Enteropathogenic Escherichia Coli Isolates Research Articles

Phenotypic diversity of type III secretion system activity in enteropathogenic Escherichia coli clinical isolates.

Introduction. Enteropathogenic Escherichia coli (EPEC) strains pose a significant threat as a leading cause of severe childhood diarrhoea in developing nations. EPEC pathogenicity relies on the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE), facilitating the secretion and translocation of bacterial effector proteins.Gap Statement. While the regulatory roles of PerC (plasmid-encoded regulator) and GrlA (global regulator of LEE-activator) in ler expression and LEE gene activation are well-documented in the EPEC prototype strain E2348/69, understanding the variability in LEE gene expression control mechanisms among clinical EPEC isolates remains an area requiring further investigation.Aim. This study aims to explore the diversity in LEE gene expression control mechanisms among clinical EPEC isolates through a comparative analysis of secretion profiles under defined growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression.Methodology. We compared T3SS-dependent secretion patterns and promoter expression in both typical EPEC (tEPEC) and atypical EPEC (aEPEC) clinical isolates under growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Additionally, we conducted promoter reporter activity assays, quantitative real-time PCR and Western blot experiments to assess gene expression activity.Results. Significant differences in T3SS-dependent secretion were observed among tEPEC and aEPEC strains, independent of LEE sequence variations or T3SS gene functionality. Notably, a clinical tEPEC isolate exhibited increased secretion levels under repressive growth conditions and in the absence of both PerC and GrlA, implicating an alternative mechanism in the activation of Ler (LEE-encoded regulator) expression.Conclusion. Our findings indicate that uncharacterized LEE regulatory mechanisms contribute to phenotypic diversity among clinical EPEC isolates, though their impact on clinical outcomes remains unknown. This challenges the conventional understanding based on reference strains and highlights the need to investigate beyond established models to comprehensively elucidate EPEC pathogenesis.

Multidrug-Resistant Enteropathogenic Escherichia coli Isolated from Diarrhoeic Calves, Milk, and Workers in Dairy Farms: A Potential Public Health Risk.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhoeagenic diseases in humans and cattle worldwide. The emergence of multidrug-resistant (MDR) EPEC from cattle sources is a public health concern. A total of 240 samples (75 diarrhoeic calves, 150 milk samples, and 15 workers) were examined for prevalence of EPEC in three dairy farms in Egypt. Antimicrobial resistance (AMR) traits were determined by antibiogram and polymerase chain reaction (PCR) detection of β-lactamase-encoding genes, plasmid-mediated quinolone resistance genes, and carbapenemase-encoding genes. The genetic relatedness of the isolates was assessed using repetitive extragenic palindromic sequence-based PCR (REP-PCR). EPEC isolates were detected in 22.7% (17/75) of diarrhoeic calves, 5.3% (8/150) of milk samples, and 20% (3/15) of worker samples. The detected serovars were O26 (5%), O111 (3.3%), O124 (1.6%), O126 (0.8%), and O55 (0.8%). AMR-EPEC (harbouring any AMR gene) was detected in 9.2% of samples. Among isolates, blaTEM was the most detected gene (39.3%), followed by blaSHV (32.1%) and blaCTX-M-1 (25%). The qnrA, qnrB, and qnrS genes were detected in 21.4%, 10.7%, and 7.1% of isolates, respectively. The blaVIM gene was detected in 14.3% of isolates. All EPEC (100%) isolates were MDR. High resistance rates were reported for ampicillin (100%), tetracycline (89.3%), cefazolin (71%), and ciprofloxacin (64.3%). Three O26 isolates and two O111 isolates showed the highest multiple-antibiotic resistance (MAR) indices (0.85–0.92); these isolates harboured blaSHV-12 and blaCTX-M-15 genes, respectively. REP-PCR genotyping showed high genetic diversity of EPEC, although isolates belonging to the same serotype or farm were clustered together. Two worker isolates (O111 and O26) showed high genetic similarity (80–95%) with diarrhoeic calf isolates of matched serotypes/farms. This may highlight potential inter-species transmission within the farm. This study highlights the potential high risk of cattle (especially diarrhoeic calves) as disseminators of MDR-EPEC and/or their AMR genes in the study area. Prohibition of non-prescribed use of antibiotics in dairy farms in Egypt is strongly warranted.

Prevalence, Antimicrobial Resistance, and Whole Genome Sequencing Analysis of Shiga Toxin-Producing Escherichia coli (STEC) and Enteropathogenic Escherichia coli (EPEC) from Imported Foods in China during 2015-2021.

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are foodborne pathogens that cause hemolytic uremic syndrome and fatal infant diarrhea, respectively, but the characterization of these bacteria from imported food in China are unknown. A total of 1577 food samples from various countries during 2015–2021 were screened for STEC and EPEC, and the obtained isolates were tested for antimicrobial resistance and whole genome sequencing analysis was performed. The prevalence of STEC and EPEC was 1.01% (16/1577) and 0.51% (8/1577), respectively. Antimicrobial resistances to tetracycline (8%), chloramphenicol (8%), ampicillin (4%), ceftazidime (4%), cefotaxime (4%), and trimethoprim-sulfamethoxazole (4%) were observed. The antimicrobial resistance phenotypes corresponded with genotypes for most strains, and some resistance genes were related to mobile genetic elements. All 16 STEC isolates were eae negative, two solely contained stx1 (stx1a or stx1c), 12 merely carried stx2 (stx2a, stx2d, or stx2e), and two had both stx1 and stx2 (stx1c + stx2b, stx1a + stx2a + stx2c). Although they were eae negative, several STEC isolates carried other adherence factors, such as iha (5/16), sab (1/16), and lpfA (8/16), and belonged to serotypes (O130:H11, O8:H19, and O100:H30) or STs (ST297, ST360), which have caused human infections. All the eight EPEC isolates were atypical EPEC; six serotypes and seven STs were found, and clinically relevant EPEC serotypes O26:H11, O103:H2, and O145:H28 were identified. Two STEC/ETEC (enterotoxigenic E. coli) hybrids and one EPEC/ETEC hybrid were observed, since they harbored sta1 and/or stb. The results revealed that food can act as a reservoir of STEC/EPEC with pathogenic potential, and had the potential ability to transfer antibiotic resistance and virulence genes.

Two Enteropathogenic Escherichia coli Strains Representing Novel Serotypes and Investigation of Their Roles in Adhesion.

Enteropathogenic Escherichia coli (EPEC), which belongs to the attaching and effacing diarrheagenic E. coli strains, is a major causative agent of life-threatening diarrhea in infants in developing countries. Most EPEC isolates correspond to certain O serotypes; however, many strains are nontypeable. Two EPEC strains, EPEC001 and EPEC080, which could not be serotyped during routine detection, were isolated. In this study, we conducted an in-depth characterization of their putative O-antigen gene clusters (O-AGCs) and also performed constructed mutagenesis of the O-AGCs for functional analysis of O-antigen (OAg) synthesis. Sequence analysis revealed that the occurrence of O-AGCs in EPEC001 and E. coli O132 may be mediated by recombination between them, and EPEC080 and E. coli O2/O50 might acquire each O-AGC from uncommon ancestors. We also indicated that OAgknockout bacteria were highly adhesive in vitro, except for the EPEC001 wzy derivative, whose adherent capability was less than that of its wild-type strain, providing direct evidence that OAg plays a key role in EPEC pathogenesis. Together, we identified two EPEC O serotypes in silico and experimentally, and we also studied the adherent capabilities of their OAgs, which highlighted the fundamental and pathogenic role of OAg in EPEC.

The first evaluation relationship of integron genes and the multidrug-resistance in class A ESBLs genes in enteropathogenic Escherichia coli strains isolated from children with diarrhea in Southwestern Iran.

Enteropathogenic Escherichia coli (EPEC) is one of the most important diarrheagenic agents among infants under 5years in developing countries. This study aimed to investigate the relationship of integron genes and class A extended-spectrum ESBLs genes in MDR E. coli strains isolated from children with diarrhea in Southwestern Iran. Totally, 321 fecal samples were collected from diarrheal children under 5years admitted to teaching hospitals of Abadan and Khorramshahr, southwest Iran. Routine bacteriological tests were performed for the identification of E. coli isolates. Multiplex PCR was used for the presence of eae, bfp, stx1, and stx2 genes to detected EPEC strains. Serogrouping was performed for EPEC strains. The EPEC isolates' antibiotic resistance pattern was determined by the disk-diffusion technique. All EPEC isolates were screened for integron and class A β-lactamase genes. Of the 14 EPEC isolates, 12 (85.7%) were found to be ESBL-positive by double disk synergy test (DDST) and PCR. In addition, blaCTX-M and blaTEM genes were detected in 83.3% (n = 10) and 58.3% (n = 7) of EPEC isolates, respectively. None of the isolates had the blaKPC gene. On the other hand, 64.2% (n = 9) and 7.1% (n = 1) were positive only for intlI and intlII genes, respectively. The results demonstrated that EPEC is one of the major causes of childhood diarrhea in our region and that the distribution of class 1 integrons and ESBLs in EPEC strains is highly prevalent. Moreover, the results revealed that continuous monitoring of the emergence and expansion of MDR in EPEC strains is necessary.

Quantitative analysis and virulence phenotypes of atypical enteropathogenic Escherichia coli (EPEC) acquired from diarrheal stool samples from a Midwest US hospital

ABSTRACT Infectious diarrhea causes approximately 179 million illnesses annually in the US. Multiplex PCR assays for enteric pathogens detect enteropathogenic Escherichia coli (EPEC) in 12–29% of diarrheal stool samples from all age groups in developed nations. The aim of this study was to isolate and characterize EPEC from diarrhea samples identified as EPEC positive by BioFire Gastrointestinal Panel (GIP). EPEC is the second most common GIP-detected pathogen, equally present in sole and mixed infections peaking during summer months. EPEC bacterial load is higher in samples with additional pathogens. EPEC-GIP-positive stool samples were cultured on MacConkey II agar and analyzed by colony PCR for eaeA and bfpA to identify and classify EPEC isolates as typical (tEPEC) or atypical (aEPEC). EPEC were not recovered from the majority of stool samples with only 61 isolates obtained from 277 samples; most were aEPEC from adults. bfpA-mRNA was severely diminished in 3 of 4 bfpA-positive isolates. HeLa and SKCO-15 epithelial cells were infected with EPEC isolates and virulence-associated phenotypes, including adherence pattern, attachment level, pedestal formation, and tight junction disruption, were assessed. All aEPEC adherence patterns were represented with diffuse adherence predominating. Attachment rates of isolates adhering with defined adherence patterns were higher than tEPEC lacking bfpA (ΔbfpA). The majority of isolates formpedestals. All but one isolate initially increases but ultimately decreases transepithelial electrical resistance of SKCO-15 monolayers, similar to ΔbfpA. Most isolates severely disrupt occludin; ZO-1 disruption is variable. Most aEPEC isolates induce more robust virulence-phenotypes in vitro than ΔbfpA, but less than tEPEC-E2348/69.

Characterization of Antimicrobial Susceptibility, Extended-Spectrum β-Lactamase Genes and Phylogenetic Groups of Enteropathogenic Escherichia coli Isolated from Patients with Diarrhea.

ObjectivesInfectious diarrhea is one of the most common causes of pediatric death worldwide and enteropathogenic Escherichia coli (EPEC) is one of the main causes. There are 2 subgroups of EPEC, typical and atypical, based on the presence or absence of bundle forming pili (bfp), of which atypical EPEC is considered less virulent, but not less pathogenic. Antimicrobial resistance towards atypical EPEC among children is growing and is considered a major problem. In this study the pattern of antibiotic resistance in clinical isolates was determined.MethodsUsing 130 isolates, antibiotic resistance patterns and phenotypes were assessed, and genotypic profiles of extended spectrum β-lactamase (ESBL) production using disc diffusion and PCR was carried out. Phylogenetic groups were analyzed using quadruplex PCR.ResultsThere were 65 E. coli isolates identified as atypical EPEC by PCR, among which the highest antibiotic resistance was towards ampicillin, followed by trimethoprim-sulfamethoxazole, and tetracycline. Multidrug resistance was detected in 44.6% of atypical EPEC isolates. Around 33% of isolates were determined to be extended spectrum β-lactamase producers, and in 90% of isolates, genes responsible for ESBL production could be detected. Moreover, the majority of atypical EPEC strains belonged to Group E, followed by Groups B1, B2 and C.ConclusionHigh rates of multidrug resistance and ESBL production among atypical EPEC isolates warrant periodical surveillance studies to select effective antibiotic treatment for patients. It is considered a critical step to manage antibiotic resistance by avoiding unnecessary prescriptions for antibiotics.

Molecular Study of Regulatory Gene (Ler) in Enteropathogenic Escherichia Coli (EPEC) of Diarrheagenic Patients

The locus of enterocyte effacement LEE-encoded regulator (Ler( is a global regulator of multiple virulence genes expression in the Enteropathogenic Escherichia coli (EPEC), including those encoding the type III secretion pathway and adhesion proteins such as intimin. Ler is central to the process of the formation of the attaching and effacing (AE) lesions. This study aimed to perform the molecular detection of Ler gene in EPEC, since there is no related previous study in Iraq. Two hundred and fifty stool specimens from children under two years of age for both sexes were collected from some Iraqi hospitals. All isolates were diagnosed according to morphological characteristics and biochemical tests. The results showed that 140 (56%) samples were identified as E.coli, while 8 (5.7%) isolates were identified as EPEC as confirmed by using VITEK 2 system. Susceptibility test was determined for all EPEC isolates to 16 different antibiotics. The results showed that 100%of these isolates were resistant to Ampicillin, Cefazolin, Ceftriaxone, Cefepime, Trimethoprim and Ceftazidime, whereas resistance values to Nitrofurantoin, Cefoxitin and Gentamicin were 66%, 40%, and 15% respectively. However 100% of the isolates were sensitive to Piperacillin, Ertapenem, Imipenem, Amikacin, Ciprofloxacin, Levofloxacin and Tigecycline. Monoplex pattern of PCR was used for detecting 16SrRNA, eae, stx1, lifA and Ler genes in EPEC. The results showed that the isolates of E.coli were positive for 16SrRNA, eae, lifA and Ler, while no bands of stx1 appeared.

Molecular Characterization and Antimicrobial Resistance of Enteropathogenic Escherichia coli in Children from Ahvaz, Iran

Background: Enteropathogenic Escherichia coli (EPEC) is one of the most important pathogens among young children worldwide. Both eae and bfp genes have been used to identify EPEC strains and categorize them into typical and atypical strains. They may be an emerging pathogen in both developing and developed countries. Objectives: This study was primarily conducted to assess the epidemiology, drug resistance, and β-lactamase distribution of EPEC, as well as the detection of efa1/lifA in atypical strains. Methods: A total of 251 E. coli strains isolated from children with diarrhea were evaluated for their EPEC pathotype by PCR for the presence of eae, stx1, stx2, and bfp genes. Serogrouping with polyvalent antisera was performed to confirm EPEC strains. Atypical EPEC-containing samples were evaluated for the efa1/lifA gene. EPEC isolates were assessed to recognize the antibiotic resistance and screened to detect extended-spectrum β-lactamases (ESBLs). Results: Enteropathogenic E. coli strains were detected in 17 (6.78%) of E. coli isolates by PCR. The prevalence of typical and atypical strains was determined at 35.3% and 64.7%. All strains were completely susceptible to colistin, imipenem, and meropenem. The prevalence of blaCTX-M and blaTEM genes was calculated at 70.58% and 58.82%, respectively. Conclusions: Enteropathogenic E. coli isolates are completely sensitive to carbapenems, and precise therapeutic strategies are required to prevent the spread of these beta-lactamase genes among diarrheagenic E. coli.

Rabbit Enteropathies on Commercial Farms in the Iberian Peninsula: Etiological Agents Identified in 2018-2019.

Simple SummaryDigestive disorders are the main cause of economic damage in rabbit farms and, usually, antibiotic treatment is the first choice to control them. Nevertheless, a broad range of infectious agents can be involved in such disorders, as we have observed in our diagnosis work as a veterinary diagnostic laboratory. In this study, a global and updated overview of the frequency of detection of those etiological agents is provided. We have seen differences depending on the age of the affected rabbits, with young rabbits (<15 days old) being the most affected by enteropathogenic Escherichia coli strains, while in preweaning and growing rabbits, a coinfection of two or three pathogens is the most prevalent situation. Clostridium spiroforme and E. coli are the main bacterial agents detected in preweaning rabbits, but enterotoxigenic Bacteroides fragilis has just appeared as a new possible emergent pathogen. Coinfections between bacteria (C. spiroforme and E. coli), parasites (Eimeria spp.), and viruses (rotavirus) are much more frequent than simple infections in growing rabbits; for this reason, complete laboratory studies are required to establish on-farm disease control measures.Digestive disorders are the main cause of economic damage to rabbit farms. This article provides a global and updated overview of the diverse etiological agents causing them, since 757 clinical cases were analyzed during 2018 and 2019—Ninety-five from young rabbits (<15 days old), 117 from preweaning rabbits (15–35 days old), and 545 from growing rabbits. Etiological diagnosis was carried out by bacteriological culture and a set of real time polymerase chain reaction (qPCR) tests for the detection of enteropathogenic Escherichia coli (EPEC), Clostridium spiroforme, C. perfringens, rotavirus A, Bacteroides fragilis, and Eimeria spp. Also, 40 EPEC and 38 non EPEC isolates were investigated for the presence of other colonization factors (afr2, ral, liftA, and paa) by qPCR. EPEC is the most prevalent agent in young rabbits, and although different virulence profiles have been found among EPEC isolates, the liftA+, ral+, and paa+ profile is the most prevalent. C. spiroforme and EPEC are the more frequently detected pathogens in preweaning rabbits, but B. fragilis appears to be a new possible emergent pathogen. In growing rabbits, diverse co-infections between C. spiroforme, Eimeria spp., EPEC, and rotavirus are much more frequent than infections due to only one of them. Other pathogens detected in very few cases are Salmonella spp. and Enterococcus hirae.

The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.

Antibiotic resistance and biofilm formation in children with Enteropathogenic Escherichia coli (EPEC) in Brazilian Amazon

Enteropathogenic Escherichia coli is an important causative agent of diarrhea in both developed and developing countries. We assessed the antibiotic resistance profile and the ability of 71 Enteropathogenic Escherichia coli (EPEC) isolates from children in the age group 6 years, or younger, to form biofilm. These children were hospitalized in Cosme and Damião Children Hospital in Porto Velho, Western Brazilian Amazon, between 2010 and 2012, with clinical symptoms of acute gastroenteritis. The highest frequency of atypical EPEC (aEPEC) isolates reached 83.1% (59/71). Most EPEC isolates presented Localized Adherence Like (LAL) pattern in HEp-2 cells (57.7% - 41/71). Biofilm production was observed in 33.8% (24/71) of EPEC isolates, and it means statistically significant association with shf gene (p = 0.0254). The highest antimicrobial resistance rates and a large number of multiresistant isolates 67.6% (48/71), regarded cefuroxime (CXM), ampicillin (AMP), trimethoprim-sulfamethoxazole (SXT) and tetracycline (TET), respectively, mainly in typical EPEC (tEPEC). Furthermore, 96% (68/71) of EPEC isolates in the present study were resistant to at least one antibiotic, whereas only 3 isolates were sensitive to all the tested drugs. Based on our findings, there was increased aEPEC identification. EPEC isolates showed high resistance rate; most strains showed multiresistance; thus, they work as warning about the continuous need of surveillance towards antimicrobial use. Besides, the ability of forming biofilm was evidenced by the EPEC isolates. This outcome is worrisome, since it is a natural resistance mechanism of bacteria.

Multi Locus VNTR (MLVA) Typing and Detection of the OI-122 Pathogenicity Island in Typical and Atypical Enteropathogenic Escherichia coli Isolated from Children with Acute Diarrhea

Background: Pathogenicity O island 122 (OI-122) associated with the severity of Enteropathogenic Escherichia coli (EPEC) disease. Objectives: The aim of this study was the characterization of OI-122 in Iranian EPEC isolates. Methods: The distribution patterns of OI-122 were investigated in 42 EPEC strains by detection of OI-122 genes and serogrouping, multi locus VNTR (MLVA) typing, and phylogenetic grouping. Results: The complete OI-122 was identified in 18.75% of tEPEC and in 3.8% of aEPEC strains, an incomplete OI-122 with various combinations of genes was found in 75% of tEPEC and 30.76% of strains aEPEC, and OI-122 genes were absent in 6.25% of tEPEC and 65.3% of aEPEC strains. nleB (52.4%) was the most frequently detected, and the prevalence of pagC, efa1/lifA, and sen genes were 19%, 16.7%, and 14.3%, respectively. The most common phylogenetic group among tEPEC and aEPEC strains were B1 (68.75% and 80.76%, respectively). The common serogroups among EPEC isolates were O128, O111, O55, O127, and O44, and 45.2% of the isolates were untypeable. Forty-one MLVA types among 42 EPEC strains were classified into 7 clonal complexes. Conclusions: The diverse distribution of OI-122 genes among MLVA clonal groups provides evidence for dynamic evolution regarding the OI-122 pathogenicity island in tEPEC and aEPEC strains. The results also indicate that the acquisition of OI-122 gene contents occurs in a modular manner.

Escherichia coli detection and identification in shellfish from southeastern Brazil

The pathogenic Escherichia coli strain may be responsible for foodborne infections in humans. This study was carried out to evaluate the presence of Escherichia coli and verify the occurrence of virulence genes in mussels (Mytella falcata) and oysters (Crassostrea brasiliana) collected from natural banks of the Estuary Lagunar Complex of Cananéia in the State of São Paulo, southeastern Brazil. A total of 150 shellfish samples (75 mussels and 75 oysters) were collected from June 2016 to February 2017. Total coliforms and Escherichia coli were determined using the most probable number (MPN) procedure. Colonies of Escherichia coli were biochemically characterized. The positive samples were submitted to molecular identification of species and posterior genetic sequencing for identifying Escherichia coli pathotypes. Escherichia coli was detected in 48 samples (32%) in concentrations ranging from <3 to <927 MPN/g. From 240 colonies tested biochemically, ninety-three were positive and submitted to molecular analyses. Fourty-eight colonies were confirmed as Escherichia coli by presenting PhoA gene and six (12.5%) were identified as enteropathogenic Escherichia coli (EPEC) by presenting eaeA gene and by sequencing of positive amplicons. Phylogenetic analyses revealed that the Brazilian samples were grouped in clades along with other EPEC isolates. These results are the first to indicate the presence of EPEC in shellfish in Brazil.

Detection and Characterization of Enteropathogenic and Shiga Toxin-Producing Escherichia coli Strains in Rattus spp. from Buenos Aires.

Enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) are pathovars of E. coli that impact human health by causing childhood diseases. In this work, 118 synanthropic rodents of the genus Rattus from Buenos Aires, Argentina were evaluated as EPEC and STEC carriers. Rectal swab samples from captured animals were evaluated by conventional PCR to detect the presence of the eae, stx1, stx2, and rfbO157 genes. Twenty-one isolates were obtained (17 EPEC isolates from seven animals and four STEC isolates from the same animal). All EPEC isolates tested negative for the presence of the bfpA gene. One EPEC isolate carried the iha gene, and five EPEC isolates carried the toxB gene. STEC isolates exhibited two different virulence profiles: stx1a/stx2a/stx2c/stx2d/saa/ehxA/subA (3/4) and stx1a/stx2a/saa/ehxA/subA (1/4). EPEC isolate serotypes included O109:H46 (7), O71:H40 (4), O71:NM (2), O138:H40 (1), O108:H21 (1), O88:H25 (1), and O76:NM (1), and STEC isolates belonged to the O108:H11 (4) serotype. Antimicrobial susceptibility testing was carried out, and resistance to tetracycline was observed in one EPEC strain. Our results demonstrate that Rattus spp. may act as carriers of EPEC and STEC strains and may be involved in the epidemiology of diarrheal disease in infancy.

Antibacterial resistance patterns of extended spectrum β-lactamase -producing enteropathogenic Escherichia coli strains isolated from children

Background and study aimThis study aimed to determine the antibacterial resistance patterns of extended spectrum β-lactamase (ESBL)-producing enteropathogenic Escherichia coli (EPEC) isolated from Iranian children and to investigate its genetic patterns. Patients and methods192 non-repeats EPEC isolates were collected from stool samples of the children with and without diarrhoea. The EPEC strains were isolated from 1355 stool specimens obtained from 247 children with diarrhoea (0–10 years old; mean age, 5.5 years) and 1108 children without any gastrointestinal symptoms (0–10 years old; mean age, 6.8 years) during the summer months in three Iranian provinces, Tehran, Ilam and Mazandaran. Strains biochemically identified as E. coli were selected and were identified by the presence of eaeA and bfpA as EPEC virulence genes. Antimicrobial susceptibilities were determined by disc diffusion method. The isolates were confirmed to be ESBL producers by the double disk synergy test (DDST). The β-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA) and insertion sequence ISEcp1 were detected by PCR method. ResultsThe highest antibiotic susceptibility was detected to imipenem (100%), followed by gentamicin (82.3%) and ciprofloxacin (79.2%). The highest resistance was detected to cefpodoxime (97.9%), trimethoprim (60.7%), and tetracycline (58.4%), respectively. Totally, 153 EPEC strains (79.7%) were ESBL-producing by DDST test. The PCR showed that 84 (43.8%) EPEC isolates were positive for ESBLs encoding genes. Among 153 ESBLs-producing EPEC, TEM was present in 9.2% of isolates. Also, CTX-M and SHV genes were detected in 7.2% and 7.8%, respectively. The SHV positive strains were associated with the highest resistance rate to tetracycline (56.5%), although the TEM and OXA were associated with the highest resistance rate to gentamicin (23.1%) and ciprofloxacin (21.4%). ConclusionsThe study revealed that 79.7% of EPEC isolates from Iranian children were ESBL-producing and were comparable with the non ESBL-producing isolates regarding susceptibility to the antibiotics.

Molecular and phenotypic typing of enteropathogenic Escherichia coli isolated in childhood acute diarrhea in Abuja, Nigeria.

Enteropathogenic Escherichia coli (EPEC) causes infectious diarrhea among children in developing countries. However, in Nigeria, due to limited laboratory resources, the genetic diversity of its virulence factors, which includes its intimin subtypes remain undefined. This study aims to evaluate the serotypes, antibiotic resistance and the intimin subtypes genes associated with EPEC strains isolated from children with acute diarrhea in Abuja, Nigeria. EPEC isolates from diarrheic children aged 0-60 months in Abuja, Nigeria were analyzed. PCR for EPEC virulence gene, Hep-2 cell Adherence and serotyping were performed. EPEC strains were further subtyped by PCR reactions for the identification of intimin subtype genes: α (alpha), β (beta), γ1 (gamma-1), and έ (epsilon). Antibiotic resistance and ESBL production was determined by CLSI guidelines. Overall, 18 (4.5%) out of 400 children with acute diarrhea had EPEC infection. Typical EPEC strains were detected in 14 (3.5%) whereas 4 (1.1%) were atypical EPEC. Fifteen (83.3%) of the EPEC isolated belonged to β intimin subtype gene, while the remaining 3 EPEC isolates possessed the intimin έ subtype. No α and γ intimin subtypes were detected. Traditional EPEC serotypes O114:H14 were detected only in tEPEC strains. Marked resistant to β-lactam agents were observed but no ESBL-producing tEPEC or aEPEC was detected. This is the first report of intimin subtype genes in Abuja, Nigeria. These data will be useful in facilitating the characterization of intimin variants of EPEC and some Shiga toxin-producing E. coli (STEC) in humans and other animal species.

Pathogenic potential and virulence genotypes of intestinal and faecal isolates of porcine post-weaning enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are frequent causes of diarrhoea in infants and in young mammals by inducing attaching effacing (AE) lesions of the intestinal epithelium. EPEC bacteria have also been implicated in cases of porcine post-weaning diarrhoea but their pathogenicity for conventional weaned pigs remains less elucidated.This present study investigates differences in pathogenic potential and virulence genotypes of intestinal and faecal isolates of EPEC from newly-weaned pigs. For this we inoculated ligated ileal loops of four weeks old weaned pigs to assess EPEC adherence to enterocytes by histology and immunohistology. Virulence gene patterns were identified by using a PCR-microarray. Intestinal EPEC isolates of sero−/intimin types O45:H11:eae-β, O49:NM:eae-β, O84:H7:eae-γ, and O123:H11:eae-β formed adherent microcolonies of EPEC with AE lesions on ileal villi more frequently than faecal isolates of O28:H28:eae-NT, O108:H9:eae-β, O145:H28:eae-γ and O157:H2:eae-β (p≤0.05). The PCR-array analysis of both groups detected all together 25 virulence genes of LEE (Locus of Enterocyte Effacement), and of non-LEE pathogenicity islands, of plasmids and phages characteristic to EPEC. Intestinal isolates carried significantly more virulence genes than faecal isolates (p≤0.05). Intestinal isolates possessed efa1, lpfA, and tsh genes most likely contributing to enterocyte adhesion while faecal isolates did not carry these genes (p≤0.05). Overall, the ileal loop model in weaned pigs combined with virulence genotyping PCR-array indicated a greater pathogenic potential of intestinal isolates over faecal isolates of porcine post-weaning EPEC. Differing virulence genotypes of the intestinal and faecal isolates as demonstrated here suggests dynamic evolutionary events within the population of porcine EPEC.

The Genetics of Enteropathogenic Escherichia coli Virulence.

In many parts of the world, enteropathogenic Escherichia coli (EPEC) are a leading cause of death in children with diarrhea. Much of what we know about the pathogenesis of EPEC infections is based on the study of one or two prototypic strains that have provided deep insight into the precise mechanisms by which EPEC colonizes the intestine, evades host immunity, and spreads from person to person. In some cases, defining the biochemical activity of the host-interacting effector proteins from these prototypic strains has led to the discovery of novel post-translational protein modifications and new understandings of biology and host-pathogen interactions. However, genomic analysis of recent EPEC isolates has revealed that the EPEC pathotype is more diverse than previously appreciated. Although by definition all strains carry the locus of enterocyte effacement, the effector repertoires of different clonal groups are quite divergent, suggesting that there is still a great deal to learn about the genetic basis of EPEC virulence.

Genetic characterization of Shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic Escherichia coli (EPEC) isolates from goat's milk and goat farm environment

The aim of this study was to characterize a collection of 44 Shiga toxin-producing (STEC) and enteropathogenic Escherichia coli (EPEC) isolated from goat milk and goat farm environment.Of the 19 STEC isolates, five (26.3%) carried the stx1 gene, four (21.1%) the stx2 gene and 10 (52.6%) presented both stx genes. Six (31.6%) STEC strains were eae-positive and belonged to serotypes related to severe human disease (O157:H7 and O5:HNM). Another seven STEC strains were of serotype O146:H21 and three of serotype O166:H28, also linked to human disease. The STEC strains isolated from goat milk were of serotypes potentially pathogenic for humans. All the 25 EPEC isolates were considered atypical (aEPEC) and one aEPEC strain was of serotype O26:H11, a serotype frequently isolated in children with diarrhea.Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 23 sequence types (ST) were detected, 14 of them newly described. Twelve STs grouped STEC isolates and 11 STs grouped EPEC isolates. Genetic typing by pulsed field gel electrophoresis (PFGE) resulted in 38 patterns which grouped in 10 clusters. Well-defined groups were also observed for strains of pathogenic serotypes.In conclusion, strains of STEC and aEPEC belonging to serotypes related to severe human disease have been detected in goat milk and the goat farm environment. Ruminants are an important reservoir of STEC strains and the role of these animals as carriers of other pathogenic types of E. coli seems to be an emerging concern.