Explore the vast range of titles available.

Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and seven Stx2 subtypes have been described in E. coli , which differed in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated Stx2h in E. coli strains isolated from wild marmots in the Qinghai-Tibetan plateau, China. Stx2h shares 91.9% nucleic acid sequence identity and 92.9% amino acid identity to the nearest Stx2 subtype. The expression of Stx2h in type strain STEC299 was inducible by mitomycin C, and culture supernatant from STEC299 was cytotoxic to Vero cells. The Stx2h converting prophage was unique in terms of insertion site and genetic composition. Whole genome-based phylo- and patho-genomic analysis revealed STEC299 was closer to other pathotypes of E. coli than STEC, and possesses virulence factors from other pathotypes. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of E. coli strains. As the emergence of new Shiga toxin genotypes and new Stx-producing pathotypes pose a great threat to the public health, Stx2h should be further included in E. coli molecular typing, and in epidemiological surveillance of E. coli infections.

Shiga toxins (Stx) produced by Shiga toxin-producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC) are ribosome-inactivating AB 5 proteins that consist of one enzymatic active A-subunit (StxA) and a pentamer of non-covalently linked B-subunits (StxB). The description of Stx as an AB 5 protein and the observation that A-subunits without their corresponding B-subunits also intoxicate eukaryotic cells, led to the question whether A- and B-subunits are produced in the bacteria in a 1:5 ratio or whether the A-subunit of the clinically most prominent subtype Stx2a is transcribed in excess revealing free A-subunits released in the bacterial environment. The aim of this study was therefore, to investigate the genetic and protein-based background for this observation in six Stx2a-encoding STEC and EHEC wildtype strains. For this purpose, transcriptional analysis of the Stx2a subunit genes, stxA2a and stxB2a , was performed by quantitative real-time PCR in one foodborne O113:H21 STEC isolate (strain TS18/08) and five HUS-associated EHEC strains with the serotypes O157:H7/H − (HUSEC003, HUSEC004), O103:H − (HUSEC008), O26:H11 (HUSEC018), and O104:H4 (LB226692). Contrary to the hypothesis that the A- and B-subunit genes are expressed in a ratio of 1:5 comparable to the holotoxin structure or in a ratio of 1:1 based on the operon structure, the results showed that stxA2a was expressed 1.90 ± 0.55-times stronger than the gene encoding the B-subunit, possibly indicating the presence of free A-subunits. In addition, strain-specific differences regarding the mRNA fold-changes of the A-subunit gene were observed. By use of native polyacrylamide gel electrophoresis and subsequent Western blot analysis, those single A-subunits were indeed detected in the culture supernatants of all six strains. To investigate whether the transcription ratios between A- and B-subunits observed are in a similar range as the amount of subunit proteins present after translation, a quantitative ELISA specific for StxA2a and StxB2a was established. Quantification of the subunits on protein level by use of ELISA revealed that the subunit ratio of StxA2a:StxB2a is 1.10 ± 0.20 for the strains HUSEC003, HUSEC004 and HUSEC008, but 4.63 ± 0.31 for the strains TS18/08, LB226692, and HUSEC018. The results of this study demonstrated that on both, the transcriptional and the translational level, the established 1:5 subunit ratio is not present in all investigated strains. In addition, the ratios observed after translation indicate that in some strains StxA2a subunits are even produced in higher amounts than B-subunits.

In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed virulence profiles and relevant phenotypes of outbreak isolates recovered in our laboratory. We analysed stool samples from 80 patients that had been submitted to the National Consulting Laboratory for Haemolytic Uraemic Syndrome in Münster, Germany, between May 23 and June 2, 2011. Isolates were screened with standard PCR for virulence genes of Shiga-toxin-producing E coli and a newly developed multiplex PCR for characteristic features of the outbreak strain ( rfb O104, fliC H4, stx 2 , and terD). Virulence profiles of the isolates were determined with PCR targeting typical virulence genes of Shiga-toxin-producing E coli and of other intestinal pathogenic E coli. We sequenced stx with Sanger sequencing and measured Shiga-toxin production, adherence to epithelial cells, and determined phylogeny and antimicrobial susceptibility. All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli ( stx 2, iha, lpf O26, lpf O113 ) and enteroaggregative E coli ( aggA, aggR, set1, pic, aap) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli, including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum β-lactamase phenotype absent in HUSEC041. Augmented adherence of the strain to intestinal epithelium might facilitate systemic absorption of Shiga toxin and could explain the high progression to haemolytic uraemic syndrome. This outbreak demonstrates that blended virulence profiles in enteric pathogens, introduced into susceptible populations, can have extreme consequences for infected people. German Federal Ministry of Education and Research, Network Zoonoses.

Porcine Shiga toxin-producing Escherichia coli (STEC) strains pose significant challenges to the pig industry. The toxins produced by these strains, particularly Shiga toxin subtype 2e (Stx2e), are associated with a range of clinical symptoms such as diarrhoea and oedema disease, which in severe cases result in death. Understanding the factors that influence the production and secretion of Stx2e is crucial to elucidate porcine STEC pathogenesis and to develop effective therapeutic strategies. Therefore, this study aimed to characterize the variability in Stx2e production among different porcine STEC strains and assess the effect of several external factors, including bile acids and antibiotics. Our results highlighted a substantial variation in extracellular Stx2e levels by porcine STEC strains. In addition, bile acids, especially the bile acid deoxycholate, exerted strain-specific effects on these extracellular Stx2e levels. Antibiotics also affected extracellular Stx2e levels with ciprofloxacin and enrofloxacin inducing a substantial increase in toxin production in certain strains. Genome analysis revealed that these strains encode a holin gene downstream of the Stx2e operon. Deleting this holin gene abolished the antibiotic-induced increase in extracellular Stx2e levels, while introducing holin expression in unresponsive strains increased the presence of Stx2e in the extracellular environment. These findings unravel a role for phage holins in Stx2e secretion and highlight the intricate interplay between genetic and environmental factors in regulating Stx2e production in porcine STEC strains. Together, our results offer insights into STEC pathogenesis.

ABSTRACT Strictly lytic phages are considered powerful tools for biocontrol of foodborne pathogens. Safety issues needed to be addressed for the biocontrol of Shiga toxin-producing Escherichia coli (STEC) include: lysogenic conversion, Shiga toxin production through phage induction, and emergence/proliferation of bacteriophage insensitive mutants (BIMs). To address these issues, two new lytic phages, vB_EcoS_Ace (Ace) and vB_EcoM_Shy (Shy), were isolated and characterized for life cycle, genome sequence and annotation, pH stability and efficacy at controlling STEC growth. Ace was efficient in controlling host planktonic cells and did not stimulate the production of the Stx prophage or Shiga toxin. A single dose of phage did not lead to the selection of BIMs. However, when reintroduced, BIMs were detected after 24 h of incubation. The gain of resistance was associated with lower virulence, as a subset of BIMs failed to agglutinate with O157-specific antibody and were more sensitive to human serum complement. BIM's biofilm formation capacity and susceptibility to disinfectants was equal to that of the wild-type strain. Overall, this work demonstrated that phage Ace is a safe biocontrol agent against STEC contamination and that the burden of BIM emergence did not represent a greater risk in environmental persistence and human pathogenicity. The safety of using phages as biocontrol agents for STEC decontamination, from a human and environmental safety point of view.

Enterohemorrhagic E. coli (EHEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. Their major virulence factor is Shiga toxin (Stx), which is encoded by bacteriophages. Of the two types of Stx, the production of Stx2, particularly that of Stx2a (a subtype of Stx2), is a major risk factor for severe EHEC infections, but the Stx2 production level is highly variable between strains. Here, we define four major and two minor subtypes of Stx2a-encoding phages according to their replication proteins. The subtypes are correlated with Stx2a titers produced by the host O157 strains, suggesting a critical role of the phage subtype in determining the Stx2a production level. We further show that one of the two subclades in the clade 8, a proposed hyper-virulent lineage of O157, carries the Stx2 phage subtype that confers the highest Stx2 production to the host strain. The presence of this subclade may explain the proposed high virulence potential of clade 8. These results provide novel insights into the variation in virulence among O157 strains and highlight the role of phage variation in determining the production level of the virulence factors that phages encode.

Porcine edema disease (ED) is caused by infection with Shiga toxin type 2 e variant (Stx2e)-producing Escherichia coli (STEC). To develop a new ED vaccine, we engineered a fusion protein, in which the Stx2e B subunit (Stx2eB) was fused to the five-stranded α-helical coiled coil domain of cartilage oligomeric matrix protein (COMP), based on our recent finding that the coiled coil strongly stabilizes the B subunit pentamer of Stx2 (Tamaki Y, Harakuni T, Arakawa T. Shiga toxin type 2 B subunit protects mice against toxin challenge when leashed and bundled by a stable pentameric coiled-coil molecule. Vaccine. 2024 Mar 7;42(7):1757–1767). Purified Stx2eB–COMP fusion protein administered to mice conferred complete protection against a lethal dose of Stx2e. However, unfused Stx2eB conferred only 10 % protection. Furthermore, when Stx2eB was fused to a trimeric or tetrameric coiled coil, a marked reduction in protective efficacy was observed, indicating the importance of “five-to-five” fusion stoichiometry. Next, we immunized weaned piglets twice with 100 μg or 10 μg of the Stx2eB–COMP fusion protein, and then orally challenged the animals with a lethal dose of STEC. The piglets immunized with the high dose were almost completely free from clinical symptoms of ED, whereas three of the six piglets administered adjuvant-only died, and the remaining surviving piglets exhibited severe ED symptoms. Although all piglets immunized with the low dose survived, they exhibited mild to moderate ED symptoms. Our findings indicate that Stx2eB is highly protective only when bundled and molecularly stabilized by the coiled coil molecule. •Shiga toxin type 2e (Stx2e) was fused to cartilage oligomeric matrix protein (COMP).•Fusion was between the Stx2eB subunit and the COMP pentameric α-helical coiled coil.•Stx2eB–COMP fusion protein, expressed in E. coli inclusion bodies, refolded in vitro.•Only the “five-to-five” chimera was protective in mice against Stx2e challenge.•Fusion protein protected piglets against orally infected Stx2e-producing E. coli.

Calf diarrhea is one of the considerable infectious diseases in calves, which results in tremendous economic losses globally. To determine the prevalence of Shiga-toxigenic E. coli (STEC) and Enterotoxigenic E. coli (ETEC) incriminated in calf diarrhea, with special reference to Shiga- toxins genes (stx1 and stx2) and enterotoxins genes (lt and sta) that govern their pathogenesis, as well as the virulence genes; eaeA (intimin) and f41(fimbrial adhesion), and the screening of their antibiogram and antimicrobial resistance genes; aadB, sul1, and bla-TEM, a total of 274 fecal samples were collected (April 2018–Feb 2019) from diarrheic calves at different farms in El-Sharqia Governorate, Egypt. The bacteriological examination revealed that the prevalence of E. coli in diarrheic calves was 28.8%. The serotyping of the isolated E. coli revealed 7 serogroups; O26, O128, O111, O125, O45, O119 and O91. Furthermore, the Congo red binding test was carried out, where 89.8% of the examined strains (n = 71) were positive. The antibiogram of the isolated strains was investigated; the majority of E. coli serotypes exhibit multidrug resistance (MDR) to four antimicrobial agents; neomycin, gentamycin, streptomycin, and amikacin. Polymerase chain reaction (PCR) was used to detect the prevalence of the virulence genes; stx1, stx2 lt, sta, f41 and eaeA, as well as the antimicrobial resistance genes; aadB, sul1, and bla-TEM. The prevalence of STEC was 20.2% (n = 16), while the prevalence of ETEC was 30.4% (n = 24). Briefly, the Shiga toxins genes; stx1 and stx2, are the most prevalent virulence genes associated with STEC, which are responsible for the pathogenesis of the disease and helped by the intimin gene (eaeA). In addition, the lt gene is the most prevalent enterotoxin gene accompanied by the ETEC strains, either alone or in combination with sta and/or f41 genes. The majority of pathogenic E. coli incriminated in calf diarrhea possesses the aadB resistance gene, followed by the sul1 gene. Enrofloxacin, florfenicol, amoxicillin-clavulanic acid, and ampicillin-sulbactam, are the most effective antimicrobial agents against the isolated STEC and ETEC strains.

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacter rodentium lysogenized with the Stx2-encoding phage Φstx2dact. This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that Φstx2dact infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (Φstx2dact) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (Φstx2dact) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.