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Disinfectants are essential for biosecurity, preventing the persistence and spread of zoonotic pathogens on farms and subsequent human infections. In this study, transcriptomics and genomics were utilised to assess the effect of disinfectant exposure on pathogenic Escherichia coli. The exposure of E. coli O157:H7 to sub-optimal concentrations of commonly used farm disinfectants elicited changes in both the transcriptome and genome. The transcriptomics identified upregulation of >300 genes and downregulation of >100 genes with functions, which included stress response, metabolism, transcription, transportation, membrane-associated and virulence genes. The phage shock protein (psp) operon was highly upregulated in response to a quaternary ammonium compound (QAC)-containing disinfectant, which has not previously been associated with a response to chemical stress. Disinfectant-adapted isolates generated by exposure to sub-lethal disinfectants levels demonstrated resistance to several common antibiotics and decreased sensitivity to biocides. Whole genome sequencing of the mutant strains indicated that they had acquired mutations in the genes associated with the upregulation of the multiple antibiotic resistance (MAR) efflux system (lon protease and marR) and topoisomerase genes (gyrA and gyrB). The disinfectant-adapted isolates also exhibited increased expression of transcription, respiration and several pH stress response genes localised in the "acid fitness island." This study demonstrated that sub-optimal disinfectant concentrations allow E. coli O157:H7 to adapt and survive disinfection and develop antibiotic resistance. These changes could have implications for disease treatment and elimination on farms. Although E. coli O157:H7 and farm disinfectants were the focus of this study, we believe these findings are also applicable to other settings, including hospitals. [ABSTRACT FROM AUTHOR]

Disinfectants are essential for biosecurity, preventing the persistence and spread of zoonotic pathogens on farms and subsequent human infections. In this study, transcriptomics and genomics were utilised to assess the effect of disinfectant exposure on pathogenic Escherichia coli. The exposure of E. coli O157:H7 to sub-optimal concentrations of commonly used farm disinfectants elicited changes in both the transcriptome and genome. The transcriptomics identified upregulation of >300 genes and downregulation of >100 genes with functions, which included stress response, metabolism, transcription, transportation, membrane-associated and virulence genes. The phage shock protein (psp) operon was highly upregulated in response to a quaternary ammonium compound (QAC)-containing disinfectant, which has not previously been associated with a response to chemical stress. Disinfectant-adapted isolates generated by exposure to sub-lethal disinfectants levels demonstrated resistance to several common antibiotics and decreased sensitivity to biocides. Whole genome sequencing of the mutant strains indicated that they had acquired mutations in the genes associated with the upregulation of the multiple antibiotic resistance (MAR) efflux system (lon protease and marR) and topoisomerase genes (gyrA and gyrB). The disinfectant-adapted isolates also exhibited increased expression of transcription, respiration and several pH stress response genes localised in the “acid fitness island.” This study demonstrated that sub-optimal disinfectant concentrations allow E. coli O157:H7 to adapt and survive disinfection and develop antibiotic resistance. These changes could have implications for disease treatment and elimination on farms. Although E. coli O157:H7 and farm disinfectants were the focus of this study, we believe these findings are also applicable to other settings, including hospitals.

ABSTRACT Escherichia coli O157:H7-adulterated food products are associated with disease outbreaks in humans. Although cattle feces are a source for E. coli O157:H7 contamination, it is unclear if human-associated outbreak isolates differentially colonize and shed in the feces of cattle from that of non-outbreak isolates. It is also unclear if phenotypes, such as biofilm formation, cell attachment, or toxin production, differentiate environmental E. coli O157:H7 isolates from those associated with human illness. The objective of this study was to compare the genotypes and phenotypes of a diverse set of E. coli O157:H7 isolates, with the intent of identifying differences that could inform cattle colonization and fecal shedding, along with virulence potential in humans. Isolates differed in attachment phenotypes on human Caco-2 cells and bovine-derived recto-anal junction squamous epithelial cells, with curli having a strong impact on attachment to the human-derived cell line. The prototypical E. coli O157 isolate EDL933 had the greatest expression of the adhesin gene iha, yet it had decreased expression of the virulence genes stx2, eae, and ehxA compared the lineage I/II isolates RM6067W and/or FRIK1989. Strong or weak biofilm production was not associated with significant differences in cattle colonization or shedding, suggesting biofilms may not play a major role in cattle colonization. No significant differences in cattle colonization and fecal shedding were detected, despite genomic and in vitro phenotypic differences. The outbreak isolate associated with the greatest incidence of hemolytic uremic syndrome, RM6067W, induced the greatest Vero cell cytotoxicity and had the greatest stx2 gene expression.IMPORTANCEFoodborne illness has major impacts on global health and imposes financial hardships on food industries. Escherichia coli serotype O157:H7 is associated with foodborne illness. Cattle feces are a source of E. coli O157:H7, and routine surveillance has led to an abundance of E. coli O157:H7 genomic data. The relationship between E. coli O157:H7 genome and phenotype is not clearly discerned for cattle colonization/shedding and improved understanding could lead to additional strategies to limit E. coli O157:H7 in the food chain. The goal of the research was to evaluate genomic and phenotypic attributes of E. coli O157:H7 associated with cattle colonization and shedding, environmental persistence, and human illness. Our results indicate variations in biofilm formation and in vitro cellular adherence was not associated with differences in cattle colonization or shedding. Overall, processes involved in cattle colonization and various phenotypes in relation to genotype are complex and remain not well understood.

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx− strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains' distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx−) evolutionary paths. [ABSTRACT FROM AUTHOR]

ABSTRACT Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

Abstract Escherichia coli O157:H7 is a foodborne pathogen that has been linked to global disease outbreaks. These diseases include hemorrhagic colitis and hemolytic uremic syndrome. It is vital to know the features that make this strain pathogenic to understand the development of disease outbreaks. In the current study, a comparative genomic analysis was carried out to determine the presence of structural and functional features of O157:H7 strains obtained from 115 National Center for Biotechnology Information database. These strains of interest were analysed in the following programs: BLAST Ring Image Generator, PlasmidFinder, ResFinder, VirulenceFinder, IslandViewer 4 and PHASTER. Five strains (ECP19–198, ECP19–798, F7508, F8952, H2495) demonstrated a great homology with Sakai because of a few regions missing. Five resistant genes were identified, however, Macrolide-associated resistance gene mdf(A) was commonly found in all genomes. Majority of the strains (97%) were positive for 15 of the virulent genes (espA, espB, espF, espJ, gad, chuA, eae, iss, nleA, nleB, nleC, ompT, tccP, terC and tir). The plasmid analysis demonstrated that the IncF group was the most prevalent in the strains analysed. The prophage and genomic island analysis showed a distribution of bacteriophages and genomic islands respectively. The results indicated that structural and functional features of the many O157:H7 strains differ and may be a result of obtaining mobile genetic elements via horizontal gene transfer. Understanding the evolution of O157:H7 strains pathogenicity in terms of their structural and functional features will enable the development of detection and control of transmission strategies.

Escherichia coli O157:H7 is a foodborne pathogen that has been linked to global disease outbreaks. These diseases include hemorrhagic colitis and hemolytic uremic syndrome. It is vital to know the features that make this strain pathogenic to understand the development of disease outbreaks. In the current study, a comparative genomic analysis was carried out to determine the presence of structural and functional features of O157:H7 strains obtained from 115 National Center for Biotechnology Information database. These strains of interest were analysed in the following programs: BLAST Ring Image Generator, PlasmidFinder, ResFinder, VirulenceFinder, IslandViewer 4 and PHASTER. Five strains (ECP19–198, ECP19–798, F7508, F8952, H2495) demonstrated a great homology with Sakai because of a few regions missing. Five resistant genes were identified, however, Macrolide-associated resistance gene mdf(A) was commonly found in all genomes. Majority of the strains (97%) were positive for 15 of the virulent genes (espA, espB, espF, espJ, gad, chuA, eae, iss, nleA, nleB, nleC, ompT, tccP, terC and tir). The plasmid analysis demonstrated that the IncF group was the most prevalent in the strains analysed. The prophage and genomic island analysis showed a distribution of bacteriophages and genomic islands respectively. The results indicated that structural and functional features of the many O157:H7 strains differ and may be a result of obtaining mobile genetic elements via horizontal gene transfer. Understanding the evolution of O157:H7 strains pathogenicity in terms of their structural and functional features will enable the development of detection and control of transmission strategies. [ABSTRACT FROM AUTHOR]

Background: Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that causes gastrointestinal infections, ranging from acute diarrhea and dysentery to life-threatening diseases such as Hemolytic Uremic Syndrome. Currently, a vaccine to prevent STEC infection is an unmet medical need. Results: We developed a chimeric protein-based vaccine targeting seven virulence factors of STEC, including the Stx2B subunit, Tir, Intimin, EspA, Cah, OmpT, and AggA proteins. Immunization of mice with this vaccine candidate elicited significant humoral and cellular immune responses against STEC. High levels of specific IgG antibodies were found in the serum and feces of immunized mice. However, specific IgA antibodies were not detected in either serum or feces. Furthermore, a significantly higher percentage of antigen-specific CD4+ T cells producing IFN-g, IL-4, and IL-17 was observed in the spleens of immunized mice. Notably, the immunized mice showed decreased shedding of STEC O157: H7 and STEC O91:H21 strains and were protected against weight loss during experimental infection. Additionally, infection with the STEC O91:H21 strain resulted in kidney damage in control unimmunized mice; however, the extent of damage was slightly lower in immunized mice. Our findings suggest that IgG antibodies induced by this vaccine candidate may have a role in inhibiting bacterial adhesion and complement-mediated killing. Conclusion: This study provides evidence that IgG responses are involved in the host defense against STEC. However, our results do not rule out that other classes of antibodies also participate in the protection against this pathogen. Additional work is needed to improve the protection conferred by our vaccine candidate and to elucidate the relevant immune responses that lead to complete protection against this pathogen. [ABSTRACT FROM AUTHOR]

Current California agricultural practices strive to comanage food safety and habitat conservation on farmland. However, the ecology of foodborne pathogens in wild bird populations, especially those avian species residing in proximity to fresh produce production fields, is not fully understood. In this repeated cross-sectional study, avifauna within agricultural lands in California were sampled over 1 year. Feces, oral swabs, and foot/feather swabs were cultured for zoonotic Salmonella spp., Escherichia coli O157:H7, and non-O157 Shiga toxin-producing E. coli (STEC) and characterized by serotyping and pulsed-field gel electrophoresis. Of 60 avian species sampled, 8 species (13.3%, bird groups of sparrows, icterids, geese, wrens, and kinglets) were positive for at least one of these foodborne pathogens. At the individual bird level, the detection of foodborne pathogens was infrequent in feces (n = 583; 0.5% Salmonella, 0.34% E. coli O157:H7, and 0.5% non-O157 STEC) and in feet/feathers (n = 401; 0.5% non-O157 STEC), and it was absent from oral swabs (n = 353). Several subtypes of public health importance were identified, including Salmonella enterica serotype Newport, E. coli O157:H7, and STEC serogroups O103 and O26. In late summer and autumn, the same STEC subtype was episodically found in several individuals of the same and different avian species, suggesting a common source of contamination in the environment. Sympatric free-range cattle shared subtypes of STEC O26 and O163 with wild geese. A limited rate of positive detection in wild birds provides insights into broad risk profile for contamination considerations but cannot preclude or predict risk on an individual farm.IMPORTANCE The shedding dynamics of foodborne pathogens by wild birds on farmland are not well characterized. This yearlong study sampled wild birds for foodborne pathogens within agricultural lands in northern California. There was a low prevalence of Salmonella spp., Escherichia coli O157:H7, and non-O157 Shiga-toxin producing E. coli (prevalence, 0.34% to 0.50%) identified in bird populations in this study. However, pathogens of public health importance (such as Salmonella Newport, E. coli O157:H7, and STEC O103 and O26) were identified in fecal samples, and two birds carried STEC on their feet or feathers. Identical pathogen strains were shared episodically among birds and between wild geese and free-range cattle. This result suggests a common source of contamination in the environment and potential transmission between species. These findings can be used to assess the risk posed by bird intrusions in produce fields and enhance policy decisions toward the comanagement of food safety and farmland habitat conservation.

BackgroundShiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that causes gastrointestinal infections, ranging from acute diarrhea and dysentery to life-threatening diseases such as Hemolytic Uremic Syndrome. Currently, a vaccine to prevent STEC infection is an unmet medical need.ResultsWe developed a chimeric protein-based vaccine targeting seven virulence factors of STEC, including the Stx2B subunit, Tir, Intimin, EspA, Cah, OmpT, and AggA proteins. Immunization of mice with this vaccine candidate elicited significant humoral and cellular immune responses against STEC. High levels of specific IgG antibodies were found in the serum and feces of immunized mice. However, specific IgA antibodies were not detected in either serum or feces. Furthermore, a significantly higher percentage of antigen-specific CD4+ T cells producing IFN-γ, IL-4, and IL-17 was observed in the spleens of immunized mice. Notably, the immunized mice showed decreased shedding of STEC O157:H7 and STEC O91:H21 strains and were protected against weight loss during experimental infection. Additionally, infection with the STEC O91:H21 strain resulted in kidney damage in control unimmunized mice; however, the extent of damage was slightly lower in immunized mice. Our findings suggest that IgG antibodies induced by this vaccine candidate may have a role in inhibiting bacterial adhesion and complement-mediated killing.ConclusionThis study provides evidence that IgG responses are involved in the host defense against STEC. However, our results do not rule out that other classes of antibodies also participate in the protection against this pathogen. Additional work is needed to improve the protection conferred by our vaccine candidate and to elucidate the relevant immune responses that lead to complete protection against this pathogen.

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx− strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx1a- and ΦStx2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains’ distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx−) evolutionary paths.

ABSTRACT: This study was conducted to assess the survival of 2 wild Shiga toxin-producing Escherichia coli strains (one serotype O157:H7 and one non-O157:H7) in ewe milk stored at different conditions and to examine the fate of the O157 strain during the manufacture and ripening of a Spanish sheep hard variety of raw milk cheese (Zamorano). The strains were selected among a population of 50 isolates, which we obtained from ewe milk, because of their high resistance to 0.3% lactic acid. Both strains were inoculated (approximately 2 log10 cfu/mL) in raw and heat-treated (low-temperature holding, LTH; 63°C/30 min) ewe milk and stored for 5 d at 6, 8, and 10°C and also according to a simulation approach for assessing the effects of failures in the cold chain. The minimum growth temperature for the O157:H7 strain in LTH and raw ewe milk was 8°C. For the non-O157:H7 strain, the lowest temperature showing bacterial growth in LTH ewe milk was 6°C, but it did not grow at any of the tested conditions in raw milk. It appears that the O157 strain was more susceptible to cold stress but was likely a better competitor than the non-O157 strain against the milk autochthonous microbiota. For manufacture of Zamorano cheese, raw milk was inoculated with approximately 3 log10 cfu/mL, and after 2 mo of ripening at 10 to 12°C, the cheeses showed the expected general characteristics for this variety. The O157:H7 strain increased 0.9 log10 cfu/g after whey drainage and during ripening and storage decreased by 2.9 log10 cfu/g. Nevertheless, its detectable level (estimated at 6.2 cfu/g) after 2 mo of ripening suggests that Zamorano cheese manufactured from raw ewe milk contaminated with E. coli O157:H7 could represent a public health concern.

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne diarrheal illness in the United States and globally, and serotype O157:H7 is frequently associated with STEC outbreaks and sporadic cases in the United States. Severe systemic diseases associated with STEC are mediated by Stx types, particularly subtype Stx2a, encoded on inducible bacteriophages. We previously identified two STEC O157:H7 clinical isolates, JH2010 and JH2012, that exhibit a large difference in virulence in a streptomycin (Str)-treated mouse model. In this study, we aimed to identify a genetic basis for the difference in virulence between those strains. Comparison of the stx2a phage sequences showed that JH2012 lacks the lytic genes S and R on the phage genome. We also demonstrated that compared to JH2012 cultures, cultures of JH2010 released more Stx2 into the supernatant and were more sensitive to bacterial lysis during growth with ciprofloxacin (Cip), an inducer of stx phages. We therefore generated an stx2a phage SR deletion mutant strain of JH2010 to determine if those genes were responsible for the high virulence of that strain. We found that deletion of the SR genes from the stx2a phage in JH2010, and another O157:H7 strain, JH2016, resulted in increased cellular retention of Stx2, but there was no difference in virulence compared to the wild-type strains. Our results indicate that the stx2a phage SR genes are involved in Stx2 localization and phage-mediated cell lysis in vitro but that they are not required in wild-type STEC strains for virulence in a mouse model. IMPORTANCE The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bacterial cell. In this study, we found that the stx2a phage lytic genes are not required for the virulence of pathogenic O157:H7 clinical isolates in a murine model of STEC infection or for release of Stx2a into the supernatant of bacterial cultures. These results point to an alternate mechanism for Stx2a release from STEC strains.

Escherichia coli O157:H7-adulterated food products are associated with disease outbreaks in humans. Although cattle feces are a source for E. coli O157:H7 contamination, it is unclear if human-associated outbreak isolates differentially colonize and shed in the feces of cattle from that of non-outbreak isolates. It is also unclear if phenotypes, such as biofilm formation, cell attachment, or toxin production, differentiate environmental E. coli O157:H7 isolates from those associated with human illness. The objective of this study was to compare the genotypes and phenotypes of a diverse set of E. coli O157:H7 isolates, with the intent of identifying differences that could inform cattle colonization and fecal shedding, along with virulence potential in humans. Isolates differed in attachment phenotypes on human Caco-2 cells and bovine-derived recto-anal junction squamous epithelial cells, with curli having a strong impact on attachment to the human-derived cell line. The prototypical E. coli O157 isolate EDL933 had the greatest expression of the adhesin gene iha , yet it had decreased expression of the virulence genes stx2 , eae , and ehxA compared the lineage I/II isolates RM6067W and/or FRIK1989. Strong or weak biofilm production was not associated with significant differences in cattle colonization or shedding, suggesting biofilms may not play a major role in cattle colonization. No significant differences in cattle colonization and fecal shedding were detected, despite genomic and in vitro phenotypic differences. The outbreak isolate associated with the greatest incidence of hemolytic uremic syndrome, RM6067W, induced the greatest Vero cell cytotoxicity and had the greatest stx2 gene expression., Importance: Foodborne illness has major impacts on global health and imposes financial hardships on food industries. Escherichia coli serotype O157:H7 is associated with foodborne illness. Cattle feces are a source of E. coli O157:H7, and routine surveillance has led to an abundance of E. coli O157:H7 genomic data. The relationship between E. coli O157:H7 genome and phenotype is not clearly discerned for cattle colonization/shedding and improved understanding could lead to additional strategies to limit E. coli O157:H7 in the food chain. The goal of the research was to evaluate genomic and phenotypic attributes of E. coli O157:H7 associated with cattle colonization and shedding, environmental persistence, and human illness. Our results indicate variations in biofilm formation and in vitro cellular adherence was not associated with differences in cattle colonization or shedding. Overall, processes involved in cattle colonization and various phenotypes in relation to genotype are complex and remain not well understood., Competing Interests: The authors declare no conflict of interest.

Shiga toxin-producing Escherichia coli, a foodborne bacterial pathogen, has been linked to a broad spectrum of clinical outcomes ranging from asymptomatic carriage to fatal hemolytic uremic syndrome (HUS). Here, we collected clinical data and STEC strains from HUS patients from 1994 through 2018, whole-genome sequencing was performed to molecularly characterize HUS-associated STEC strains, statistical analysis was conducted to identify bacterial genetic factors associated with severe outcomes in HUS patients. O157:H7 was the most predominant serotype (57%) among 54 HUS-associated STEC strains, followed by O121:H19 (19%) and O26:H11 (7%). Notably, some non-predominant serotypes such as O59:H17 (2%) and O109:H21 (2%) also caused HUS. All O157:H7 strains with one exception belonged to clade 8. During follow-up at a median of 4 years, 41% of the patients had renal sequelae. Fifty-nine virulence genes were found to be statistically associated with severe renal sequelae, these genes encoded type II and type III secretion system effectors, chaperones, and other factors. Notably, virulence genes associated with severe clinical outcomes were significantly more prevalent in O157:H7 strains. In contrast, genes related to mild symptoms were evenly distributed across all serotypes. The whole-genome phylogeny indicated high genomic diversity among HUS-STEC strains. No distinct cluster was found between HUS and non-HUS STEC strains. The current study showed that O157:H7 remains the main cause of STEC-associated HUS, despite the rising importance of other non-O157 serotypes. Besides, O157:H7 is associated with severe renal sequelae in the follow-up, which could be a risk factor for long-term prognosis in HUS patients.

The human colonic mucus is mainly composed of mucins, which are highly glycosylated proteins. The normal commensal colonic microbiota has mucolytic activity and is capable of releasing the monosaccharides contained in mucins, which can then be used as carbon sources by pathogens such as Enterohemorrhagic Escherichia coli (EHEC). EHEC can regulate the expression of some of its virulence factors through environmental sensing of mucus-derived sugars, but its implications regarding its main virulence factor, Shiga toxin type 2 (Stx2), among others, remain unknown. In the present work, we have studied the effects of five of the most abundant mucolytic activity-derived sugars, Fucose (L-Fucose), Galactose (D-Galactose), N-Gal (N-acetylgalactosamine), NANA (N-Acetyl-Neuraminic Acid) and NAG (N-Acetyl-D-Glucosamine) on EHEC growth, adhesion to epithelial colonic cells (HCT-8), and Stx2 production and translocation across a polarized HCT-8 monolayer. We found that bacterial growth was maximum when using NAG and NANA compared to Galactose, Fucose or N-Gal, and that EHEC adhesion was inhibited regardless of the metabolite used. On the other hand, Stx2 production was enhanced when using NAG and inhibited with the rest of the metabolites, whilst Stx2 translocation was only enhanced when using NANA, and this increase occurred only through the transcellular route. Overall, this study provides insights on the influence of the commensal microbiota on the pathogenicity of E. coli O157:H7, helping to identify favorable intestinal environments for the development of severe disease. [ABSTRACT FROM AUTHOR]

This study was conducted to assess the survival of 2 wild Shiga toxin-producing Escherichia coli strains (one serotype O157:H7 and one non-O157:H7) in ewe milk stored at different conditions and to examine the fate of the O157 strain during the manufacture and ripening of a Spanish sheep hard variety of raw milk cheese (Zamorano). The strains were selected among a population of 50 isolates, which we obtained from ewe milk, because of their high resistance to 0.3% lactic acid. Both strains were inoculated (approximately 2 log 10 cfu/mL) in raw and heat-treated (low-temperature holding, LTH; 63°C/30 min) ewe milk and stored for 5 d at 6, 8, and 10°C and also according to a simulation approach for assessing the effects of failures in the cold chain. The minimum growth temperature for the O157:H7 strain in LTH and raw ewe milk was 8°C. For the non-O157:H7 strain, the lowest temperature showing bacterial growth in LTH ewe milk was 6°C, but it did not grow at any of the tested conditions in raw milk. It appears that the O157 strain was more susceptible to cold stress but was likely a better competitor than the non-O157 strain against the milk autochthonous microbiota. For manufacture of Zamorano cheese, raw milk was inoculated with approximately 3 log 10 cfu/mL, and after 2 mo of ripening at 10 to 12°C, the cheeses showed the expected general characteristics for this variety. The O157:H7 strain increased 0.9 log 10 cfu/g after whey drainage and during ripening and storage decreased by 2.9 log 10 cfu/g. Nevertheless, its detectable level (estimated at 6.2 cfu/g) after 2 mo of ripening suggests that Zamorano cheese manufactured from raw ewe milk contaminated with E. coli O157:H7 could represent a public health concern. [ABSTRACT FROM AUTHOR]

Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms.IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.

The human colonic mucus is mainly composed of mucins, which are highly glycosylated proteins. The normal commensal colonic microbiota has mucolytic activity and is capable of releasing the monosaccharides contained in mucins, which can then be used as carbon sources by pathogens such as Enterohemorrhagic Escherichia coli (EHEC). EHEC can regulate the expression of some of its virulence factors through environmental sensing of mucus-derived sugars, but its implications regarding its main virulence factor, Shiga toxin type 2 (Stx2), among others, remain unknown. In the present work, we have studied the effects of five of the most abundant mucolytic activity-derived sugars, Fucose (L-Fucose), Galactose (D-Galactose), N-Gal (N-acetyl-galactosamine), NANA (N-Acetyl-Neuraminic Acid) and NAG (N-Acetyl-D-Glucosamine) on EHEC growth, adhesion to epithelial colonic cells (HCT-8), and Stx2 production and translocation across a polarized HCT-8 monolayer. We found that bacterial growth was maximum when using NAG and NANA compared to Galactose, Fucose or N-Gal, and that EHEC adhesion was inhibited regardless of the metabolite used. On the other hand, Stx2 production was enhanced when using NAG and inhibited with the rest of the metabolites, whilst Stx2 translocation was only enhanced when using NANA, and this increase occurred only through the transcellular route. Overall, this study provides insights on the influence of the commensal microbiota on the pathogenicity of E. coli O157:H7, helping to identify favorable intestinal environments for the development of severe disease.

• Antimicrobial properties of CNMA against EHEC bacteria were determined. • CNMA antibacterial effect was resolved on physiological and gene expression level. • CNMA activity induce (p)ppGpp alarmones synthesis via RelA function. • Elevated (p)ppGpp attenuate Stx prophage development and Shiga toxin synthesis. • CNMA antimicrobial activity in vivo was evaluated in G. mellonella. Compounds of natural origin are potent source of drugs with unique mechanisms of action. Among phytochemicals, trans -cinnamaldehyde (t- CA) exhibits a wide range of biological activity, thus has been used for centuries to fight bacterial and fungal infections. However, the molecular basis of these properties has not been fully covered. Considering that difficult-to-control infections are becoming a rising global problem, there is a need to elucidate the molecular potential of t- CA. To evaluate the antibacterial activity of t -CA against Shiga-toxigenic E. coli strains and elucidate its mechanism of action based on the inhibition of the virulence factor expression. The antimicrobial potential of t -CA was assessed with two-fold microdilution and time-kill assays. Further evaluation included bioluminescence suppression assays, quantification of reactive oxygen species (ROS) and assessment of NAD+/NADH ratios. Morphological changes post t- CA exposure were examined using transmission electron microscopy. RNA sequencing and radiolabeling of nucleotides elucidated the metabolic alterations induced by t- CA. Toxin expression level was monitored through the application of fusion proteins, monitoring of bacteriophage development, and fluorescence microscopy studies. Lastly, the therapeutic efficacy in vivo was assessed using Galleria mellonella infection model. A comprehensive study of t- CA's bioactivity showed unique properties affecting bacterial metabolism and morphology, resulting in significant bacterial cell deformation and effective virulence inhibition. Elucidation of the underlying mechanisms indicated that t- CA activates the global regulatory system, the stringent response, manifested by its alarmone, (p)ppGpp, overproduction mediated by the RelA enzyme, thereby inhibiting bacterial proliferation. Intriguingly, t- CA effectively downregulates Shiga toxin gene expression via alarmone molecules, indicating its potential for therapeutic effect. In vivo validation demonstrated a significant improvement in larval survival rates post- t- CA treatment with 50 mg/kg (p < 0.05), akin to the efficacy observed with azithromycin, thus indicating its effectiveness against EHEC infections (p < 0.05). Collectively, these results reveal the robust antibacterial capabilities of t- CA, warranting its further exploration as a viable anti-infective agent. [Display omitted] [ABSTRACT FROM AUTHOR]

A laboratory-acquired E. coli O157:H7 infection with associated severe sequelae including hemolytic uremic syndrome occurred in an individual working in the laboratory with a mixture of nalidixic acid-resistant (NalR) O157:H7 mutant strains in a soil-biochar blend. The patient was hospitalized and treated with an intravenous combination of metronidazole and levofloxacin. The present study investigated the source of this severe laboratory acquired infection and further examined the influence of the antibiotics used during treatment on the expression and production of Shiga toxin. Genomes of two Stx2a-and eae -positive O157:H7 strains isolated from the patient's stool were sequenced along with two pairs of the wt strains and their derived NalR mutants used in the laboratory experiments. High-resolution SNP typing determined the strains' individual genetic relatedness and unambiguously identified the two laboratory-derived NalR mutant strains as the source of the researcher's life-threatening disease, rather than a conceivable ingestion of unrelated O157:H7 isolates circulating at the same time. It was further confirmed that in sublethal doses, the antibiotics increased toxin expression and production. Our results support a simultaneous co-infection with clinical strains in the laboratory, which were the causative agents of previous O157:H7 outbreaks, and further that the administration of antibiotics may have impacted the outcome of the infection. [ABSTRACT FROM AUTHOR]

A laboratory-acquired E. coli O157:H7 infection with associated severe sequelae including hemolytic uremic syndrome occurred in an individual working in the laboratory with a mixture of nalidixic acid-resistant (NalR) O157:H7 mutant strains in a soil-biochar blend. The patient was hospitalized and treated with an intravenous combination of metronidazole and levofloxacin. The present study investigated the source of this severe laboratory acquired infection and further examined the influence of the antibiotics used during treatment on the expression and production of Shiga toxin. Genomes of two Stx2a-and eae-positive O157:H7 strains isolated from the patient’s stool were sequenced along with two pairs of the wt strains and their derived NalR mutants used in the laboratory experiments. High-resolution SNP typing determined the strains’ individual genetic relatedness and unambiguously identified the two laboratory-derived NalR mutant strains as the source of the researcher’s life-threatening disease, rather than a conceivable ingestion of unrelated O157:H7 isolates circulating at the same time. It was further confirmed that in sublethal doses, the antibiotics increased toxin expression and production. Our results support a simultaneous co-infection with clinical strains in the laboratory, which were the causative agents of previous O157:H7 outbreaks, and further that the administration of antibiotics may have impacted the outcome of the infection.

Prophage-encoded Escherichia coli O157:H7 transcription factor (TF), PchE, inhibits biofilm formation and attachment to cultured epithelial cells by reducing curli fimbriae expression and increasing flagella expression. To identify pchE regulators that might be used in intervention strategies to reduce environmental persistence or host infections, we performed a computational search of O157:H7 strain PA20 pchE promoter sequences for binding sites used by known TFs. A common site shared by MarA/SoxS/Rob TFs was identified and the typical MarA/Rob inducers, salicylate and decanoate, were tested for biofilm and motility effects. Sodium salicylate, a proven biofilm inhibitor, but not sodium decanoate, strongly reduced O157:H7 biofilms by a pchE-independent mechanism. Both salicylate and decanoate enhanced O157:H7 motility dependent on pchE using media and incubation temperatures optimum for culturing human epithelial cells. However, induction of pchE by salicylate did not activate the SOS response. MarA/SoxS/Rob inducers provide new potential agents for controlling O157:H7 interactions with the host and its persistence in the environment. [ABSTRACT FROM AUTHOR]

Cattle are carriers, without clinical manifestations, of enterohemorrhagic Escherichia coli (EHEC) O157:H7 responsible for life-threatening infections in humans. A better identification of factors playing a role in maintaining persistence of such strains in cattle is required to develop more effective control measures. Hence, we conducted a study to identify farms with a persistent circulation of EHEC O157:H7. The EHEC O157:H7 herd status of 13 farms, which had previously provided bovine EHEC O157:H7 carriers at slaughter was investigated. Two farms were still housing positive young bulls, and this was true over a 1-year period. Only one fecal sample could be considered from a supershedder, and 60% of the carriers shed concentrations below 10 MPN/g. Moreover, EHEC O157:H7 represented minor subpopulations of E. coli. PFGE analysis of the EHEC O157:H7 strains showed that persistent circulation was due either to the persistence of a few predominant strains or to the repeated exposure of cattle to various strains. Finally, we compared fecal microbial communities of shedders (S) (n = 24) and non-shedders (NS) (n = 28), including 43 young bulls and nine cows, from one farm. Regarding alpha diversity, no significant difference between S vs. NS young bulls (n = 43) was observed. At the genus level, we identified 10 amplicon sequence variant (ASV) indicators of the S or NS groups. The bacterial indicators of S belonged to the family XIII UCG-001, Slackia, and Campylobacter genera, and Ruminococcaceae NK4A21A, Lachnospiraceae-UGC-010, and Lachnospiraceae-GCA-900066575 groups. The NS group indicator ASVs were affiliated to Pirellulaceae-1088-a5 gut group, Anaerovibrio, Victivallis, and Sellimonas genera. In conclusion, the characteristics enhancing the persistence of some predominant strains observed here should be explored further, and studies focused on mechanisms of competition among E. coli strains are also needed.

Shiga toxin-producing Escherichia coli, a foodborne bacterial pathogen, has been linked to a broad spectrum of clinical outcomes ranging from asymptomatic carriage to fatal hemolytic uremic syndrome (HUS). Here, we collected clinical data and STEC strains from HUS patients from 1994 through 2018, whole-genome sequencing was performed to molecularly characterize HUS-associated STEC strains, statistical analysis was conducted to identify bacterial genetic factors associated with severe outcomes in HUS patients. O157:H7 was the most predominant serotype (57%) among 54 HUS-associated STEC strains, followed by O121:H19 (19%) and O26:H11 (7%). Notably, some non-predominant serotypes such as O59:H17 (2%) and O109:H21 (2%) also caused HUS. All O157:H7 strains with one exception belonged to clade 8. During follow-up at a median of 4 years, 41% of the patients had renal sequelae. Fifty-nine virulence genes were found to be statistically associated with severe renal sequelae, these genes encoded type II and type III secretion system effectors, chaperones, and other factors. Notably, virulence genes associated with severe clinical outcomes were significantly more prevalent in O157:H7 strains. In contrast, genes related to mild symptoms were evenly distributed across all serotypes. The whole-genome phylogeny indicated high genomic diversity among HUS-STEC strains. No distinct cluster was found between HUS and non-HUS STEC strains. The current study showed that O157:H7 remains the main cause of STEC-associated HUS, despite the rising importance of other non-O157 serotypes. Besides, O157:H7 is associated with severe renal sequelae in the follow-up, which could be a risk factor for long-term prognosis in HUS patients. [ABSTRACT FROM AUTHOR]

Abstract Background Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC O157 encounters varied microenvironments during infection, and can efficiently adapt to these using the two-component system (TCS). Recently, a functional TCS, RstAB, has been implicated in the regulation of virulence of several bacterial pathogens. However, the regulatory function of RstAB in EHEC O157 is poorly understood. This study aimed at providing insights into the global effects of RstA on gene expression in EHEC O157. Results In the present study, we analyzed gene expression differences between the EHEC O157 wild-type strain and a ΔrstA mutant using RNA-seq technology. Genes with differential expression in the ΔrstA mutant compared to that in the wild-type strain were identified and grouped into clusters of orthologous categories. RstA promoted EHEC O157 LEE gene expression, adhesion in vitro, and colonization in vivo by indirect regulation. We also found that RstA could bind directly to the promoter region of hdeA and yeaI to enhance acid tolerance and decrease biofilm formation by modulating the concentration of c-di-GMP. Conclusions In summary, the RstAB TCS in EHEC O157 plays a major role in the regulation of virulence, acid tolerance, and biofilm formation. We clarified the regulatory function of RstA, providing an insight into mechanisms that may be potential drug targets for treatment of EHEC O157-related infections.

Abstract Background Shiga toxin-producing Escherichia coli (STEC) are emerging foodborne pathogens that are public health concern. Cattle have been identified as the major STEC reservoir. In the present study, we investigated the prevalence and characteristics of STEC strains in beef cattle from a commercial farm in Sichuan province, China. Results Among 120 beef cattle fecal samples, stx genes were positive in 90% of samples, as assessed using TaqMan real-time PCR, and 87 (72.5%) samples were confirmed to yield at least one STEC isolate by culture using four selective agars, MacConkey, CHROMagar™ ECC, modified Rainbow® Agar O157, and CHROMagar™ STEC, from which 31, 32, 91, and 73 STEC strains were recovered, respectively. A total of 126 STEC isolates were selected and further characterized. Seventeen different O:H serotypes were identified, all of which belonged to the non-O157 serotypes. One stx 1 subtype (stx 1a) and three stx 2 subtypes (stx 2a, stx 2c, and stx 2d) were present among these isolates. The intimin encoding gene eae, and other adherence-associated genes (iha, saa, and paa) were present in 37, 125, 74, and 30 STEC isolates, respectively. Twenty-three isolates carried the virulence gene subA, and only one harbored both cnf1 and cnf2 genes. Three plasmid-origin virulence genes (ehxA, espP, and katP) were present in 111, 111, and 7 isolates, respectively. The 126 STEC isolates were divided into 49 pulsed-field gel electrophoresis (PFGE) patterns. Conclusions Our study showed that the joint use of the selective MacConkey and modified Rainbow® Agar O157 agars increased the recovery frequency of non-O157 STEC strains in animal feces, which could be applied to other samples and in regular STEC surveillance. Moreover, the results revealed high genetic diversity of non-O157 STEC strains in beef cattle, some of which might have the potential to cause human diseases.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 (O157) is a major foodborne pathogen that causes severe illness in humans worldwide. The genome of O157 contains 177 genomic islands known as O islands (OIs), including Shiga toxin-converting phages (OI-45 and OI-93) and the locus for enterocyte effacement (LEE) pathogenicity island (OI-148). However, most genes in OIs are uncharacterized and code for unknown functions. In this study, we demonstrated, for the first time, that OI-9 encodes a novel transcriptional activator, Z0346 (named OvrB), which is required for bacterial adherence to host cells and LEE gene expression in O157. OvrB directly binds to the promoter region of LEE1 and activates the transcription of ler (encoding a master regulator of LEE genes), which in turn activates LEE1–5 genes to promote O157 adherence. Furthermore, mouse oral infection assays showed that OvrB promotes O157 colonization in the mouse intestine. Finally, OvrB is shown to be a widespread transcriptional activator of virulence genes in other enterohemorrhagic and enteropathogenic Escherichia coli serotypes. Our work significantly expands the understanding of bacterial virulence control and provides new evidence suggesting that horizontally transferred regulator genes mediate LEE gene expression.

Super-shed (SS) Escherichia coli O157 (E. coli O157) demonstrate a strong, aggregative, locus of enterocyte effacement (LEE)-independent adherence phenotype on bovine recto-anal junction squamous epithelial (RSE) cells, and harbor polymorphisms in non-LEE-adherence-related loci, including in the type 1 fimbriae operon. To elucidate the role of type 1 fimbriae in strain- and host-specific adherence, we evaluated the entire Fim operon (FimB-H) and its adhesion (FimH) deletion mutants in four E. coli O157 strains, SS17, SS52, SS77 and EDL933, and evaluated the adherence phenotype in bovine RSE and human HEp-2 adherence assays. Consistent with the prevailing dogma that fimH expression is genetically switched off in E. coli O157, the ΔfimHSS52, ΔfimB-HSS52, ΔfimB-HSS17, and ΔfimHSS77 mutants remained unchanged in adherence phenotype to RSE cells. In contrast, the ΔfimHSS17 and ΔfimB-HSS77 mutants changed from a wild-type strong and aggregative, to a moderate and diffuse adherence phenotype, while both ΔfimHEDL933 and ΔfimB-HEDL933 mutants demonstrated enhanced binding to RSE cells (p < 0.05). Additionally, both ΔfimHSS17 and ΔfimHEDL933 were non-adherent to HEp-2 cells (p < 0.05). Complementation of the mutant strains with their respective wild-type genes restored parental phenotypes. Microscopy revealed that the SS17 and EDL933 strains indeed carry type 1 fimbriae-like structures shorter than those seen in uropathogenic E. coli. Taken together, these results provide compelling evidence for a strain and host cell type-dependent role of fimH and the fim operon in E. coli O157 adherence that needs to be further evaluated.

A total of 482 veal cutlet, 555 ground veal, and 540 ground beef samples were purchased from retail establishments in the mid-Atlantic region of the United States over a noncontiguous 2-year period between 2014 and 2017. Samples (325 g each) were individually enriched and screened via real-time PCR for all seven regulated serogroups of Shiga toxin–producing Escherichia coli (STEC). Presumptive STEC-positive samples were subjected to serogroup-specific immunomagnetic separation and plated onto selective media. Up to five isolates typical for STEC from each sample were analyzed via multiplex PCR for both the virulence genes (i.e., eae, stx1 and/or stx2, and ehxA) and serogroup-specific gene(s) for the seven regulated STEC serogroups. The recovery rates of non-O157 STEC from veal cutlets (3.94%, 19 of 482 samples) and ground veal (7.03%, 39 of 555 samples) were significantly higher (P < 0.05) than that from ground beef (0.93%, 5 of 540 samples). In contrast, only a single isolate of STEC O157:H7 was recovered; this isolate originated from 1 (0.18%) of 555 samples of ground veal. Recovery rates for STEC were not associated with state, season, packaging type, or store type (P > 0.05) but were associated with brand and fat content (P < 0.05). Pulsed-field subtyping of the 270 viable and confirmed STEC isolates from the 64 total samples testing positive revealed 78 pulsotypes (50 to 80% similarity) belonging to 39 pulsogroups, with ≥90% similarity among pulsotypes within pulsogroups. Multiple isolates from 43 (67.7%) of 64 samples testing positive had an indistinguishable pulsotype. STEC serotypes O26 and O103 were the most prevalent serogroups in beef and veal, respectively. These findings support related findings from regulatory sampling studies over the past decade and confirm that recovery rates for the regulated STEC serogroups are higher for raw veal than for raw beef samples, as was observed in the present study of meat purchased at food retailers in the mid-Atlantic region of the United States. Higher recovery rates of regulated STEC serogroups were found in raw veal than in beef. Recovery of STEC was not associated with state, season, packaging, or store type. Recovery of STEC was associated with brand and fat content. The most prevalent STEC serogroups in beef and veal were O26 and O103, respectively. STEC with the same pulsotype had the same virulence profile. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli is an important pathogen and microorganism of the normal intestinal flora of humans and animals. One of the important serotypes of E. coli is O157: H7. Because of the excessive and arbitrary use of antibiotics, multiple drug resistance has increased against these organisms. The main problem in treating infections caused by E. coli is its dependence on the administration of a large number of common antibiotics and the resistance of some strains to antibiotics. Phage therapy refers to the therapeutic use of phages to eliminate bacterial infections. In the first step, it is necessary to separate and identify bacteriophages that affect the target bacteria. Therefore, the present study was performed to isolate the phage that was effective on enterohemorrhagic E. coli isolates. Materials & Methods: In this study, after collection of sewage samples, bacteriophages were isolated by filtration and enrichment in an enterohemorrhagic E. coli overnight culture. The presence of bacteriophage was detected by plaque observation in a double layer agar and confirmed by TEM electron microscopy. Results and Conclusion: The results of the observation with electron microscopy revealed the presence of bacteriophage with the appearance of the Cystoviridae, Myoviridae and Podoviridae families. Unfortunately, although the titration of phages and molecular study were not performed in the current study due to the lack of budget, we found antibacterial activity of isolated phages using plaque formation observation, and the presence of phages belonging to Cystoviridae, Myoviridae and Podoviridae families was confirmed by TEM microscopy. Therefore, the effective phage against O157: H7 was successfully identified, isolated, and purified. [ABSTRACT FROM AUTHOR]

Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC , the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance., Competing Interests: The authors declare no conflict of interest.

In this study we compared nine Shiga toxin (Stx)-producing Escherichia coli O157:H7 patient isolates for Stx levels, stx-phage insertion site(s), and pathogenicity in a streptomycin (Str)-treated mouse model. The strains encoded stx2a, stx1a and stx2a, or stx2a and stx2c. All of the strains elaborated 105-106 cytotoxic doses 50% (CD50) into the supernatant after growth in vitro as measured on Vero cells, and showed variable levels of increased toxin production after growth with sub-inhibitory levels of ciprofloxacin (Cip). The stx2a+stx2c+ isolates were 90–100% lethal for Str-treated BALB/c mice, though one isolate, JH2013, had a delayed time-to-death. The stx2a+ isolate was avirulent. Both an stx2a and a recA deletion mutant of one of the stx2a+stx2c+ strains, JH2010, exhibited at least a three-log decrease in cytotoxicity in vitro and both were avirulent in the mice. Stool from Str-treated mice infected with the highly virulent isolates were 10- to 100-fold more cytotoxic than feces from mice infected with the clinical isolate, JH2012, that made only Stx2a. Taken together these findings demonstrate that the stx2a-phage from JH2010 induces to higher levels in vivo than does the phage from JH2012. The stx1a+stx2a+ clinical isolates were avirulent and neutralization of Stx1 in stool from mice infected with those strains indicated that the toxin produced in vivo was primarily Stx1a. Treatment of mice infected with Stx1a+Stx2a+ isolates with Cip resulted in an increase in Stx2a production in vivo and lethality in the mice. Our data suggest that high levels of Stx2a in stool are predictive of virulence in mice.

Abstract Background Rapid and accurate identification of Verotoxigenic Escherichia coli (VTEC) O157:H7 is dependent on well-established, standardized and highly discriminatory typing methods. Currently, conventional subtyping tests for foodborne bacterial pathogen surveillance are rapidly being replaced with whole-genome sequencing (WGS) in public health laboratories. The capacity of WGS to revolutionize global foodborne disease surveillance has positioned this tool to become the new gold standard; however, to ensure evidence standards for public health decision making can still be achieved, the performance of WGS must be thoroughly validated against current gold standard methods prior to implementation. Here we aim to verify the performance of WGS in comparison to pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) for eight retrospective outbreaks of VTEC O157:H7 from the Canadian perspective. Since real-time implementation and routine use of WGS in public health laboratories is highly reliant on standardized data analysis tools, we also provide a comparative analysis of two popular methodologies for WGS analyses; an in-house developed single nucleotide variant phylogenomics (SNVPhyl) pipeline and the BioNumerics whole genome multilocus sequence typing (wgMLST) tool. To provide a useful and consistent starting point for examining laboratory-based surveillance data for VTEC O157:H7 in Canada, we also aim to describe the number of genetic differences observed among outbreak-associated isolates. Results WGS provided enhanced resolution over traditional subtyping methods, and accurately distinguished outbreak-related isolates from non-outbreak related isolates with high epidemiological concordance. WGS also illuminated potential linkages between sporadic cases of illness and contaminated food, and isolates spanning multiple years. The topologies generated by SNVPhyl and wgMLST were highly congruent with strong statistical support. Few genetic differences were observed among outbreak-related isolates (≤5 SNVs/

Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157: H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stxconverting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains. [ABSTRACT FROM AUTHOR]

Signs and symptoms of Shiga toxin-producing Escherichia coli (STEC) serogroup O157:H7 infection range from mild gastrointestinal to bloody diarrhea and hemolytic uremic syndrome (HUS). We assessed the association between Shiga toxin gene (stx) subtype and disease severity for »3,000 patients with STEC O157:H7 in England during 2009-2019. Odds of bloody diarrhea, HUS, or both, were significantly higher for patients infected with STEC O157:H7 possessing stx2a only or stx2a combined with other stx subtypes. Odds of severe signs/symptoms were significantly higher for isolates encoding stx2a only and belonging to sublineage Ic and lineage I/II than for those encoding stx2a only and belonging to sublineage IIb, indicating that stx2a is not the only driver causing HUS. Strains of STEC O157:H7 that had stx1a were also significantly more associated with severe disease than strains with stx2c only. This finding confounds public health risk assessment algorithms based on detection of stx2 as a predictor of severe disease. [ABSTRACT FROM AUTHOR]

Intestinal inflammation can cause impaired epithelial barrier function and disrupt immune homeostasis, which increases the risks of developing many highly fatal diseases. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes intestinal infections worldwide and is a major pathogen that induces intestinal inflammation. Various antibacterial peptides have been described as having the potential to suppress and treat pathogen-induced intestinal inflammation. Cecropin A (1–8)-LL37 (17–30) (C-L), a novel hybrid peptide designed in our laboratory that combines the active center of C with the core functional region of L, shows superior antibacterial properties and minimized cytotoxicity compared to its parental peptides. Herein, to examine whether C-L could inhibit pathogen-induced intestinal inflammation, we investigated the anti-inflammatory effects of C-L in EHEC O157:H7-infected mice. C-L treatment improved the microbiota composition and microbial community balance in mouse intestines. The hybrid peptide exhibited improved anti-inflammatory effects than did the antibiotic, enrofloxacin. Hybrid peptide treated infected mice demonstrated reduced clinical signs of inflammation, reduced weight loss, reduced expression of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interferon-gamma (IFN-γ)], reduced apoptosis, and reduced markers of jejunal epithelial barrier function. The peptide also affected the MyD88–nuclear factor κB signaling pathway, thereby modulating inflammatory responses upon EHEC stimulation. Collectively, these findings suggest that the novel hybrid peptide C-L could be developed into a new anti-inflammatory agent for use in animals or humans. [ABSTRACT FROM AUTHOR]

In this study we compared nine Shiga toxin (Stx)-producing Escherichia coli O157:H7 patient isolates for Stx levels, stx -phage insertion site(s), and pathogenicity in a streptomycin (Str)-treated mouse model. The strains encoded stx 2a, stx 1a and stx 2a, or stx 2a and stx 2c. All of the strains elaborated 105-106 cytotoxic doses 50% (CD50) into the supernatant after growth in vitro as measured on Vero cells, and showed variable levels of increased toxin production after growth with sub-inhibitory levels of ciprofloxacin (Cip). The stx 2a+ stx 2c+ isolates were 90–100% lethal for Str-treated BALB/c mice, though one isolate, JH2013, had a delayed time-to-death. The stx 2a+ isolate was avirulent. Both an stx 2a and a recA deletion mutant of one of the stx 2a+ stx 2c+ strains, JH2010, exhibited at least a three-log decrease in cytotoxicity in vitro and both were avirulent in the mice. Stool from Str-treated mice infected with the highly virulent isolates were 10- to 100-fold more cytotoxic than feces from mice infected with the clinical isolate, JH2012, that made only Stx2a. Taken together these findings demonstrate that the stx 2a-phage from JH2010 induces to higher levels in vivo than does the phage from JH2012. The stx 1a+ stx 2a+ clinical isolates were avirulent and neutralization of Stx1 in stool from mice infected with those strains indicated that the toxin produced in vivo was primarily Stx1a. Treatment of mice infected with Stx1a+Stx2a+ isolates with Cip resulted in an increase in Stx2a production in vivo and lethality in the mice. Our data suggest that high levels of Stx2a in stool are predictive of virulence in mice. [ABSTRACT FROM AUTHOR]

ABSTRACT Here we exploit the natural properties of a synthetic nanoparticle (NP) scaffold as a subunit vaccine against enterohemorrhagic Escherichia coli (EHEC). Two EHEC-specific immunogenic antigens, namely, LomW and EscC, either alone or in combination, were covalently linked on the surface of gold nanoparticles (AuNPs) and used to immunize mice prior to challenge with EHEC O157:H7 strain 86-24. LomW is a putative outer membrane protein encoded in bacteriophage BP-933W, while EscC is a structural type III secretion system protein which forms a ring in the outer membrane. The resulting AuNP preparations, AuNP-LomW and AuNP-EscC, showed that the nanoparticles were able to incorporate the antigens, forming stable formulations that retained robust immunogenicity in vivo after subcutaneous immunization. When administered subcutaneously, AuNP-LomW or AuNP-EscC or a combination containing equivalent amounts of both candidates resulted in higher IgG titers in serum and secretory IgA titers in feces. The serum IgG titers correlated with a significant reduction in EHEC intestinal colonization after 3 days postinoculation. In addition, we showed that serum from antigen-coated AuNP-immunized mice resulted in a reduction of adherence to human intestinal epithelial cells for EHEC, as well as for two other E. coli pathotypes (enteropathogenic E. coli [EPEC], encoding EscC, and enteroaggregative E. coli [EAEC], encoding LomW). Further, the serum had antigen-specific bactericidal properties, engaging the classical complement pathway. Overall, our results demonstrate the immunogenicity and stability of a novel nanovaccine against EHEC. These results also strengthen the prospect of development of a synthetic nanoparticle vaccine conjugated to E. coli antigens as a promising platform against other enteric pathogens. IMPORTANCE Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis, which can progress to hemolytic uremic syndrome. These complications represent a serious global public health problem that requires laborious public health interventions and safety control measures to combat recurrent outbreaks worldwide. Today, there are no effective interventions for the control of EHEC infections, and, in fact, the use of antibiotics is counterindicated for EHEC disease. Therefore, a viable alternative for the prevention of human infections is the development of vaccines; however, no such vaccines are approved for human use. In this study, we developed a novel gold nanoparticle platform which acts as a scaffold for the delivery of various antigens, representing a nanovaccine technology which can be applied to several disease models.

The effect of UV-C radiation technique on survival of Escherichia coli O157:H7 inoculated the freshwater Nile tilapia (Oreochromis niloticus) and marine Japanese threadfin bream (Nemipterus japonicas) fillets were investigated. Tilapia samples (average weight and length are 150 ± 10 g, 15 ± 5 cm, respectively) and threadfin bream (average weight and length are 120 ± 5 g and 15 ± 2 cm, respectively) were purchased from local fish market in 2018 and filleted. The fish fillet samples were inoculated with Escherichia coli O157:H7 strain, and subjected to UV treatment (15 W) at four different distances (12, 24, 30 and 45 cm) from the UV lamps that emitted monochromatic UV irradiation at 253.7 nm. Each UV-C radiation treatment was performed for 5, 10, 15, and 20 min. Results showed that the maximum reduction of E. coli O157:H7 of 1.82 logs CFU/g in threadfin bream samples treated for 20 min treatment at 45 cm. whereas in tilapia samples maximum reduction of E. coli O157:H7 was 1.1 logs CFU/g treated for 15 min at 24 cm. In conclusion, this study recommends that UV-C radiation could be used as a successful technique to improve the safety of fish fillets from fresh water and marine sources. [ABSTRACT FROM AUTHOR]

We have reported that bicarbonate (NaHCO3) potentiates the activity of aminoglycosides in Escherichia coli, but the action mechanism was not identified. To eventually understand how NaHCO3 can potentiate antibiotics, we thought that a rational first step was to examine the effect of NaHCO3 separately and to inspect initial gene expression changes triggered by it. In this work, we started by confirming that NaHCO3 can reduce the number of viable E. coli bacteria. We then investigated, via RNAseq, gene expression changes induced by NaHCO3. There were upregulated and downregulated genes, among the top upregulated genes c. 10‐fold increase in expression) was tnaA, the gene encoding tryptophanase, the enzyme that degrades tryptophan to indole. Considering that higher expression of tnaA likely led to increases in indole, we tested the effect of indole and found both growth inhibition and synergy with NaHCO3. We suggest that indole may participate in growth inhibition of E. coli. The RNAseq analysis also revealed upregulation (≥4‐fold) of genes encoding proteins for the acquisition of iron and downregulation (≥16‐fold) of genes encoding iron‐sulphur‐holding proteins; hence NaHCO3 apparently triggered also an iron‐deficit response. We suggest that iron deficiency may also be involved in growth inhibition by NaHCO3. Significance and Impact of the Study: Bicarbonate (NaHCO3) can enhance the activity of various antibiotics. This work investigated its action mechanism. We carried out a transcriptional analysis in Escherichia coli with the aim of defining initial bacterial changes potentially linked to the enhancing activity of NaHCO3. Our approach differed from the longer term exposure to NaHCO3 recently used by other researchers, who noticed changes in the bacterial proton motive force. Based on our analysis, we propose two routes possibly linked to the effect of NaHCO3. Conceivably, those routes are potential targets that could be manipulated by alternative means to augment the effect of antibiotics. Significance and Impact of the Study: Bicarbonate (NaHCO3) can enhance the activity of various antibiotics. This work investigated its action mechanism. We carried out a transcriptional analysis in Escherichia coli with the aim of defining initial bacterial changes potentially linked to the enhancing activity of NaHCO3. Our approach differed from the longer term exposure to NaHCO3 recently used by other researchers, who noticed changes in the bacterial proton motive force. Based on our analysis, we propose two routes possibly linked to the effect of NaHCO3. Conceivably, those routes are potential targets that could be manipulated by alternative means to augment the effect of antibiotics. [ABSTRACT FROM AUTHOR]

We identified the mucus-activatable Shiga toxin genotype stx2d in the most common hemolytic uremic syndrome–associated Escherichia coli serotype, O157:H7. stx2d was detected in a strain isolated from a 2-year-old boy with bloody diarrhea in Spain, and whole-genome sequencing was used to confirm and fully characterize the strain.

Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E. coli responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coli O157:H7 outside the bovine gut. The E. coli O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coli MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra-intestinal environments was analyzed. E. coli MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 15°C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E. coli MC2 possessed the genetic information encoding 14 adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HCP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E. coli MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coli strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15°C. E. coli MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coli to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coli MC2 in the farm environment.

مقدمه: اغلب سویه‌های اشریشیاکلی می‌تواند موجب بیماری‌های روده‌ای و خارج روده‌ای شوند. بیماری‌های ناشی از Enterohemorragic Escherichia coli (EHEC) سویه‌ی O157:H7 رو به افزایش است؛ به طوری که امروزه، در کشورهای توسعه یافته، به عنوان سومین عامل ایجاد اسهال قرار گرفته است. هدف از این مطالعه، بررسی ژن‌های حدت eae، hly و 7flich‌ در باکتری اشریشیاکلی جدا شده از نمونه‌های اسهال بود. روش‌ها: پس از جمع آوری 55 نمونه‌ی اسهال انسانی از بیمارستان‌های شهر تهران، آزمایش‌های مختلف میکروبی و شیمیایی انجام و باکتری اشریشیاکلی جداسازی و DNA جدایه‌ها با استفاده از روش Multiplex polymerase chain reaction (Multiplex PCR) استخراج گردید. یافته‌ها: از مجموع 55 نمونه‌ی انسانی، 34 نمونه (8/61 درصد) دارای ژن 7flich، 11 نمونه (0/20 درصد) دارای ژنhly و 4 نمونه (2/7 درصد) دارای ژن eaeA بود. نتیجه‌گیری: تفاوت مشاهده شده در میزان شیوع در این تحقیق نسبت به سایر تحقیقات می‌تواند به علت سن افراد، نوع نمونه، محل جغرافیایی، نوع رژیم غذایی، روش‌های شناسایی و جدا سازی باکتری، وضعیت بهداشت فردی و حتی فصول سال باشد.

Background: Rapid and accurate identification of Verotoxigenic Escherichia coli (VTEC) O157:H7 is dependent on well-established, standardized and highly discriminatory typing methods. Currently, conventional subtyping tests for foodborne bacterial pathogen surveillance are rapidly being replaced with whole-genome sequencing (WGS) in public health laboratories. The capacity of WGS to revolutionize global foodborne disease surveillance has positioned this tool to become the new gold standard; however, to ensure evidence standards for public health decision making can still be achieved, the performance of WGS must be thoroughly validated against current gold standard methods prior to implementation. Here we aim to verify the performance of WGS in comparison to pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) for eight retrospective outbreaks of VTEC O157:H7 from the Canadian perspective. Since real-time implementation and routine use of WGS in public health laboratories is highly reliant on standardized data analysis tools, we also provide a comparative analysis of two popular methodologies for WGS analyses; an in-house developed single nucleotide variant phylogenomics (SNVPhyl) pipeline and the BioNumerics whole genome multilocus sequence typing (wgMLST) tool. To provide a useful and consistent starting point for examining laboratory-based surveillance data for VTEC O157:H7 in Canada, we also aim to describe the number of genetic differences observed among outbreak-associated isolates. Results: WGS provided enhanced resolution over traditional subtyping methods, and accurately distinguished outbreak-related isolates from non-outbreak related isolates with high epidemiological concordance. WGS also illuminated potential linkages between sporadic cases of illness and contaminated food, and isolates spanning multiple years. The topologies generated by SNVPhyl and wgMLST were highly congruent with strong statistical support. Few genetic differences were observed among outbreak-related isolates (≤5 SNVs/ < 10 wgMLST alleles) unless the outbreak was suspected to be multi-strain. Conclusions: This study validates the superiority of WGS and indicates the BioNumerics wgMLST schema is suitable for surveillance and cluster detection of VTEC O157:H7. These findings will provide a useful and consistent starting point for examining WGS data for prospective laboratory-based surveillance of VTEC O157:H7, but however, the data will continue to be interpreted according to context and in combination with epidemiological and food safety evidence to inform public-health decision making in Canada. [ABSTRACT FROM AUTHOR]

Escherichia coli O157:H7 is a zoonotic pathogen of global importance and the serotype of Shiga toxin-producing E. coli (STEC) most frequently associated with Hemolytic Uremic Syndrome (HUS) in humans. The main STEC reservoir is cattle. Vaccination of calves with the carboxy-terminal fraction of Intimin γ (IntC280) and EspB can reduce E. coli O157:H7 fecal shedding after experimental challenge. Shiga toxin (Stx) exerts local immunosuppressive effects in the bovine intestine and Stx2B fused to Brucella lumazine synthase (BLS-Stx2B) induces Stx2-neutralizing antibodies. To determine if an immune response against Stx could improve a vaccine’s effect on fecal shedding, groups of calves were immunized with EspB + IntC280, with EspB + IntC280 + BLS-Stx2B, or kept as controls. At 24 days post vaccination calves were challenged with E. coli O157:H7. Shedding of E. coli O157:H7 was assessed in recto-anal mucosal swabs by direct plating and enrichment followed by immunomagnetic separation and multiplex PCR. Calves were euthanized 15 days after the challenge and intestinal segments were obtained to assess mucosal antibodies. Vaccination induced a significant increase of IntC280 and EspB specific antibodies in serum and intestinal mucosa in both vaccinated groups. Antibodies against Stx2B were detected in serum and intestinal mucosa of animals vaccinated with 3 antigens. Sera and intestinal homogenates were able to neutralize Stx2 verocytotoxicity compared to the control and the 2-antigens vaccinated group. Both vaccines reduced E. coli O157:H7 shedding compared to the control group. The addition of Stx2B to the vaccine formulation did not result in a superior level of protection compared to the one conferred by IntC280 and EspB alone. It remains to be determined if the inclusion of Stx2B in the vaccine alters E. coli O157:H7 shedding patterns in the long term and after recurrent low dose exposure as occurring in cattle herds. [ABSTRACT FROM AUTHOR]