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Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

Among the nine clades of Shiga toxin (Stx)-producing Escherichia coli O157:H7, clade 8 is thought to be highly pathogenic, as it causes severe disease more often than other clades. Two subclades have been proposed, but there are conflicting reports on intersubclade differences in Stx2 levels, although Stx2 production is a risk factor for severe disease development. The global population structure of clade 8 has also yet to be fully elucidated. Here, we present genome analyses of a global clade 8 strain set (n=510), including 147 Japanese strains sequenced in this study. The complete genome sequences of 18 of the 147 strains were determined to perform detailed clade-wide genome analyses together with 17 publicly available closed genomes. Intraclade variations in Stx2 production level and disease severity were also re-evaluated within the phylogenetic context. Based on phylogenomic analysis, clade 8 was divided into four lineages corresponding to the previously proposed SNP genotypes (SGs): SG8_30, SG8_31A, SG8_31B and SG8_32. SG8_30 and the common ancestor of the other SGs were first separated, with SG8_31A and SG8_31B emerging from the latter and SG8_32 emerging from SG8_31B. Comparison of 35 closed genomes revealed the overall structure of chromosomes and pO157 virulence plasmids and the prophage contents to be well conserved. However, Stx2a phages exhibit notable genomic diversity, even though all are integrated into the argW locus, indicating that subtype changes in Stx2a phage occurred from the γ subtype to its variant (γ_v1) in SG8_31A and from γ to δ in SG8_31B and SG8_32 via replacement of parts or almost entire phage genomes, respectively. We further show that SG8_30 strains (all carrying γ Stx2a phages) produce significantly higher levels of Stx2 and cause severe disease more frequently than SG8_32 strains (all carrying δ Stx2a phages). Clear conclusions on SG8_31A and SG8_31B cannot be made due to the small number of strains available, but as SG8_31A (carrying γ_v1 Stx2a phages) contains strains that produce much more Stx2 than SG8_30 strains, attention should also be paid to this SG.

Locus for Enterocyte Effacement (LEE)-positive Shiga-toxigenic Escherichia coli (STEC) contributes to many global foodborne diseases, with infection characterized by severe gastrointestinal symptoms, including bloody diarrhea. The incidence of LEE-negative STEC-mediated disease is also increasing globally. Subtilase cytotoxin (SubAB) is released by some LEE-negative STEC strains. It cleaves BiP, which is a chaperone protein located in the endoplasmic reticulum (ER), thereby causing apoptosis induced by ER stress. To date, the apoptotic signaling pathway mediated by SubAB has not been identified. In the current study, RNA-seq analysis showed that SubAB significantly induced the expression of Kelch domain containing 7B (KLHDC7B). We explored the role of KLHDC7B in the SubAB-induced apoptotic pathway. SubAB-induced KLHDC7B mRNA expression was increased after 12 h of incubation of toxin with HeLa cells. KLHDC7B expression was downregulated by knockdown of PKR-like endoplasmic reticulum kinase (PERK), CEBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and CEBP β (CEBPB). KLHDC7B knockdown suppressed SubAB-stimulated CHOP expression, poly(ADP-ribose) polymerase (PARP) cleavage, and cytotoxicity. The over-expressed KLHDC7B was localized to the nucleus and cytosolic fractions. Next, we used RNA-seq to analyze the effect of KLHDC7B knockdown on apoptosis induced by SubAB, and found that the gene encoding for the pro-apoptotic Bcl-2 family protein, Harakiri (HRK), was upregulated in SubAB-treated control cells. However, this effect was not observed in SubAB-treated KLHDC7B-knockdown cells. Therefore, we identified the pathway through which SubAB-induced KLHDC7B regulates HRK expression, which is essential for apoptosis in toxin-mediated ER stress.

Enterohemorrhagic Escherichia coli (EHEC) causes severe human diseases worldwide. The type three secretion system and effector proteins are essential for EHEC infection, and are encoded by the locus of enterocyte effacement (LEE). RNA-binding protein Hfq is essential for small regulatory RNA (sRNA)-mediated regulation at a post-transcriptional level and full virulence of many pathogenic bacteria. Although two early studies indicated that Hfq represses LEE expression by post-transcriptionally controlling the expression of genes grlRA and/or ler, both of which encode LEE regulators mediating a positive regulatory loop, the detailed molecular mechanism and biological significance remain unclear. Herein, we show that LEE overexpression was caused by defective RNA-binding activity of the Hfq distal face, which post-transcriptionally represses grlA and ler expression. In vitro analyses revealed that the Hfq distal face directly binds near the translational initiation site of grlA and ler mRNAs, and inhibits their translation. Taken together, we conclude that Hfq inhibits grlA and ler translation by binding their mRNAs through the distal face in an sRNA-independent manner. Additionally, we show that Hfq-mediated repression of LEE is critical for normal EHEC growth because all suppressor mutations that restored the growth defect in the hfq mutant abolished hfq deletion-induced overexpression of LEE.

Escherichia coli (E. coli) has multiple copies of the chaperone-usher (CU) pili operon in five fimbria groups: CU pili, curli, type IV pili, type III secretion pili, and type IV secretion pili. Commensal E. coli K-12 contains 12 CU pili operons. Among these operons, Sfm is expressed by the sfmACDHF operon. Transcriptome analyses, reporter assays, and chromatin immunoprecipitation PCR analyses reported that FimZ directly binds to and activates the sfmA promoter, transcribing sfmACDHF. In addition, FimZ regularly induces constant cell elongation in E. coli, which is required for F-type ATPase function. The bacterial two-hybrid system showed a specific interaction between FimZ and the α subunit of the cytoplasmic F

Enterohemorrhagic

To develop subtyping methods for Shiga toxin (Stx)1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g genes for epidemiological analyses of Shiga toxin-producing Escherichia coli (STEC), we developed 10 simplex real-time polymerase chain reaction (PCR) assays with reference to 284 valid stx sequences and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates and recombinant plasmids, respectively. Three stx

Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.

Subtilase cytotoxin (SubAB) is an AB

In June 2020, a large-scale food poisoning outbreak involving about 3000 elementary and junior high school students occurred in Yashio, Saitama, Japan. A school lunch was the only food stuff ingested by all of the patients. Escherichia coli serotype O7:H4 carrying the astA gene for enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1) was detected in faecal specimens from the patients, and sample inspection revealed its presence in a seaweed salad and red seaweed (Gigartina tenella) as one of the raw materials. Analysis of the antibiotic sensitivity of the isolates revealed resistance to ampicillin and cefotaxime. All isolates were confirmed to be of the same origin by pulsed-field gel electrophoresis after digestion with the restriction enzyme XbaI, and single nucleotide polymorphism analysis using whole genome sequencing. To our knowledge, this is the first report of a large-scale food poisoning caused by E. coli O7:H4, which lacks well-characterized virulence genes other than astA.

Hypermutable simple sequence repeats (SSRs) are major drivers of phase variation in Campylobacter jejuni. The presence of multiple SSR-mediated phase-variable genes encoding enzymes that modify surface structures, including capsular polysaccharide (CPS) and lipooligosaccharide (LOS), generates extreme cell surface diversity within bacterial populations, thereby promoting adaptation to selective pressures in host environments. Therefore, genetically controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. Here, we show that natural “cotransformation” is an effective method for C. jejuni genome editing. Cotransformation is a trait of naturally competent bacteria that causes uptake/integration of multiple different DNA molecules, which has been recently adapted to multiplex genome editing by natural transformation (MuGENT), a method for introducing multiple mutations into the genomes of these bacteria. We found that cotransformation efficiently occurred in C. jejuni. To examine the feasibility of MuGENT in C. jejuni, we “locked” different polyG SSR tracts in strain NCTC11168 (which are located in the biosynthetic CPS/LOS gene clusters) into either the ON or OFF configurations. This approach, termed “MuGENT-SSR,” enabled the generation of all eight edits within 2 weeks and the identification of a phase-locked strain with a highly stable type of Penner serotyping, a CPS-based serotyping scheme. Furthermore, extensive genome editing of this strain by MuGENT-SSR identified a phase-variable gene that determines the Penner serotype of NCTC11168. Thus, MuGENT-SSR provides a platform for genetic and phenotypic engineering of genetically unstable C. jejuni, making it a reliable approach for elucidating the mechanisms underlying phase-variable expression of specific phenotypes. IMPORTANCE Campylobacter jejuni is the leading bacterial cause of foodborne gastroenteritis in developed countries and occasionally progresses to the autoimmune disease Guillain-Barré syndrome. A relatively large number of hypermutable simple sequence repeat (SSR) tracts in the C. jejuni genome markedly decreases its phenotypic stability through reversible changes in the ON or OFF expression states of the genes in which they reside, a phenomenon called phase variation. Thus, controlling SSR-mediated phase variation can be important for achieving stable and reproducible research on C. jejuni. In this study, we developed a feasible and effective approach for genetically manipulate multiple SSR tracts in the C. jejuni genome using natural cotransformation, a trait of naturally transformable bacterial species that causes the uptake and integration of multiple different DNA molecules. This approach will greatly help to improve the genetic and phenotypic stability of C. jejuni to enable diverse applications in research and development.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Although most cases of STEC infection in humans are due to O157 and non-O157 serogroups, there are also reports of infection with STEC strains that cannot be serologically classified into any O serogroup (O-serogroup untypeable [OUT]). Recently, it has become clear that even OUT strains can be subclassified based on the diversity of O-antigen biosynthesis gene cluster (O-AGC) sequences. Cattle are thought to be a major reservoir of STEC strains belonging to various serotypes; however, the internal composition of OUT STEC strains in cattle remains unknown. In this study, we screened 366 STEC strains isolated from healthy cattle by using multiplex PCR kits including primers that targeted novel O-AGC types (Og types) found in OUT E. coli and Shigella strains in previous studies. Interestingly, 94 (25.7%) of these strains could be classified into 13 novel Og types. Genomic analysis revealed that the results of the in silico serotyping of novel Og-type strains were perfectly consistent with those of the PCR experiment. In addition, it was revealed that a dual Og8+OgSB17-type strain carried two types of O-AGCs from E. coli O8 and Shigella boydii type 17 tandemly inserted at the locus, with both antigens expressed on the cell surface. The results of this comprehensive analysis of cattle-derived STEC strains may help improve our understanding of the strains circulating in the environment. Additionally, the DNA-based serotyping systems used in this study could be used in future epidemiological studies and risk assessments of other STEC strains.

Shiga-toxigenic Escherichia coli (STEC) infection causes severe bloody diarrhea, renal failure, and hemolytic uremic syndrome. Recent studies showed global increases in Locus for Enterocyte Effacement (LEE)-negative STEC infection. Some LEE-negative STEC produce Subtilase cytotoxin (SubAB), which cleaves endoplasmic reticulum (ER) chaperone protein BiP, inducing ER stress and apoptotic cell death. In this study, we report that SubAB induces expression of a novel form of Lipocalin-2 (LCN2), and describe its biological activity and effects on apoptotic cell death. SubAB induced expression of a novel LCN2, which was regulated by PRKR-like endoplasmic reticulum kinase via the C/EBP homologous protein pathway. SubAB-induced novel-sized LCN2 was not secreted into the culture supernatant. Increased intracellular iron level by addition of holo-transferrin or FeCl3 suppressed SubAB-induced PARP cleavage. Normal-sized FLAG-tagged LCN2 suppressed STEC growth, but this effect was not seen in the presence of SubAB- or tunicamycin-induced unglycosylated FLAG-tagged LCN2. Our study demonstrates that SubAB-induced novel-sized LCN2 does not have anti-STEC activity, suggesting that SubAB plays a crucial role in the survival of LEE-negative STEC as well as inducing apoptosis of the host cells.

The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.

Since the Shiga toxin-producing enteroaggregative Escherichia coli (Stx-EAEC) O104:H4 strain caused a massive outbreak across Europe in 2011, the importance of Stx-EAEC has attracted attention from a public health perspective. Two Stx-EAEC O86 isolates were obtained from patients with severe symptoms in Japan in 1999 and 2015. To characterize the phylogeny and pathogenic potential of these Stx-EAEC O86 isolates, whole-genome sequence analyses were performed by short-and long-read sequencing. Among genetically diverse E. coli O86, the Stx-EAEC O86 isolates were clustered with the EAEC O86:H27 ST3570 subgroup. Strikingly, there were only two loci with single nucleotide polymorphisms (SNPs) between the Stx2a phage of a Japanese O86:H27 isolate and that of the European epidemic-related Stx-EAEC O104:H4 isolate. These results provide evidence of global distribution of epidemic-related Stx2a phages among various lineages of E. coli with few mutations.

The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.

Aim Recently eculizumab, a monoclonal antibody to C5, was found to improve the disease course of atypical haemolytic uraemic syndrome (aHUS) and has been recommended as the first line treatment by an international consensus guideline. However, several practical issues in the use of eculizumab for the acute phase of aHUS have yet to be resolved. Methods Children who received eculizumab with diagnosis of aHUS between March 2010 and December 2015 at Tokyo Metropolitan Children's Medical Center were enrolled. aHUS was diagnosed according to the haemolytic uraemic syndrome (HUS) criteria after excluding Shiga toxin-inducing Escherichia coli (STEC) -associated HUS and thrombocytopaenic purpura. We retrieved and analyzed data from the electronic medical records at our institution. Results We reviewed four patients with suspected aHUS. Eculizumab was discontinued in one patient in whom STEC-HUS was later diagnosed. Treatment was continued in the remaining three patients without recurrence. Practical issues included difficulty in diagnosing aHUS, particularly in the acute phase, risk of infection by encapsulated organisms, especially Neisseria meningitis, and infusion reaction. In addition to issues relating to the acute phase, discontinuing eculizumab in stable patients in the chronic phase must be considered. Conclusion Eculizumab, the first line treatment for children with aHUS, is usually effective. However, certain problems associated with its use require caution to be exercised. As clinical information on eculizumab are still very limited, and the rationale for its long-term use has yet to be established, physicians are advised to exercise care when using eculizumab to manage aHUS.

Shiga toxigenic Escherichia coli (STEC) are responsible for a worldwide foodborne disease, which is characterized by severe bloody diarrhea and hemolytic uremic syndrome (HUS). Subtilase cytotoxin (SubAB) is a novel AB5 toxin, which is produced by Locus for Enterocyte Effacement (LEE)-negative STEC. Cleavage of the BiP protein by SubAB induces endoplasmic reticulum (ER) stress, followed by induction of cytotoxicity in vitro or lethal severe hemorrhagic inflammation in mice. Here we found that steroids and diacylglycerol (DAG) analogues (e.g., bryostatin 1, Ingenol-3-angelate) inhibited SubAB cytotoxicity. In addition, steroid-induced Bcl-xL expression was a key step in the inhibition of SubAB cytotoxicity. Bcl-xL knockdown increased SubAB-induced apoptosis in steroid-treated HeLa cells, whereas SubAB-induced cytotoxicity was suppressed in Bcl-xL overexpressing cells. In contrast, DAG analogues suppressed SubAB activity independent of Bcl-xL expression at early time points. Addition of Shiga toxin 2 (Stx2) with SubAB to cells enhanced cytotoxicity even in the presence of steroids. In contrast, DAG analogues suppressed cytotoxicity seen in the presence of both toxins. Here, we show the mechanism by which steroids and DAG analogues protect cells against SubAB toxin produced by LEE-negative STEC.

SUMMARYA nationwide study of Shiga toxin-producingEscherichia coli(STEC) was performed to determine the prevalence, characteristics and risk factors for fecal shedding of STEC among cattle in Japan. Information on rearing practices was also collected to identify risk factors for fecal shedding of STEC. STEC was isolated from 24·1% of samples (133/551) collected from 59·1% of farms (65/110). Bayesian clustering using the virulence marker profiles of the isolates subdivided the isolates into four genetically distinct groups, two of which corresponded toeae- orsaa-positive STEC, which can cause severe disease in human. Both STEC groups exhibited characteristic phylogeny and virulence marker profiles. It is noteworthy that the tellurite resistance gene was not detected in allsaa-positive STEC isolates, suggesting that the standard isolation method using tellurite might lead to an underestimation of the prevalence ofsaa-positive STEC. A multivariate logistic regression model using epidemiological information revealed a significantly (P< 0·01) high odds ratio on STEC fecal shedding in tie-stall housing and a low odds ratio in flat feed box and mechanical ventilation. Information on isolate characteristics of the two major pathotypes and risk factors in rearing practices will facilitate the development of preventative measures for STEC fecal shedding from cattle.

Phages and plasmids play important roles in bacterial evolution and diversification. Although many draft genomes have been generated, phage and plasmid genomes are usually fragmented, limiting our understanding of their dynamics. Here, we performed a systematic analysis of 239 draft genomes and 7 complete genomes of Shiga toxin (Stx)-producing Escherichia coli O145:H28, the major virulence factors of which are encoded by prophages (PPs) or plasmids. The results indicated that PPs are more stably maintained than plasmids. A set of ancestrally acquired PPs was well conserved, while various PPs, including Stx phages, were acquired by multiple sublineages. In contrast, gains and losses of a wide range of plasmids have frequently occurred across the O145:H28 lineage, and only the virulence plasmid was well conserved. The different dynamics of PPs and plasmids have differentially impacted the pangenome of O145:H28, with high proportions of PP- and plasmid-associated genes in the variably present and rare gene fractions, respectively. The dynamics of PPs and plasmids have also strongly impacted virulence gene repertoires, such as the highly variable distribution of stx genes and the high conservation of a set of type III secretion effectors, which probably represents the core effectors of O145:H28 and the genes on the virulence plasmid in the entire O145:H28 population. These results provide detailed insights into the dynamics of PPs and plasmids, and show the application of genomic analyses using a large set of draft genomes and appropriately selected complete genomes.

Phages and plasmids play important roles in bacterial evolution and diversification. Although many draft genomes have been generated, phage and plasmid genomes are usually fragmented, limiting our understanding of their dynamics. Here, we performed a systematic analysis of 239 draft genomes and 7 complete genomes of Shiga toxin (Stx)-producing Escherichia coli O145:H28, the major virulence factors of which are encoded by prophages (PPs) or plasmids. The results indicated that PPs are more stably maintained than plasmids. A set of ancestrally acquired PPs was well conserved, while various PPs, including Stx phages, were acquired by multiple sublineages. In contrast, gains and losses of a wide range of plasmids have frequently occurred across the O145:H28 lineage, and only the virulence plasmid was well conserved. The different dynamics of PPs and plasmids have differentially impacted the pangenome of O145:H28, with high proportions of PP- and plasmid-associated genes in the variably present and rare gene fractions, respectively. The dynamics of PPs and plasmids have also strongly impacted virulence gene repertoires, such as the highly variable distribution of stx genes and the high conservation of a set of type III secretion effectors, which probably represents the core effectors of O145:H28 and the genes on the virulence plasmid in the entire O145:H28 population. These results provide detailed insights into the dynamics of PPs and plasmids, and show the application of genomic analyses using a large set of draft genomes and appropriately selected complete genomes.

We present the complete genome sequence of an enterohemorrhagic Escherichia coli O111:H8 strain. This strain was isolated from a hemolytic-uremic syndrome patient and was responsible for a large outbreak associated with the consumption of raw beef in 2011.

Due to the potential of enterohemorrhagic Escherichia coli (EHEC) serogroup O157 to cause large food borne outbreaks, national and international surveillance is necessary. For developing an effective method of molecular surveillance, a conventional method, multilocus variable-number tandem-repeat analysis (MLVA), and whole-genome sequencing (WGS) analysis were compared. WGS of 369 isolates of EHEC O157 belonging to 7 major MLVA types and their relatives were subjected to comprehensive in silico typing, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) analyses. The typing resolution was the highest in cgSNP analysis. However, determination of the sequence of the mismatch repair protein gene mutS is necessary because spontaneous deletion of the gene could lead to a hypermutator phenotype. MLVA had sufficient typing resolution for a short-term outbreak investigation and had advantages in rapidity and high throughput. cgMLST showed less typing resolution than cgSNP, but it is less time-consuming and does not require as much computer power. Therefore, cgMLST is suitable for comparisons using large data sets (e.g., international comparison using public databases). In conclusion, screening using MLVA followed by cgMLST and cgSNP analyses would provide the highest typing resolution and improve the accuracy and cost-effectiveness of EHEC O157 surveillance. IMPORTANCE Intensive surveillance for enterohemorrhagic Escherichia coli (EHEC) serogroup O157 is important to detect outbreaks and to prevent the spread of the bacterium. Recent advances in sequencing technology made molecular surveillance using whole-genome sequence (WGS) realistic. To develop rapid, high-throughput, and cost-effective typing methods for real-time surveillance, typing resolution of WGS and a conventional typing method, multilocus variable-number tandem-repeat analysis (MLVA), was evaluated. Nation-level systematic comparison of MLVA, core genome single nucleotide polymorphism (cgSNP), and core genome multilocus sequence typing (cgMLST) indicated that a combination of WGS and MLVA is a realistic approach to improve EHEC O157 surveillance.

To characterise the dissemination patterns of uropathogenicEscherichia coli(UPEC) in a community, we conducted a study utilising molecular and fundamental descriptive epidemiology. The subjects, consisted of women having community-acquired acute urinary tract infection (UTI), were enrolled in the study from 2011 to 2012. UPEC isolates were subjected to antibacterial-susceptibility testing, O serogrouping, phylotyping, multilocus-sequence typing with phylogenetic-tree analysis and pulsed-field-gel electrophoresis (PFGE). From the 209 unique positive urinary samples 166 UPEC were isolated, of which 129 were fully susceptible to the tested antibiotics. Of the 53 sequence types (STs), the four most prevalent STs (ST95, ST131, ST73 and ST357) accounted for 60% of all UPEC strains. Antimicrobial resistance was less frequently observed for ST95 and ST73 than for the others. A majority of rare STs and a few common STs constituted the diversity pattern within the population structure, which was composed of the two phylogenetically distinct clades. Eleven genetically closely related groups were determined by PFGE, which accounted for 42 of the 166 UPEC isolates, without overt geo-temporal clustering. Our results indicate that a few major lineages of UPEC, selected by unidentified factors, are disseminated in this community and contribute to a large fraction of acute UTIs.

This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections.

To evaluate the potential public health risk caused by secondary Shiga toxin–producing Escherichia coli (STEC) infections in Japan, we investigated the prevalence and characteristics of STEC isolated from healthy adults during 2010–2012. Although prevalence among healthy adults was high, most STEC organisms displayed characteristics rarely found in isolates from symptomatic patients.

In Escherichia coli , more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli .

Subtilase cytotoxin (SubAB) is mainly produced by locus of enterocyte effacement (LEE)-negative strains of Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress. This stress causes activation of ER stress sensor proteins and induction of caspase-dependent apoptosis. We found that SubAB induces stress granules (SG) in various cells. Aim of this study was to explore the mechanism by which SubAB induced SG formation. Here, we show that SubAB-induced SG formation is regulated by activation of double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK). The culture supernatant of STEC O113:H21 dramatically induced SG in Caco2 cells, although subAB knockout STEC O113:H21 culture supernatant did not. Treatment with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and lysosomal inhibitors, NH(4)Cl and chloroquine, suppressed SubAB-induced SG formation, which was enhanced by PKC and PKD inhibitors. SubAB attenuated the level of PKD1 phosphorylation. Depletion of PKCδ and PKD1 by siRNA promoted SG formation in response to SubAB. Furthermore, death-associated protein 1 (DAP1) knockdown increased basal phospho-PKD1(S916) and suppressed SG formation by SubAB. However, SG formation by an ER stress inducer, Thapsigargin, was not inhibited in PMA-treated cells. Our findings show that SubAB-induced SG formation is regulated by the PERK/DAP1 signalling pathway, which may be modulated by PKCδ/PKD1, and different from the signal transduction pathway that results in Thapsigargin-induced SG formation.

Shiga toxin family members have recently been classified using a new nomenclature into three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g). To develop screening methods for Stx genes, including all of these subtype genes, and Escherichia coli O26-, O111-, and O157-specific genes in laboratory investigations of Shiga toxin-producing E. coli (STEC) foodborne cases, we developed multiplex real-time PCR assays and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates, recombinant plasmids, and food enrichment cultures and by performing STEC spiking experiments with beef and sprout enrichment cultures. In addition, we evaluated the relationship between the recovery rates of the target strains by direct plating and immunomagnetic separation and the cycle threshold (CT) values of the real-time PCR assays for the Stx subtypes and STEC O26, O111, and O157 serogroups. All three stx1- and seven stx2-subtype genes were detected by real-time PCR with high sensitivity and specificity, and the quantitative accuracy of this assay was confirmed using control plasmids and STEC spiking experiments. The results of the STEC spiking experiments suggest that it is not routinely possible to isolate STEC from enrichment cultures with real-time PCR CT values greater than 30 by direct plating on MacConkey agar, although highly selective media and immunomagnetic beads were able to isolate the inoculated strains from the enrichment cultures. These data suggest that CT values obtained from the highly quantitative real-time PCR assays developed in this study provide useful information to develop effective isolation strategies for STEC from food samples. The real-time PCR assays developed here are expected to aid in investigations of infections or outbreaks caused by STEC harboring any of the stx-subtype genes in the new Stx nomenclature, as well as STEC O26, O111, and O157.

The O serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and enhancing phylogenetic studies. In particular, the identification of strains of the same O serogroup is essential in outbreak investigations and surveillance. In a previous study, we analyzed the O-antigen biosynthesis gene cluster in all known E. coli O serogroups (A. Iguchi et al., DNA Res, 22:101–107, 2015, http://dx.doi.org/10.1093/dnares/dsu043 ). Based on those results, we have arranged 162 PCR primer pairs for the identification or classification of O serogroups. Of these, 147 pairs were used to identify 147 individual O serogroups with unique O-antigen biosynthesis genes, and the other 15 pairs were used to identify 15 groups of strains (Gp1 to Gp15). Each of these groups consisted of strains with identical or very similar O-antigen biosynthesis genes, and the groups represented a total of 35 individual O serogroups. We then used the 162 primer pairs to create 20 multiplex PCR sets. Each set contained six to nine primer pairs that amplify products of markedly different sizes. This genetic methodology ( E. coli O-genotyping PCR) allowed for comprehensive, rapid, and low-cost typing. Validation of the PCR system using O-serogroup references and wild strains showed that the correct O serogroups were specifically and accurately identified for 100% (182/182) and 90.8% (522/575) of references and wild strains, respectively. The PCR-based system reported here might be a promising tool for the subtyping of E. coli strains for epidemiological studies as well as for the surveillance of pathogenic E. coli during outbreaks.

SUMMARYA large outbreak of enterohaemorrhagic Escherichia coli (EHEC) O111 and O157 occurred in Japan in April 2011. We conducted an unmatched case-control study and trace-back investigation to determine the source of EHEC O111 infection and risk factors for severe complications. Pulsed-field gel electrophoresis was performed to help define cases. A total of 86 individuals met the case definition. Of these, 40% experienced haemolytic uraemic syndrome (HUS), 24% acute encephalopathy, and 6% died. Illness was significantly associated with eating the raw beef dish yukhoe (odds ratio 19·64, 95% confidence interval 7·03–54·83), the likely food vehicle. EHEC O111 and its closely related stx-negative variants were found in the beef. HUS occurred most frequently in individuals aged 5–9 years, and this age group was significantly associated with acute encephalopathy. The prevalence of HUS and acute encephalopathy was higher than in previous non-O157-related outbreaks, indicating a high risk of severe complications.

A key virulence factor of enterohaemorrhagic Escherichia coli (EHEC) is the bacteriophage-encoded Shiga toxin (Stx). Stxs are classified into two types, Stx1 and Stx2, and Stx2-producing strains are thought to cause more severe infections than strains producing only Stx1. Although O26 : H11 is the second most prevalent EHEC following O157 : H7, the majority of O26 : H11 strains produce Stx1 alone. However, Stx2-producing O26 strains have increasingly been detected worldwide. Through a large-scale genome analysis, we present a global phylogenetic overview and evolutionary timescale for E. coli O26 : H11. The origin of O26 has been estimated to be 415 years ago. Sequence type 21C1 (ST21C1), one of the two sublineages of ST21, the most predominant O26 : H11 lineage worldwide, emerged 213 years ago from one of the three ST29 sublineages (ST29C2). The other ST21 lineage (ST21C2) emerged 95 years ago from ST21C1. Increases in population size occurred in the late 20th century for all of the O26 lineages,but most remarkably for ST21C2. Analysis of the distribution of stx2-positive strains revealed the recent and repeated acquisition of the stx2 gene in multiple lineages of O26, both in ST21 and ST29. Other major EHEC virulence genes, such as type III secretion system effector genes and plasmid-encoded virulence genes, were well conserved in ST21 compared to ST29. In addition, more antimicrobial-resistance genes have accumulated in the ST21C1 lineage. Although current attention is focused on several highly virulent ST29 clones that have acquired the stx2 gene, there is also a considerable risk that the ST21 lineage could yield highly virulent clones. KEYWORDS: Shiga toxin; antimicrobial-resistance gene; enterohaemorrhagic Escherichia coli; genome evolution; population structure PMID: 29208163 PMCID: PMC5729918 DOI: 10.1099/mgen.0.000141, A key virulence factor of enterohaemorrhagic Escherichia coli (EHEC) is the bacteriophage-encoded Shiga toxin (Stx). Stxs are classified into two types, Stx1 and Stx2, and Stx2-producing strains are thought to cause more severe infections than strains producing only Stx1. Although O26 : H11 is the second most prevalent EHEC following O157 : H7, the majority of O26 : H11 strains produce Stx1 alone. However, Stx2-producing O26 strains have increasingly been detected worldwide. Through a large-scale genome analysis, we present a global phylogenetic overview and evolutionary timescale for E. coli O26 : H11. The origin of O26 has been estimated to be 415 years ago. Sequence type 21C1 (ST21C1), one of the two sublineages of ST21, the most predominant O26 : H11 lineage worldwide, emerged 213 years ago from one of the three ST29 sublineages (ST29C2). The other ST21 lineage (ST21C2) emerged 95 years ago from ST21C1. Increases in population size occurred in the late 20th century for all of the O26 lineages, but most remarkably for ST21C2. Analysis of the distribution of stx2-positive strains revealed the recent and repeated acquisition of the stx2 gene in multiple lineages of O26, both in ST21 and ST29. Other major EHEC virulence genes, such as type III secretion system effector genes and plasmid-encoded virulence genes, were well conserved in ST21 compared to ST29. In addition, more antimicrobial-resistance genes have accumulated in the ST21C1 lineage. Although current attention is focused on several highly virulent ST29 clones that have acquired the stx2 gene, there is also a considerable risk that the ST21 lineage could yield highly virulent clones.

Shiga toxigenic Escherichia coli (STEC) are responsible for a worldwide foodborne disease, which is characterized by severe bloody diarrhea and hemolytic uremic syndrome (HUS). Subtilase cytotoxin (SubAB) is a novel AB5 toxin, which is produced by Locus for Enterocyte Effacement (LEE)-negative STEC. Cleavage of the BiP protein by SubAB induces endoplasmic reticulum (ER) stress, followed by induction of cytotoxicity in vitro or lethal severe hemorrhagic inflammation in mice. Here we found that steroids and diacylglycerol (DAG) analogues (e.g., bryostatin 1, Ingenol-3-angelate) inhibited SubAB cytotoxicity. In addition, steroid-induced Bcl-xL expression was a key step in the inhibition of SubAB cytotoxicity. Bcl-xL knockdown increased SubAB-induced apoptosis in steroid-treated HeLa cells, whereas SubAB-induced cytotoxicity was suppressed in Bcl-xL overexpressing cells. In contrast, DAG analogues suppressed SubAB activity independent of Bcl-xL expression at early time points. Addition of Shiga toxin 2 (Stx2) with SubAB to cells enhanced cytotoxicity even in the presence of steroids. In contrast, DAG analogues suppressed cytotoxicity seen in the presence of both toxins. Here, we show the mechanism by which steroids and DAG analogues protect cells against SubAB toxin produced by LEE-negative STEC.

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx 2 and stx -negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx -negative E. coli O111 isolates were closely related to EHEC O111 stx 2 isolates. Although the EHEC O157 strains had diverse stx gene profiles ( stx 1 , stx 2 , and stx 1 stx 2 ), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx 2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx 2 to an stx -negative strain may have occurred during infection.