out to determine the serological characteristics and virulence-associated genes of 32 Escherichia coli strains isolated from different meat products. Serotyping of somatic (O) and flagellar (H) antigens reveled that 3 strains were typed into 2 serogroups; O121:H19 (2 strains) and O148:H8 (1 strain), while the other 29 strains were not agglutinated with any serum. For molecular characterization, multiplex PCR has been performed by combining seven primer pairs specific for enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli (EIEC). The targets selected for each group were the genes encoding heat-labile (LT) and heat-stable (ST) toxins for ETEC isolates, eae, stx1 and stx2 for EHEC isolates, eae and bfpA for EPEC isolates, and ipaH for EIEC isolates. This facilitates simultaneous identification of the four different categories of diarrheagenic E. coli in a single reaction. Amongst the 32 E. coli strains tested, eleven (37.5%) were potentially diarrheagenic. Five of which (15.63%) were ETEC, three (9.38%) were EHEC, two (6.26%) were EPEC, and one strain (3.13%) was EIEC. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations that may be present among high numbers of E. coli contaminating meat products which do not necessarily correlate with disease.

Distribution of espM and espT among enteropathogenic and enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) translocate dozens of type III secretion system effectors, including the WxxxE effectors Map, EspM and EspT that activate Rho GTPases. While map, which is carried on the LEE pathogenicity island, is absolutely conserved among EPEC and EHEC strains, the prevalence of espM and espT is not known. Here we report the results of a large screen aimed at determining the prevalence of espM and espT among clinical EPEC and EHEC isolates. The results suggest that espM, detected in 51 % of the tested strains, is more commonly found in EPEC and EHEC serogroups that are linked to severe human infections. In contrast, espT was absent from all the EHEC isolates and was found in only 1.8 % of the tested EPEC strains. Further characterization of the virulence gene repertoire of the espT-positive strains led to the identification of a new ζ2 intimin variant. All the espT-positive strains but two contained the tccP gene. espT was first found in Citrobacter rodentium and later in silico in EPEC E110019, which is of particular interest as this strain was responsible for a particularly severe diarrhoeal outbreak in Finland in 1987 that affected 650 individuals in a school complex and an additional 137 associated household members. Comparing the protein sequences of EspT to that of E110019 showed a high level of conservation, with only three strains encoding EspT that differed in 6 amino acids. At present, it is not clear why espT is so rare, and what impact EspM and EspT have on EPEC and EHEC infection.

Genetic Characterization of Antibiotic Resistance in EnteropathogenicEscherichia coliCarrying Extended-Spectrum β-Lactamases Recovered from Diarrhoeic Rabbits

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended-spectrum beta-lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K-:H2) was observed among 17 EPEC strains, the O92:K-serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K-serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim-sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The bla(TEM) gene was demonstrated in the majority of ampicillin-resistant isolates. The aac(3)-II or aac(3)-IV genes were detected in the four gentamicin-resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin-resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline-resistant isolates. A total of nine EPEC isolates showed the phenotype SXT-resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.