DNA adenine methylase, not the PstI restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coli.

We previously reported a distinct methylome between the two Shiga toxin-producing Escherichia coli (STEC) O145:H28 strains linked to the 2010 U.S. lettuce-associated outbreak (RM13514) and the 2007 Belgium ice cream-associated outbreak (RM13516), respectively. This difference was thought to be attributed to a prophage encoded type II restriction-modification system (PstI R-M) in RM13514. Here, we characterized this PstI R-M system in comparison to DNA adenine methylase (Dam), a highly conserved enzyme in γ proteobacteria, by functional genomics. Deficiency in Dam led to a differential expression of over 1000 genes in RM13514, whereas deficiency in PstI R-M only impacted a few genes transcriptionally. Dam regulated genes involved in diverse functions, whereas PstI R-M regulated genes mostly encoding transporters and adhesins. Dam regulated a large number of genes located on prophages, pathogenicity islands, and plasmids, including Shiga toxin genes, type III secretion system (TTSS) genes, and enterohemolysin genes. Production of Stx2 in dam mutant was significantly higher than in RM13514, supporting a role of Dam in maintaining lysogeny of Stx2-prophage. However, following mitomycin C treatment, Stx2 in RM13514 was significantly higher than that of dam or PstI R-M deletion mutant, implying that both Dam and PstI R-M contributed to maximum Stx2 production.

Application of bacteriophages in simultaneously controlling Escherichia coli O157:H7 and extended-spectrum beta-lactamase producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of public health concern and frequently isolated from raw beef products. Bacteriophage-based methods have been increasingly exploited to control bacterial contamination in meats. Here, we describe the isolation, characterization, and application of a lytic phage PE37 for the simultaneous bio-control of STEC O157:H7 and ESBLEC. Phage PE37, isolated from the bovine intestine, was morphologically characterized as a member of the Myoviridae family, with a broad host range and great stability under various stress conditions. Sequencing analysis revealed that the genomic DNA of phage PE37 contains genes that contribute to virion structure, replication, assembly, and host lysis. PE37 significantly reduced the viable counts of STEC O157:H7 by 4.9 and 2.6 log CFU/mL in broth after 6 h of incubation at 25 and 8 °C, respectively. Application of phage PE37 to raw beef artificially contaminated with STEC O157:H7 resulted in significant reductions in the viable counts by 2.3 and 0.9 log CFU/piece after 24 h of storage at 25 and 8 °C, respectively. Treatment of raw beef contaminated with a bacterial cocktail of STEC O157:H7 and ESBLEC with PE37 also significantly decreased the viable counts of the bacterial mixture by 1.4 and 1.0 log CFU/piece after 24 h of incubation at 25 and 8 °C, respectively. These findings suggest that bacteriophage PE37 may be a potential bio-agent for controlling STEC O157:H7 and ESBLEC contamination in raw beef.

Shiga Toxin-Producing Escherichia coli Infection in Jönköping County, Sweden: Occurrence and Molecular Characteristics in Correlation With Clinical Symptoms and Duration of stx Shedding.

Shiga toxin-producing Escherichia coli (STEC) cause bloody diarrhea (BD), hemorrhagic colitis (HC), and even hemolytic uremic syndrome (HUS). In Nordic countries, STEC are widely spread and usually associated with gastrointestinal symptoms and HUS. The objective of this study was to investigate the occurrence of STEC in Swedish patients over 10 years of age from 2003 through 2015, and to analyze the correlation of critical STEC virulence factors with clinical symptoms and duration of stx shedding. Diarrheal stool samples were screened for presence of stx by real-time PCR. All STEC isolates were characterized by DNA microarray assay and PCR to determine serogenotypes, stx subtypes, and presence of intimin gene eae and enterohaemolysin gene ehxA. Multilocus sequencing typing (MLST) was used to assess phylogenetic relationships. Clinical features were collected and analyzed using data from the routine infection control measures in the county. A total of 14,550 samples were enrolled in this 12-years period study, and 175 (1.2%) stools were stx positive by real-time PCR. The overall incidence of STEC infection was 4.9 cases per 100,000 person-years during the project period. Seventy-five isolates, with one isolate per sample were recovered, among which 43 were from non-bloody stools, 32 from BD, and 3 out of the 75 STEC positive patients developed HUS. The presence of stx2 in both stools and isolates were associated with BD (p = 0.008, p = 0.05), and the presence of eae in isolates was related to BD (p = 0.008). The predominant serogenotypes associated with BD were O157:H7, O26:H11, O121:H19, and O103:H2. Isolates from HUS were O104:H4 and O98: H21 serotypes. Phylogenetic analysis revealed our strains were highly diverse, and showed close relatedness to HUS-associated STEC collection strains. In conclusion, the presence of stx2 in stool was related to BD already at the initial diagnostic procedure, thus could be used as risk predictor at an early stage. STEC isolates with stx2 and eae were significantly associated with BD. The predominant serotypes associated with BD were O157:H7, O26:H11, O121:H19, and O103:H2. Nevertheless, the pathogenic potential of other serotypes and genotypes should not be neglected.

Molecular characterisation of blaOXA-48 carbapenemase-, extended-spectrum ß-lactamase- and Shiga toxin-producing Escherichia coli isolated from farm piglets in India.

OBJECTIVES: The aim of this study was to characterise carbapenemase-, extended-spectrum ß-lactamase (ESBL)- and Shiga toxin-producing Escherichia coli isolated from farm piglets in India. METHODS: Faecal samples (n=741) from 10 organised pig farms, including non-diarrhoeic (n=546) and diarrhoeic (n=195) piglets, were processed for isolation of carbapenem-resistant and ESBL-producing E. coli. RESULTS: A total of 27 and 243 isolates were phenotypically confirmed as carbapenem-resistant and ESBL-producers, respectively. The meropenem minimum inhibitory concentration (MIC) of carbapenem-resistant isolates ranged from 8-128µg/mL. On genotypic screening of the 27 carbapenem-resistant isolates, 3 isolates were positive for the blaOXA-48 carbapenemase gene; no other carbapenemase genes were detected. The 243 ESBL-producing isolates were positive for blaCTX-M-1 (n=135), qnrA (n=92), qnrB (n=112), qnrS (n=49), tetA (n=42), tetB (n=45) and sul1 (n=43). The Shiga toxin virulence markers stx1 and stx2 were detected in 41 and 38 of the 243 phenotypic ESBL-producing isolates, respectively. Multilocus sequence typing (MLST) of blaOXA-48-positive E. coli isolates showed ST10- and ST5053-like sequence types. CONCLUSION: This is the first report on the presence of blaOXA-48-carrying E. coli in piglets in India, which pose a potential risk to public health.

Identification of the newly identified subtilase cytotoxin-encoding gene (subAB2-2) among clinical Shiga toxin-producing Escherichia coli isolates.

Subtilase cytotoxin (SubAB) is an important virulence factor of eae-negative Shiga toxin-producing Escherichia coli (STEC). Three variants of SubAB-encoding genes have been reported in the literature; however, the newly described subAB variant (subAB2-2) was found only in STEC strains from deer meat, sheep, and some wild animals. In this study, subAB variants were detected by PCR and DNA sequencing in 5 out of 12 (41.6%) eae-negative STEC strains isolated from patients. Most subAB-positive STEC strains (80%) harbored the subAB1 gene. The subAB2-2 gene was detected for the first time in the clinical STEC O128:H2 strain. Other virulence genes including stx1a, stx1c, stx2b, ehxA, and tia were also detected in this strain. The DNA sequence analyses of the subAB1 and subAB2-2 genes of the clinical STEC strains showed 99% and 100% identity to those of the reference strains 98NK2 and LM27558stx2, respectively. This is the first report on the detection of the subAB2-2 gene in a clinical STEC isolate.

Cytotoxic and apoptotic effects of recombinant subtilase cytotoxin variants of shiga toxin-producing Escherichia coli.

In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB1 and the chromosome-encoded SubAB2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1 exhibited cytotoxic effects in the absence of the respective B1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1 alone induced apoptosis, while the B1 subunit alone did not induce cell death.

Extended-spectrum beta-lactamase-producing Shiga toxin gene (stx1)-positive Escherichia coli O91:H14 carrying blaCTX-M-15 on an IncI1-ST31 plasmid isolated from a human patient in Germany.

In 2011, the Shiga toxin- and extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli O104:H4 caused a serious outbreak of gastroenteritis in Germany. This strain carried bla(CTX-M-15) and bla(TEM-1) on an IncI1-ST31 plasmid. During screening of individuals at risk for acquisition of the epidemic E. coli O104:H4, we isolated another ESBL-producing and Shiga toxin-positive E. coli belonging to serotype O91:H14 from feces of a human patient. Interestingly, the patient also carried a further ESBL-producing but Shiga toxin-negative E. coli. Both strains harbored bla(CTX-M-15) and bla(TEM-1) on an IncI1-ST31 plasmid, which was indistinguishable regarding size and plasmid restriction pattern from the plasmid of the epidemic E. coli O104:H4 strain. The patient had traveled to India 6 months prior to the isolation of the E. coli strains. This is the first report of an ESBL-producing, Shiga toxin-positive E. coli of serogroup O91. Our data suggest a high propensity of the IncI1-ST31 plasmid to spread in the human and/or animal population.

Cyclic-di-GMP signalling and biofilm-related properties of the Shiga toxin-producing 2011 German outbreak Escherichia coli O104:H4.

In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)--producing the biofilm-promoting second messenger c-di-GMP--that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS.

Contribution of urease to colonization by Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen with a low infectious dose that colonizes the colon in humans and can cause severe clinical manifestations such as hemolytic-uremic syndrome. The urease enzyme, encoded in the STEC chromosome, has been demonstrated to act as a virulence factor in other bacterial pathogens. The NH(3) produced as urease hydrolyzes urea can aid in buffering bacteria in acidic environments as well as provide an easily assimilated source of nitrogen that bacteria can use to gain a metabolic advantage over intact microflora. Here, we explore the role of urease in STEC pathogenicity. The STEC urease enzyme exhibited maximum activity near neutral pH and during the stationary-growth phase. Experiments altering growth conditions performed with three phylogenetically distinct urease-positive strains demonstrated that the STEC ure gene cluster is inducible by neither urea nor pH but does respond to nitrogen availability. Quantitative reverse transcription-PCR (qRT-PCR) data indicate that nitrogen inhibits the transcriptional response. The deletion of the ure gene locus was constructed in STEC strain 88-0643, and the ure mutant was used with the wild-type strain in competition experiments in mouse models to examine the contribution of urease. The wild-type strain was twice as likely to survive passage through the acidic stomach and demonstrated an enhanced ability to colonize the intestinal tract compared to the ure mutant strain. These in vivo experiments reveal that, although the benefit STEC gains from urease expression is modest and not absolutely required for colonization, urease can contribute to the pathogenicity of STEC.

CTX-M-15 extended-spectrum ß-lactamase in a shiga toxin-producing Escherichia coli isolate of serotype O111:H8.

We report the discovery of a CTX-M-15-producing Escherichia coli (STEC) of serogroup O111:H8, a major serotype responsible for human enterohemorrhagic Escherichia coli (EHEC) infections. In line with the recent CTX-M-15/O104:H4 E. coli outbreak, these data may reflect an accelerating spread of resistance to expanded-spectrum cephalosporins within the E. coli population, including STEC isolates.

Functional and phylogenetic analysis of ureD in Shiga toxin-producing Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG, the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD, encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro. We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.

Outbreak caused by cad-negative Shiga toxin-producing Escherichia coli O111, Oklahoma.

An outbreak of severe diarrheal illness was recently reported in northeastern Oklahoma, and Shiga toxin-producing Escherichia coli serotype O111 was identified as the etiological agent. Our results indicated that this isolate is unable to decarboxylate lysine, a characteristic that is shared with other outbreak-linked O111 isolates. Therefore, further investigation is recommended to determine whether the lysine decarboxylase test could be used to identify a subset of pathogenic E. coli, particularly Shiga toxin-producing E. coli O111 isolates, that have the potential of causing human infections and outbreaks.

Molecular subtyping and distribution of the serine protease from shiga toxin-producing Escherichia coli among atypical enteropathogenic E. coli strains.

Atypical enteropathogenic Escherichia coli (aEPEC) and Shiga toxin-producing E. coli (STEC) were examined to determine the prevalence and sequence of espP, which encodes a serine protease. These analyses indicated shared espP sequence types between the two E. coli pathotypes and thus provide further insights into the evolution of aEPEC and STEC.

Serine protease espP subtype alpha, but not beta or gamma, of Shiga toxin-producing Escherichia coli is associated with highly pathogenic serogroups.

Besides Shiga toxins (Stx), Stx-producing Escherichia coli (STEC) harbour several other putative virulence factors, including the serine protease EspP. We have investigated 214 STEC strains from Austria belonging to 61 different serotypes from humans, animals, and food for the presence of this serine protease gene and have determined the espP subtypes and their association with clinical outcome. espP was detected in 121 (57%) out of 214 strains. Sixty-five of 68 strains (96%) of non-sorbitol-fermenting (NSF) O157:H7/NM (NM, non-motile) were positive for espP, while none of 8 SF E. coli O157:NM isolates contained this gene. All 9 strains of serotype O145:NM and 17 of 21 strains (81%) of serotype O26:H11/NM were positive for espP. Nineteen STEC serogroups including O103 and O111 serogroups--considered to be highly pathogenic--were completely negative for espP. Only 5 of 12 strains isolated from patients suffering from haemolytic uraemic syndrome (HUS) were espP-positive (all serogroup NSF O157) as well as 28 of 39 strains from patients with bloody diarrhoea, 40 of 63 strains from patients with non-bloody diarrhoea, and 15 of 19 strains from asymptomatic patients. In O157:H7/NM, O26:H11/NM, and O145:NM only espP subtype alpha was found, whereas in most of the other non-O157 serogroups, subtypes beta and gamma were found. Subtype delta was not detected in our strain collection. Regarding the espP subtypes, only subtype alpha, but not beta and gamma, were found in HUS patients. Moreover, we could demonstrate that espP, and in particular subtype alpha, is associated with highly pathogenic serogroups.

Subtilase cytotoxin, produced by Shiga-toxigenic Escherichia coli, transiently inhibits protein synthesis of Vero cells via degradation of BiP and induces cell cycle arrest at G1 by downregulation of cyclin D1.

Subtilase cytotoxin (SubAB) is a AB(5) type toxin produced by Shiga-toxigenic Escherichia coli, which exhibits cytotoxicity to Vero cells. SubAB B subunit binds to toxin receptors on the cell surface, whereas the A subunit is a subtilase-like serine protease that specifically cleaves chaperone BiP/Grp78. As noted previously, SubAB caused inhibition of protein synthesis. We now show that the inhibition of protein synthesis was transient and occurred as a result of ER stress induced by cleavage of BiP; it was closely associated with phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha). The phosphorylation of PERK and eIF2alpha was maximal at 30-60 min and then returned to the control level. Protein synthesis after treatment of cells with SubAB was suppressed for 2 h and recovered, followed by induction of stress-inducible C/EBP-homologous protein (CHOP). BiP degradation continued, however, even after protein synthesis recovered. SubAB-treated cells showed cell cycle arrest in G1 phase, which may result from cyclin D1 downregulation caused by both SubAB-induced translational inhibition and continuous prolonged proteasomal degradation.