Pathogenic E. coli Extracts Nutrients from Infected Host Cells Utilizing Injectisome Components.

Microbiota and intestinal epithelium restrict pathogen growth by rapid nutrient consumption. We investigated how pathogens circumvent this obstacle to colonize the host. Utilizing enteropathogenic E. coli (EPEC), we show that host-attached bacteria obtain nutrients from infected host cell in a process we termed host nutrient extraction (HNE). We identified an inner-membrane protein complex, henceforth termed CORE, as necessary and sufficient for HNE. The CORE is a key component of the EPEC injectisome, however, here we show that it supports the formation of an alternative structure, composed of membranous nanotubes, protruding from the EPEC surface to directly contact the host. The injectisome and flagellum are evolutionarily related, both containing conserved COREs. Remarkably, CORE complexes of diverse ancestries, including distant flagellar COREs, could rescue HNE capacity of EPEC lacking its native CORE. Our results support the notion that HNE is a widespread virulence strategy, enabling pathogens to thrive in competitive niches.

Human Enteric α-Defensin 5 Promotes Shigella Infection by Enhancing Bacterial Adhesion and Invasion.

Shigella is a Gram-negative bacterium that causes bacillary dysentery worldwide. It invades the intestinal epithelium to elicit intense inflammation and tissue damage, yet the underlying mechanisms of its host selectivity and low infectious inoculum remain perplexing. Here, we report that Shigella co-opts human α-defensin 5 (HD5), a host defense peptide important for intestinal homeostasis and innate immunity, to enhance its adhesion to and invasion of mucosal tissues. HD5 promoted Shigella infection in vitro in a structure-dependent manner. Shigella, commonly devoid of an effective host-adhesion apparatus, preferentially targeted HD5 to augment its ability to colonize the intestinal epithelium through interactions with multiple bacterial membrane proteins. HD5 exacerbated infectivity and Shigella-induced pathology in a culture of human colorectal tissues and three animal models. Our findings illuminate how Shigella exploits innate immunity by turning HD5 into a virulence factor for infection, unveiling a mechanism of action for this highly proficient human pathogen.

The bacterial virulence factor NleA undergoes host-mediated O-linked glycosylation.

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form "attaching and effacing" lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection.

Enteropathogenic Escherichia coli modulates the virulence and pathogenicity of Entamoeba dispar.

Amoebiasis is a disease caused by Entamoeba histolytica, affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions.

O26 Polysaccharides as Key Players in Enteropathogenic E. coli Immune Evasion and Vaccine Development.

Enteropathogenic Escherichia coli (EPEC) produce a capsule of polysaccharides identical to those composing the O-antigen polysaccharide of its LPS (lipopolysaccharide) molecules. In light of this, the impact of O26 polysaccharides on the immune evasion mechanisms of capsulated O26 EPEC compared to non-capsulated enterohemorrhagic Escherichia coli (EHEC) was investigated. Our findings reveal that there was no significant difference between the levels in EPEC and EHEC of rhamnose (2.8:2.5), a molecule considered to be a PAMP (Pathogen Associated Molecular Patterns). However, the levels of glucose (10:1.69), heptose (3.6:0.89) and N-acetylglucosamine (4.5:2.10), were significantly higher in EPEC than EHEC, respectively. It was also observed that the presence of a capsule in EPEC inhibited the deposition of C3b on the bacterial surface and protected the pathogen against lysis by the complement system. In addition, the presence of a capsule also protected EPEC against phagocytosis by macrophages. However, the immune evasion provided by the capsule was overcome in the presence of anti-O26 polysaccharide antibodies, and additionally, these antibodies were able to inhibit O26 EPEC adhesion to human epithelial cells. Finally, the results indicate that O26 polysaccharides can generate an effective humoral immune response, making them promising antigens for the development of a vaccine against capsulated O26 E. coli.

EPEC-induced activation of the Ca2+ transporter TRPV2 leads to pyroptotic cell death.

The enteropathogenic Escherichia coli (EPEC) type III secretion system effector Tir, which mediates intimate bacterial attachment to epithelial cells, also triggers Ca2+ influx followed by LPS entry and caspase-4-dependent pyroptosis, which could be antagonized by the effector NleF. Here we reveal the mechanism by which EPEC induces Ca2+ influx. We show that in the intestinal epithelial cell line SNU-C5, Tir activates the mechano/osmosensitive cation channel TRPV2 which triggers extracellular Ca2+ influx. Tir-induced Ca2+ influx could be blocked by siRNA silencing of TRPV2, pre-treatment with the TRPV2 inhibitor SET2 or by growing cells in low osmolality medium. Pharmacological activation of TRPV2 in the absence of Tir failed to initiate caspase-4-dependent cell death, confirming the necessity of Tir. Consistent with the model implicating activation on translocation of TRPV2 from the ER to plasma membrane, inhibition of protein trafficking by either brefeldin A or the effector NleA prevented TRPV2 activation and cell death. While infection with EPECΔnleA triggered pyroptotic cell death, this could be prevented by NleF. Taken together this study shows that while integration of Tir into the plasma membrane activates TRPV2, EPEC uses NleA to inhibit TRPV2 trafficking and NleF to inhibit caspase-4 and pyroptosis.

Small, Dynamic Clusters of Tir-Intimin Seed Actin Polymerization.

The understanding of actin pedestal formation by enteropathogenic Escherichia coli (EPEC) relies mainly on static ensemble information obtained from cell lysates. Here, the dynamic nature of signaling components on the subsecond timescale, which resemble phase condensates, is demonstrated. Unlike in vitro phase condensates, transfected intimin receptor (Tir) and downstream component form clusters 200 nm in diameter that are spaced ≈500 nm on average, indicating cellular regulation. On supported lipid bilayers with diffusive intimin, Tir-expressing fibroblasts formed Tir-intimin clusters even without Tir tyrosines, although Tir tyrosine phosphorylation is necessary for actin polymerization from clusters. Single-molecule tracking showed that Tir is diffusive in the clusters and exchanges with Tir in the plasma membrane. Further, Nck and N-WASP bind to the clusters and exchange with cytoplasmic molecules. Tir has a similar cluster lifetime to Nck, but longer than that of N-WASP. Actin polymerization from the clusters requires N-WASP binding, involved Arp2/3 activation, and stabilized N-WASP clusters. These dynamic properties are distinct from larger in vitro systems and do not depend significantly upon crosslinking. Thus, Tir-intimin clusters in the plasma membrane are limited in size by exchange and enhance signaling needed for actin polymerization that enables strong and stable bacterial attachment to host cells.

Does pre-incubation in selective-enrichment media improve the detection of diarrheagenic Escherichia coli using the RIDA®GENE PCR?

We aimed to investigate whether a selective pre-PCR enrichment step improves test performance of RIDA®GENE EHEC/EPEC to detect diarrheagenic Escherichia coli from stool samples. Each of the 250 stool samples was analyzed for the presence of stx1/2 and eae both with and without pre-PCR enrichment in selective broth. In comparison to a reference method, sensitivities for stx1/2 and eae with and without pre-PCR enrichment were 84% (95%CI 70-93) and 89% (stx1/2, 95%CI 76-96), and 71% (95%CI 58-81) and 72% (eae, 95%CI 60-82), respectively. Specificity exceeded 97% for both methods and target genes. In summary, pre-PCR broth enrichment did not improve test performance.

Enteropathogenic Escherichia coli is a predominant pathotype in healthy pigs in Hubei Province of China.

AIM: This study aims to investigate the prevalence of intestinal pathogenic Escherichia coli (InPEC) in healthy pig-related samples and evaluate the potential virulence of the InPEC strains. METHODS AND RESULTS: A multiplex PCR method was established to identify different pathotypes of InPEC. A total of 800 rectal swab samples and 296 pork samples were collected from pig farms and slaughterhouses in Hubei province, China. From these samples, a total of 21 InPEC strains were isolated, including 19 enteropathogenic E. coli (EPEC) and 2 shiga toxin-producing E. coli (STEC) strains. By whole-genome sequencing and in silico typing, it was shown that the sequence types and serotypes were diverse among the strains. Antimicrobial susceptibility assays showed that 90.48% of the strains were multi-drug resistant. The virulence of the strains was first evaluated using the Galleria mellonella larvae model, which showed that most of the strains possessed medium to high pathogenicity. A moderately virulent EPEC isolate was further selected to characterize its pathogenicity using a mouse model, which suggested that it could cause significant diarrhea. Bioluminescence imaging (BLI) was then used to investigate the colonization dynamics of this EPEC isolate, which showed that the EPEC strain could colonize the mouse cecum for up to 5 days.

Molecular and Genomic Analysis of the Virulence Factors and Potential Transmission of Hybrid Enteropathogenic and Enterotoxigenic Escherichia coli (EPEC/ETEC) Strains Isolated in South Korea.

Hybrid strains Escherichia coli acquires genetic characteristics from multiple pathotypes and is speculated to be more virulent; however, understanding their pathogenicity is elusive. Here, we performed genome-based characterization of the hybrid of enteropathogenic (EPEC) and enterotoxigenic E. coli (ETEC), the strains that cause diarrhea and mortality in children. The virulence genes in the strains isolated from different sources in the South Korea were identified, and their phylogenetic positions were analyzed. The EPEC/ETEC hybrid strains harbored eae and est encoding E. coli attaching and effacing lesions and heat-stable enterotoxins of EPEC and ETEC, respectively. Genome-wide phylogeny revealed that all hybrids (n = 6) were closely related to EPEC strains, implying the potential acquisition of ETEC virulence genes during ETEC/EPEC hybrid emergence. The hybrids represented diverse serotypes (O153:H19 (n = 3), O49:H10 (n = 2), and O71:H19 (n = 1)) and sequence types (ST546, n = 4; ST785, n = 2). Furthermore, heat-stable toxin-encoding plasmids possessing estA and various other virulence genes and transporters, including nleH2, hlyA, hlyB, hlyC, hlyD, espC, espP, phage endopeptidase Rz, and phage holin, were identified. These findings provide insights into understanding the pathogenicity of EPEC/ETEC hybrid strains and may aid in comparative studies, virulence characterization, and understanding evolutionary biology.

Insights into the structure-activity relationship of a type III secretion system inhibitor, aurodox.

Aurodox was originally isolated in 1972 as a linear polyketide compound exhibiting antibacterial activity against Gram-positive bacteria. We have since identified aurodox as a specific inhibitor of the bacterial type III secretion system (T3SS) using our original screening system for inhibition of T3SS-mediated hemolysis in enteropathogenic Escherichia coli (EPEC). In this research, we synthesized 15 derivatives of aurodox and evaluated EPEC T3SS inhibitory activity as well as antibacterial activity against EPEC. One of the derivatives was highly selective for T3SS inhibition, equivalent to that of aurodox, but without exhibiting antibacterial activity (69-fold selectivity). This work revealed the structure-activity relationship for the inhibition of T3SS by aurodox and suggests that the target of T3SS is distinct from the target for antibacterial activity.

Genetic and phylogenetic characterization of Shiga toxin-producing Escherichia coli and enteropathogenic E. coli from livestock in Jiangsu by using whole-genome sequencing.

AIMS: There are knowledge gaps regarding STEC and EPEC strains in livestock in Jiangsu, China. This study aimed to evaluate the potential public health significance of STEC and EPEC strains isolated from livestock by determining the serotypes, virulence profiles, and genetic relationship with international STEC strains. METHODS AND RESULTS: A total of 68 STEC and 37 EPEC strains were obtained from 231 faecal sheep samples and 70 faecal cattle samples. By using whole-genome sequencing (WGS) analysis, all STEC belonged to 15 O: H serotypes, and the most prevalent serotypes were O6:H10 (19.1%), O155:H21 (14.7%), and O21:H25 (10.3%). The main Shiga toxin gene subtypes detected were stx1c (41.2%), stx1a (26.5%), stx2b (14.7%), and stx2k (14.7%). Only the STEC from cattle carried eae gene. Other adherence-associated or toxin-related genes, including lpfA (70.6%), iha (48.5%), subA (54.4%), and ehxA (33.8%), were found in STEC. All EPEC strains were bfpA-negative, and the predominant eae variants were eae-ß1 (62.2%), eae-ζ (21.6%), and eae-θ (8.1%). The core-genome multi-locus sequence typing (cgMLST) analysis revealed nine scattered clusters in STEC and one dominant cluster in EPEC. The strains with the same serotypes, including O22:H8 and O43:H2 in the two towns, possessed a closely genomic distance. The core genome single-nucleotide polymorphism (cgSNP) showed that part of STEC strains in this study was clustered with isolates possessing the same serotypes from the Netherlands, Sweden, and Xinjiang of China. Five serotypes of STEC isolates were associated with the clinical STEC strains from databases. CONCLUSION: This study provided the diverse serotypes and the virulence genes profiles in STEC and EPEC strains. Local strains possessed widely diverse and scattered clusters by cgMLST. Closely genomic correlation with clinical isolates displayed that part of the STEC strains may threaten to public health. SIGNIFICANCE AND IMPACT OF THE STUDY: Non-O157 STEC strains act as important pathogens for human infections. This study supports the increased surveillance work of non-O157 STEC rather than just O157 STEC in this region.

Bacteriophages: A possible solution to combat enteropathogenic Escherichia coli infections in neonatal goats.

Due to awareness and benefits of goat rearing in developing economies, goats' significance is increasing. Unfortunately, these ruminants are threatened via multiple bacterial pathogens such as enteropathogenic Escherichia coli (EPEC). In goat kids and lambs, EPEC causes gastrointestinal disease leading to substantial economic losses for farmers and may also pose a threat to public health via the spread of zoonotic diseases. Management of infection is primarily based on antibiotics, but the need for new therapeutic measures as an alternative to antibiotics is becoming vital because of the advent of antimicrobial resistance (AMR). The prevalence of EPEC was established using bfpA gene, uspA gene and Stx1 gene, followed by phylogenetic analysis using Stx1 gene. The lytic activity of the isolated putative coliphages was tested on multi-drug resistant strains of EPEC. It was observed that a PCR based approach is more effective and rapid as compared to phenotypic tests of Escherichia coli virulence. It was also established that the isolated bacteriophages exhibited potent antibacterial efficacy in vitro, with some of the isolates (16%) detected as T4 and T4-like phages based on gp23 gene. Hence, bacteriophages as therapeutic agents may be explored as an alternative to antibiotics in managing public, livestock and environmental health in this era of AMR.

Tracking the Transmission of Antimicrobial-Resistant Non-O157 Escherichia coli and Salmonella Isolates at the Interface of Food Animals and Fresh Produce from Agriculture Operations Using Whole-Genome Sequencing.

An increasing number of outbreaks are caused by foodborne pathogens such as Escherichia coli and Salmonella, which often harbor antimicrobial resistance (AMR) genes. We previously demonstrated the transmission of pathogens from animal operations to produce fields on sustainable farms, which illustrated an urgent need to develop and implement novel prevention methods and remediation practices such as the vegetative buffer zone (VBZ) to prevent this movement. The focus of this study was to use whole-genome sequencing (WGS) to characterize the AMR, virulence, and single-nucleotide polymorphism profile of 15 Salmonella and 128 E. coli isolates collected from small-scale dairy and poultry farms on a research station in North Carolina. Phenotypically, seven E. coli and three Salmonella isolates displayed resistance to antibiotics such as tetracycline (n = 4), ampicillin (n = 4), nalidixic acid (n = 3), chloramphenicol (n = 2), sulfisoxazole (n = 1), and streptomycin (n = 1). A single E. coli isolate was found to be resistant to five different antibiotic class types and possessed the blaTEM-150 resistance gene. Virulence genes that facilitate toxin production and cell invasion were identified. Mauve analysis of the E. coli isolates identified seven clusters (dairy-six and poultry-one) indicating that transmission is occurring from animal operations to fresh produce fields and the surrounding environment when the VBZ is denudated. This suggests that the VBZ is a useful barrier to reducing the transmission of enteric pathogens in agricultural systems. Our study demonstrates the prevalence of AMR and virulence genes on small-scale sustainable farms and highlights the advantage of using WGS to assess the impact of the VBZ to reduce the transmission of E. coli and Salmonella.

The Assembly of Flagella in Enteropathogenic Escherichia coli Requires the Presence of a Functional Type III Secretion System.

In enteropathogenic Escherichia coli (EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (ΔespA, ΔespB, ΔespD, ΔescC, ΔescN, and ΔescV), and in secreting effector molecules (ΔsepD and ΔsepL) were tested for flagella production under several growth conditions. The ΔespA mutant did not produce flagella in any condition tested, although fliC was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco's minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The ΔqseB and ΔqseC mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of fliC transcription. The ΔgrlR, ΔperA, Δler, Δhns, and Δfis mutants produced low levels of flagella, suggesting these regulators are activators of fliC expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs.

Comparative Genomics of Atypical Enteropathogenic Escherichia coli from Kittens and Children Identifies Bacterial Factors Associated with Virulence in Kittens.

Typical enteropathogenic Escherichia coli (tEPEC) is a leading cause of diarrhea and associated death in children worldwide. Atypical EPEC (aEPEC) lacks the plasmid encoding bundle-forming pili and is considered less virulent, but the molecular mechanism of virulence is poorly understood. We recently identified kittens as a host for aEPEC where intestinal epithelial colonization was associated with diarrheal disease and death. The purposes of this study were to (i) determine the genomic similarity between kitten aEPEC and human aEPEC isolates and (ii) identify genotypic or phenotypic traits associated with virulence in kitten aEPEC. We observed no differences between kitten and human aEPEC in core genome content or gene cluster sequence identities, and no distinguishing genomic content was observed between aEPEC isolates from kittens with nonclinical colonization (NC) versus those with lethal infection (LI). Variation in adherence patterns and ability to aggregate actin in cultured cells mirrored descriptions of human aEPEC. The aEPEC isolated from kittens with LI were significantly more motile than isolates from kittens with NC. Kittens may serve as a reservoir for aEPEC that is indistinguishable from human aEPEC isolates and may provide a needed comparative animal model for the study of aEPEC pathogenesis. Motility seems to be an important factor in pathogenesis of LI associated with aEPEC in kittens.

Enteropathogenic Escherichia coli Infection in Cancer and Immunosuppressed Patients.

BACKGROUND: The role of enteropathogenic Escherichia coli (EPEC) as a cause of diarrhea in cancer and immunocompromised patients is controversial. Quantitation of fecal bacterial loads has been proposed as a method to differentiate colonized from truly infected patients. METHODS: We studied 77 adult cancer and immunosuppressed patients with diarrhea and EPEC identified in stools by FilmArray, 25 patients with pathogen-negative diarrhea, and 21 healthy adults without diarrhea. Stools were studied by quantitative polymerase chain reaction (qRT-PCR) for EPEC genes eaeA and lifA/efa-1 and strains characterized for virulence factors and adherence to human intestinal enteroids (HIEs). RESULTS: Patients with EPEC were more likely to have community-acquired diarrhea (odds ratio, 3.82 [95% confidence interval, 1.5-10.0]; P = .008) compared with pathogen-negative cases. Although EPEC was identified in 3 of 21 (14%) healthy subjects by qPCR, the bacterial burden was low compared to patients with diarrhea (≤55 vs median, 6 × 104 bacteria/mg stool; P < .001). Among EPEC patients, the bacterial burden was higher in those who were immunosuppressed (median, 6.7 × 103 vs 55 bacteria/mg; P < .001) and those with fecal lifA/ifa-1 (median, 5 × 104 vs 120 bacteria/mg; P = .015). Response to antimicrobial therapy was seen in 44 of 48 (92%) patients with EPEC as the sole pathogen. Antimicrobial resistance was common and strains exhibited distinct patterns of adherence with variable cytotoxicity when studied in HIEs. Cancer care was delayed in 13% of patients. CONCLUSIONS: Immunosuppressed cancer patients with EPEC-associated diarrhea carry high burden of EPEC with strains that are resistant to antibiotics, exhibit novel patterns of adherence when studied in HIEs, and interfere with cancer care.

Role of the SOS Response in the Generation of Antibiotic Resistance In Vivo.

The SOS response to DNA damage is a conserved stress response in Gram-negative and Gram-positive bacteria. Although this pathway has been studied for years, its relevance is still not familiar to many working in the fields of clinical antibiotic resistance and stewardship. Under some conditions, the SOS response favors DNA repair and preserves the genetic integrity of the organism. On the other hand, the SOS response also includes induction of error-prone DNA polymerases, which can increase the rate of mutation, called the mutator phenotype or "hypermutation." As a result, mutations can occur in genes conferring antibiotic resistance, increasing the acquisition of resistance to antibiotics. Almost all of the work on the SOS response has been on bacteria exposed to stressors in vitro. In this study, we sought to quantitate the effects of SOS-inducing drugs in vivo, in comparison with the same drugs in vitro. We used a rabbit model of intestinal infection with enteropathogenic Escherichia coli strain E22. SOS-inducing drugs triggered the mutator phenotype response in vivo as well as in vitro. Exposure of E. coli strain E22 to ciprofloxacin or zidovudine, both of which induce the SOS response in vitro, resulted in increased antibiotic resistance to 3 antibiotics: rifampin, minocycline, and fosfomycin. Zinc was able to inhibit the SOS-induced emergence of antibiotic resistance in vivo, as previously observed in vitro. Our findings may have relevance in reducing the emergence of resistance to new antimicrobial drugs.

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 5 years 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 5 years 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 ß-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.

Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2.

AIM: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in Belgium and to comparing them with human EHEC after whole genome sequencing. METHODS AND RESULTS: Ten EHEC and 21 EPEC O80 identified by PCR between 2009 and 2018 from faeces, intestinal content and a kidney of diarrhoeic or septicaemic calves were genome sequenced and compared to 19 human EHEC identified between 2008 and 2019. They all belonged to the O80:H2 serotype and ST301, harboured the eaeξ gene, and 23 of the 29 EHEC contained the stx2d gene. Phylogenetically, they were distributed in two major sub-lineages: one comprised a majority of bovine EPEC whereas the second one comprised a majority of stx2d bovine and human EHEC. CONCLUSIONS: Not only EPEC but also EHEC O80:H2 are present in diarrhoeic and septicaemic calves in Belgium and are genetically related to human EHEC. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the need to assess cattle as potential source of contamination of humans by EHEC O80:H2 and to understand the evolution of bovine and human EHEC and EPEC O80:H2.