Structural and Functional Insights into Host Death Domains Inactivation by the Bacterial Arginine GlcNAcyltransferase Effector.

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.

Cross-talk between QseBC and PmrAB two-component systems is crucial for regulation of motility and colistin resistance in Enteropathogenic Escherichia coli.

The quorum sensing two-component system (TCS) QseBC has been linked to virulence, motility and metabolism regulation in multiple Gram-negative pathogens, including Enterohaemorrhagic Escherichia coli (EHEC), Uropathogenic E. coli (UPEC) and Salmonella enterica. In EHEC, the sensor histidine kinase (HK) QseC detects the quorum sensing signalling molecule AI-3 and also acts as an adrenergic sensor binding host epinephrine and norepinephrine. Downstream changes in gene expression are mediated by phosphorylation of its cognate response regulator (RR) QseB, and 'cross-talks' with non-cognate regulators KdpE and QseF to activate motility and virulence. In UPEC, cross-talk between QseBC and TCS PmrAB is crucial in the regulation and phosphorylation of QseB RR that acts as a repressor of multiple pathways, including motility. Here, we investigated QseBC regulation of motility in the atypical Enteropathogenic E. coli (EPEC) strain O125ac:H6, causative agent of persistent diarrhoea in children, and its possible cross-talk with the KdpDE and PmrAB TCS. We showed that in EPEC QseB acts as a repressor of genes involved in motility, virulence and stress response, and in absence of QseC HK, QseB is likely activated by the non-cognate PmrB HK, similarly to UPEC. We show that in absence of QseC, phosphorylated QseB activates its own expression, and is responsible for the low motility phenotypes seen in a QseC deletion mutant. Furthermore, we showed that KdpD HK regulates motility in an independent manner to QseBC and through a third unidentified party different to its own response regulator KdpE. We showed that PmrAB has a role in iron adaptation independent to QseBC. Finally, we showed that QseB is the responsible for activation of colistin and polymyxin B resistance genes while PmrA RR acts by preventing QseB activation of these resistance genes.

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.

Molecular Identification of Escherichia coli Pathotypes and their Antimicrobial Resistance Profiles in Stool Samples Isolated from Patients in the West of Iran.

BACKGROUND: Gastroenteritis refers to an infection in the stomach and small intestine that may be caused by bacteria, viruses, and other pathogenic agents. Most strains of Escherichia coli (E. coli) in the gastrointestinal system have shared a symbiotic relationship with humans, but some serotypes are pathogenic. This study aimed to identify E. coli pathotypes isolated from stool samples and determine the antibiotic resistance profiles of these pathotypes in the west of Iran. METHODS: The study was conducted on 106 samples of diarrheal feces which were sent to Imam Reza laboratory. First E. coli was detected and then the DNA was extracted. Next, the antibiotic sensitivity test was performed by the disk diffusion method. The E. coli pathotypes were qualitatively detected using the Amplisense Escherichioses-FRT PCR kit after DNA extraction from E. coli isolated in the stool sample. RESULTS: In this study, out of 106 E. coli-positive samples, pathogenic E. coli were detected in 62 samples including 5 samples (8.1%) which only contained the EPEC pathotype, 10 samples (16.1%) contained only the EAEC pathotype, and 12 samples (19.4%) had only the EHEC pathotype. ETEC and EIEC were not isolated from any of the samples. The sensitivity to Meropenem (97%) and Gentamicin (96.2%) showed the highest frequency among the samples. The highest level of resistance was related to Amoxicillin (93.4%) and Ampicillin (78%). CONCLUSIONS: The epidemiological results show that the predominant pathotype among all isolates is EHEC and most antibiotic resistances were related to Amoxicillin and Ampicillin. Finally, a comprehensive molecular diagnosis of E. coli pathotypes, investigation of their incidence, and antibiogram profiles will help to determine better diagnostic and therapeutic measures for managing diarrheal diseases.

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.

Characterization of diarrheagenic Escherichia coli from different cattle production systems in Brazil.

Diarrheagenic E. coli (DEC) can cause severe diarrhea and is a public health concern worldwide. Cattle are an important reservoir for this group of pathogens, and once introduced into the abattoir environment, these microorganisms can contaminate consumer products. This study aimed to characterize the distribution of DEC [Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)] from extensive and intensive cattle production systems in Brazil. Samples (n = 919) were collected from animal feces (n = 200), carcasses (n = 600), meat cuts (n = 90), employee feces (n = 9), and slaughterhouse water (n = 20). Virulence genes were detected by PCR in 10% of animal samples (94/919), with STEC (n = 81) as the higher prevalence, followed by EIEC (n = 8), and lastly EPEC (n = 5). Animals raised in an extensive system had a higher prevalence of STEC (average 48%, sd = 2.04) when compared to animals raised in an intensive system (23%, sd = 1.95) (Chi-square test, P < 0.001). From these animals, most STEC isolates only harbored stx2 (58%), and 7% were STEC LEE-positive isolates that were further identified as O157:H7. This study provides further evidence that cattle are potential sources of DEC, especially STEC, and that potentially pathogenic E. coli isolates are widely distributed in feces and carcasses during the slaughter process.

[Molecular characterization of diarrheagenic Escherichia coli from an outpatient pediatric population with diarrhea attended in two hospitals from Buenos Aires, Argentina]. / Caracterización molecular de Escherichia coli diarreogénica proveniente de población pediátrica ambulatoria con diarrea, atendida en dos hospitales de Buenos Aires, Argentina.

Diarrheagenic Escherichia coli comprises a heterogeneous group of pathotypes or pathogenic variants that share phenotypic characteristics with marked differences in virulence genes, colonization sites, pathogenesis, clinical presentation, and epidemiology of infection. The most studied pathotypes are Shiga toxin-producing E.coli (STEC), enterotoxigenic E.coli (ETEC), enteropathogenic E.coli (EPEC), enteroaggregative E.coli (EAEC), and enteroinvasive E.coli (EIEC). The objective of the study was to characterize the isolates of diarrheagenic E.coli from an outpatient pediatric population with diarrhea attended in two public hospitals from Buenos Aires, Argentina. Diarrheagenic E.coli pathotypes were investigated by amplifying characteristic virulence gene fragments: intimin (eae), heat-labile toxin (lt), heat-stable toxins (stp, sth), invasion plasmid antigen H (ipaH), transcriptional activator R (aggR) and Shiga toxins (stx1, stx2). Molecular subtyping of isolates was performed using PFGE (XbaI). Diarrheagenic E.coli was detected in 14% (84/601) of cases. The EAEC pathotype was prevalent, while ETEC, STEC, EPEC and EIEC were found in a lower proportion. EAEC isolates exhibited a high degree of genetic diversity. All pathotypes were found in children under 5years of age, while only EAEC, EIEC and ETEC were detected in the older population. Future studies that include the characterization of isolates from a greater number of genes and populations from other geographical areas will be necessary to determine the relevance of diarrheagenic E.coli in Argentina.

Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1Cr, NleG7Cr, and NleG8Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleGCr knockout strains, we show that NleG7Cr contributes to bacterial survival during enteric infection while NleG1Cr promotes the expression of diarrheal symptoms and NleG8Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8Ec) effector, which shares 60% identity with NleG8Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8Ec does not complement the ΔnleG8Cr phenotype during infection, revealing functional diversification between these NleG effectors.

Epidemiology of Enteroaggregative, Enteropathogenic, and Shiga Toxin-Producing Escherichia coli Among Children Aged <5 Years in 3 Countries in Africa, 2015-2018: Vaccine Impact on Diarrhea in Africa (VIDA) Study.

BACKGROUND: To address knowledge gaps regarding diarrheagenic Escherichia coli (DEC) in Africa, we assessed the clinical and epidemiological features of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), and Shiga toxin-producing E. coli (STEC) positive children with moderate-to-severe diarrhea (MSD) in Mali, The Gambia, and Kenya. METHODS: Between May 2015 and July 2018, children aged 0-59 months with medically attended MSD and matched controls without diarrhea were enrolled. Stools were tested conventionally using culture and multiplex polymerase chain reaction (PCR), and by quantitative PCR (qPCR). We assessed DEC detection by site, age, clinical characteristics, and enteric coinfection. RESULTS: Among 4840 children with MSD and 6213 matched controls enrolled, 4836 cases and 1 control per case were tested using qPCR. Of the DEC detected with TAC, 61.1% were EAEC, 25.3% atypical EPEC (aEPEC), 22.4% typical EPEC (tEPEC), and 7.2% STEC. Detection was higher in controls than in MSD cases for EAEC (63.9% vs 58.3%, P < .01), aEPEC (27.3% vs 23.3%, P < .01), and STEC (9.3% vs 5.1%, P < .01). EAEC and tEPEC were more frequent in children aged <23 months, aEPEC was similar across age strata, and STEC increased with age. No association between nutritional status at follow-up and DEC pathotypes was found. DEC coinfection with Shigella/enteroinvasive E. coli was more common among cases (P < .01). CONCLUSIONS: No significant association was detected between EAEC, tEPEC, aEPEC, or STEC and MSD using either conventional assay or TAC. Genomic analysis may provide a better definition of the virulence factors associated with diarrheal disease.

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.

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.

Oral Administration with Live Attenuated Citrobacter rodentium Protects Immunocompromised Mice from Lethal Infection.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important causative agents for foodborne diseases worldwide. Besides antibiotic treatment, vaccination has been deemed as the most effective strategy for preventing EPEC- and EHEC-caused foodborne illnesses. Despite substantial progress made in identifying promising antigens and efficacious vaccines, no vaccines against EPEC or EHEC have yet been licensed. Mice are inherently resistant to EPEC and EHEC infections; infection with Citrobacter rodentium (CR), the murine equivalent of EPEC and EHEC, in mice has been widely used as a model to study bacterial pathogenesis and develop novel vaccine strategies. Mirroring the severe outcomes of EPEC and EHEC infections in immunocompromised populations, immunocompromised mouse strains such as interleukin-22 knockout (Il22-/-) are susceptible to CR infection with severe clinical symptoms and mortality. Live attenuated bacterial vaccine strategies have been scarcely investigated for EPEC and EHEC infections, in particular in immunocompromised populations associated with severe outcomes. Here we examined whether live attenuated CR strain with rational genetic manipulation generates protective immunity against lethal CR infection in the susceptible Il22-/- mice. Our results demonstrate that oral administration of live ΔespFΔushA strain promotes efficient systemic and humoral immunity against a wide range of CR virulence determinants, thus protecting otherwise lethal CR infection, even in immunocompromised Il22-/- mice. This provides a proof of concept of live attenuated vaccination strategy for preventing CR infection in immunocompromised hosts associated with more severe symptoms and lethality.

Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains.

How pathogens evolve their virulence to humans in nature is a scientific issue of great medical and biological importance. Shiga toxin (Stx)-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are the major foodborne pathogens that can cause hemolytic uremic syndrome and infantile diarrhea, respectively. The locus of enterocyte effacement (LEE)-encoded type 3 secretion system (T3SS) is the major virulence determinant of EPEC and is also possessed by major STEC lineages. Cattle are thought to be the primary reservoir of STEC and EPEC. However, genome sequences of bovine commensal E. coli are limited, and the emerging process of STEC and EPEC is largely unknown. Here, we performed a large-scale genomic comparison of bovine commensal E. coli with human commensal and clinical strains, including EPEC and STEC, at a global level. The analyses identified two distinct lineages, in which bovine and human commensal strains are enriched, respectively, and revealed that STEC and EPEC strains have emerged in multiple sublineages of the bovine-associated lineage. In addition to the bovine-associated lineage-specific genes, including fimbriae, capsule, and nutrition utilization genes, specific virulence gene communities have been accumulated in stx- and LEE-positive strains, respectively, with notable overlaps of community members. Functional associations of these genes probably confer benefits to these E. coli strains in inhabiting and/or adapting to the bovine intestinal environment and drive their evolution to highly virulent human pathogens under the bovine-adapted genetic background. Our data highlight the importance of large-scale genome sequencing of animal strains in the studies of zoonotic pathogens.

Enhanced protein translocation to mammalian cells by expression of EtgA transglycosylase in a synthetic injector E. coli strain.

BACKGROUND: Bacterial type III secretion systems (T3SSs) assemble a multiprotein complex termed the injectisome, which acts as a molecular syringe for translocation of specific effector proteins into the cytoplasm of host cells. The use of injectisomes for delivery of therapeutic proteins into mammalian cells is attractive for biomedical applications. With that aim, we previously generated a non-pathogenic Escherichia coli strain, called Synthetic Injector E. coli (SIEC), which assembles functional injectisomes from enteropathogenic E. coli (EPEC). The assembly of injectisomes in EPEC is assisted by the lytic transglycosylase EtgA, which degrades the peptidoglycan layer. As SIEC lacks EtgA, we investigated whether expression of this transglycosylase enhances the protein translocation capacity of the engineered bacterium. RESULTS: The etgA gene from EPEC was integrated into the SIEC chromosome under the control of the inducible tac promoter, generating the strain SIEC-eEtgA. The controlled expression of EtgA had no effect on the growth or viability of bacteria. Upon induction, injectisome assembly was ~ 30% greater in SIEC-eEtgA than in the parental strain, as determined by the level of T3SS translocon proteins, the hemolytic activity of the bacterial strain, and the impairment in flagellar motility. The functionality of SIEC-eEtgA injectisomes was evaluated in a derivative strain carrying a synthetic operon (eLEE5), which was capable of delivering Tir effector protein into the cytoplasm of HeLa cells triggering F-actin polymerization beneath the attached bacterium. Lastly, using ß-lactamase as a reporter of T3SS-protein injection, we determined that the protein translocation capacity was ~ 65% higher in the SIEC-EtgA strain than in the parental SIEC strain. CONCLUSIONS: We demonstrate that EtgA enhances the assembly of functional injectisomes in a synthetic injector E. coli strain, enabling the translocation of greater amounts of proteins into the cytoplasm of mammalian cells. Accordingly, EtgA expression may boost the protein translocation of SIEC strains programmed as living biotherapeutics.

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.

Virulence markers, adhesion and biofilm formation of Escherichia coli strains isolated from drinking water supplies of north Paraná State, Brazil.

Waterborne diseases are a major public health problem responsible for a high number of deaths worldwide, of which Escherichia coli is a major agent of contamination. This study investigates the occurrence of different diarrheagenic E. coli (DEC) pathotypes and its relationship with adherence patterns and biofilm formation. Between 2012 and 2014, a total of 1,780 drinking water samples were collected from different rural communities and urban water systems of north Paraná State. A total of 14% were positive for E. coli and 250 non-duplicate E. coli isolates were obtained. Between the E. coli isolates, 28 (11.2%) harbored DEC-associated genes, 10.7% being classified as Shiga toxin-producing E. coli (STEC), 64.3% enteroaggregative E. coli (EAEC) and 25% atypical enteropathogenic E. coli (aEPEC). The aggregative adherence (AA) was the predominant adherence pattern (84%), significantly associated with biofilm formation (p < 0.0001). On the other hand, the AA pattern and biofilm formation were not significantly associated to DEC pathotypes (p > 0.05). Therefore, we proposed that the AA pattern and biofilm formation in E. coli isolated from drinking water supplies could be associated with adherence and colonization of abiotic surfaces, such as pipes, leading to persistence and resistance to treatment or disinfection.

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.

Map of Enteropathogenic Escherichia coli Targets Mitochondria and Triggers DRP-1-Mediated Mitochondrial Fission and Cell Apoptosis in Bovine Mastitis.

Bovine mastitis seriously affects bovine health and dairy product quality. Escherichia coli is the most important pathogen in the environment and dairy products. Enteropathogenic Escherichia coli (EPEC) is a zoonotic pathogen, which seriously threatens the health of people and dairy cows. We recently reported that E. coli can induce endogenous apoptosis in bovine mammary epithelial cells. However, the mechanism of EPEC-damaged mitochondria and -induced bovine mastitis is unclear. In this study, we found that EPEC can induce DRP-1-dependent mitochondrial fission and apoptosis. This was verified by the application of Mdivi, a DRP-1 inhibitor. Meanwhile, in order to verify the role of the Map virulence factor in EPEC-induced bovine mastitis, we constructed a map mutant, complementary strain, and recombinant plasmid MapHis. In the present study, we find that Map induced DRP-1-mediated mitochondrial fission, resulting in mitochondrial dysfunction and apoptosis. These inferences were further verified in vivo by establishing a mouse mastitis model. After the map gene was knocked out, breast inflammation and apoptosis in mice were significantly alleviated. All results show that EPEC targets mitochondria by secreting the Map virulence factor to induce DRP-1-mediated mitochondrial fission, mitochondrial dysfunction, and endogenous apoptosis in bovine mastitis.

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.