The global population structure and evolutionary history of the acquisition of major virulence factor-encoding genetic elements in Shiga toxin-producing Escherichia coli O121:H19.

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.

Rapid and Accurate Diagnosis Based on Real-Time PCR Cycle Threshold Value for the Identification of Campylobacter jejuni, astA Gene-Positive Escherichia coli, and eae Gene-Positive E. coli.

We previously developed a multiplex real-time PCR assay (Rapid Foodborne Bacterial Screening 24 ver.5, [RFBS24 ver.5]) for simultaneous detection of 24 foodborne bacterial targets. Here, to overcome the discrepancy of the results from RFBS24 ver.5 and bacterial culture methods (BC), we analyzed 246 human clinical samples from 49 gastroenteritis outbreaks using RFBS24 ver.5 and evaluated the correlation between the cycle threshold (CT) value of RFBS24 ver.5 and the BC results. The results showed that the RFBS24 ver.5 was more sensitive than BC for Campylobacter jejuni and Escherichia coli harboring astA or eae, with positive predictive values (PPV) of 45.5-87.0% and a kappa coefficient (KC) of 0.60-0.92, respectively. The CTs were significantly different between BC-positive and -negative samples (p ＜ 0.01). All RFBS24 ver.5-positive samples were BC-positive under the lower confidence interval (CI) limit of 95% or 99% for the CT of the BC-negative samples. We set the 95% or 99% CI lower limit to the determination CT (d-CT) to discriminate for assured BC-positive results (d-CTs: 27.42-30.86), and subsequently the PPVs (94.7%-100.0%) and KCs (0.89-0.95) of the 3 targets were increased. Together, we concluded that the implication of a d-CT-based approach would be a valuable tool for rapid and accurate diagnoses using the RFBS24 ver.5 system.

Detection of pathogenic Yersinia enterocolitica in pet Djungarian hamsters in Japan.

The prevalence of Yersinia enterocolitica (Y. enterocolitica) and Yersinia pseudotuberculosis was examined in 151 pet animals including 108 rodents, 39 rabbits and four sugar gliders from 13 pet stores in the Yamaguchi Prefecture, Japan. Y. enterocolitica serogroup O:3 biotype 3 negative for the Voges-Proskauer reaction (O:3/3 variant VP-) was isolated from five Djungarian hamsters (Phodopus sungorus) raised at the same pet store. These pathogenic Y. enterocolitica isolates carried the virulence genes, yadA, ail and virF, and were shown to be clonal by pulsed-field gel electrophoresis with NotI digestion. This is a first report of pathogenic Y. enterocolitica O:3/3 variant VP- in pet Djungarian hamsters in Japan.

Spontaneous mutations in Streptococcus pyogenes isolates from streptococcal toxic shock syndrome patients play roles in virulence.

Streptococcus pyogenes (group A Streptococcus; GAS) is a widespread human pathogen and causes streptococcal toxic shock syndrome (STSS). STSS isolates have been previously shown to have high frequency mutations in the csrS/csrR (covS/covR) and/or rgg (ropB) genes, which are negative regulators of virulence. However, these mutations were found at somewhat low frequencies in emm1-genotyped isolates, the most prevalent STSS genotype. In this study, we sought to detect causal mutations of enhanced virulence in emm1 isolates lacking mutation(s) in the csrS/csrR and rgg genes. Three mutations associated with elevated virulence were found in the sic (a virulence gene) promoter, the csrR promoter, and the rocA gene (a csrR positive regulator). In vivo contribution of the sic promoter and rocA mutations to pathogenicity and lethality was confirmed in a GAS mouse model. Frequency of the sic promoter mutation was significantly higher in STSS emm1 isolates than in non-invasive STSS isolates; the rocA gene mutation frequency was not significantly different among STSS and non-STSS isolates. STSS emm1 isolates possessed a high frequency mutation in the sic promoter. Thus, this mutation may play a role in the dynamics of virulence and STSS pathogenesis.

An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

Distribution of IS629 and stx genotypes among enterohemorrhagic Escherichia coli O157 isolates in Yamaguchi Prefecture, Japan, 2004-2013.

Patterns of insertion sequence (IS)629, norV genotype, and Shiga toxin (Stx) genotype distribution were investigated amongst 203 enterohemorrhagic Escherichia coli O157 isolates collected in Yamaguchi Prefecture, Japan, between 2004 and 2013. A total of 114 IS629 patterns were identified; these were divided into eight IS groups (A-H). Ninety isolates carried an intact norV gene, whereas 113 isolates carried a norV with a 204-bp deletion. Other than one isolate from IS group G, all isolates with an intact norV belonged to groups A-F, whereas isolates with a mutant norV belonged to IS groups G and H. Seven stx genotypes were identified, and of those, stx1a/stx2a was predominant (n=105), followed by stx2c (n=32) and stx2a (n=27). The stx1a/stx2a genotype was associated with the mutant norV isolates, whereas isolates with an intact norV had the stx2c genotype. Therefore, certain combinations of IS type and stx genotype appear to be more frequent among O157 clades which may be useful for detection of predominant subtypes in the interest of public health.

Detection of CMY-2 AmpC ß-lactamase-producing enterohemorrhagic Escherichia coli O157:H7 from outbreak strains in a nursery school in Japan.

In 2013, an outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 occurred in a nursery school in Japan. The outbreak affected 12 school children and five members of their families. All 17 isolates obtained from these individuals were found to be clonal, as determined by pulsed-field gel electrophoresis analysis and multilocus variable number tandem repeat analysis. The antimicrobial susceptibility profiles of the isolates to 20 drugs were examined, with three isolates showing resistance to the extended-spectrum cephalosporins (ESC) and cephamycin, including cefotaxime, ceftazidime, and cefminox. The resistant isolates carried the blaCMY-2 AmpC ß-lactamase gene. It is proposed that the ESC-resistant EHEC O157:H7 isolates might have acquired the resistance plasmid encoding the blaCMY-2 gene during human to human infection in the nursery school.

Biochemical Features and Virulence Gene Profiles of Non-O157/O26 Enterohemorrhagic Escherichia coli Strains from Humans in the Yamaguchi Prefecture, Japan.

The biochemical features and virulence gene profiles of 37 enterohemorrhagic Escherichia coli (EHEC) strains belonging to serogroups other than O157 and O26 (non-O157/O26 EHEC) were investigated. All strains were isolated from humans between 2002 and 2013 in the Yamaguchi Prefecture. Serogroup O111 strains were the most common, followed by O103, O121, and O145. Most strains (84%) were negative for sorbose fermentation, whereas only 1 and 2 were negative for sorbitol and rhamnose fermentation, respectively. Two strains lacked ß-D-glucuronidase activity. Shiga toxin (stx) subtyping revealed 6 genotypes:stx1a (n = 20), stx1a + stx2a (n = 8), stx2a (n = 4), stx2b (n = 3), stx2a + stx2c (n = 1), and stx2a + stx2d (n = 1). Polymerase chain reaction screening of other toxin and adherence genes showed that astA, subA, and cdtB were present in 5, 2, and 2 strains, respectively. The intimin gene eae was present in 30 strains (81%). Of the 7 eae-negative strains, saa and eibG were found in 3 and 2 strains, respectively; no adherence factors were detected in the remaining 2 strains. The antimicrobial susceptibility profiles of the strains to 12 drugs were examined and 11 strains (30%) showed resistance to 1 or more drugs. Our results revealed that non-O157/O26 EHEC strains exhibit various biochemical phenotypes and carry several toxins and adherence factor genes.

Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR.

Phylogenetic Clades 6 and 8 of Enterohemorrhagic Escherichia coli O157:H7 With Particular stx Subtypes are More Frequently Found in Isolates From Hemolytic Uremic Syndrome Patients Than From Asymptomatic Carriers.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe diseases such as bloody diarrhea and hemolytic uremic syndrome (HUS). Although EHEC O157:H7 strains have exhibited high genetic variability, their abilities to cause human diseases have not been fully examined. METHODS: Clade typing and stx subtyping of EHEC O157:H7 strains, which were isolated in Japan during 1999-2011 from 269 HUS patients and 387 asymptomatic carriers (ACs) and showed distinct pulsed-field gel electrophoresis patterns, were performed to determine relationships between specific lineages and clinical presentation. RESULTS: Clades 6 and 8 strains were more frequently found among the isolates from HUS cases than those from ACs (P = .00062 for clade 6, P < .0001 for clade 8). All clade 6 strains isolated from HUS patients harbored stx2a and/or stx2c, whereas all clade 8 strains harbored either stx2a or stx2a/stx2c. However, clade 7 strains were predominantly found among the AC isolates but less frequently found among the HUS isolates, suggesting a significant association between clade 7 and AC (P < .0001). Logistic regression analysis revealed that 0-9 year old age is a significant predictor of the association between clade 8 and HUS. We also found an intact norV gene, which encodes for a nitric oxide reductase that inhibits Shiga toxin activity under anaerobic condition, in all clades 1-3 isolates but not in clades 4-8 isolates. CONCLUSIONS: Early detection of EHEC O157:H7 strains that belonged to clades 6/8 and harbored specific stx subtypes may be important for defining the risk of disease progression in EHEC-infected 0- to 9-year-old children.

Prevalence and epidemiological relationship of CMY-2 AmpC ß-lactamase and CTX-M extended-spectrum ß-lactamase-producing Escherichia coli isolates from broiler farms in Japan.

To evaluate the prevalence of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae in broiler chickens, 41 rectal samples taken from 4 commercial farms were examined. Desoxycholate hydrogen sulfide lactose agars, supplemented with either 4 µg/ml cefotaxime or 16 µg/ml ceftazidime, were used to screen ESC-resistant bacteria. ESC-resistant bacteria were isolated from all samples. Of the 164 ESC-resistant bacteria (included 4 isolates per a sample), 163 were Escherichia coli, while 1 isolate was identified as Enterobacter cloacae. Extended-spectrum ß-lactamase (ESBL) genes and plasmid-mediated AmpC ß-lactamase genes in the isolates were determined by PCR and sequencing. One AmpC ß-lactamase gene, bla(CMY-2) (66%), and 4 ESBL genes, bla(CTX-M-1) (26%), bla(CTX-M-55) (10%), bla(SHV-5) (4%) and bla(CTX-M-2) (3%), were detected in the E. coli isolates. The epidemiological relationship of the CMY-2 and CTX-M ß-lactamase-producing isolates among the farms was analyzed by pulsed-field gel electrophoresis using the XbaI restriction enzyme. Forty-one (Y1-Y41) and 14 (X1-X14) clusters were found in the CMY-2 and CTX-M-carrying E. coli isolates, respectively. Some clusters included isolates derived from more than 1 farm, indicating some cross-contamination of clonal strains and spread of CMY-2 AmpC ß-lactamase or CTX-M ESBL among the farms.

Emergence of Salmonella enterica serovar infantis harboring IncI1 plasmid with bla(CTX-M-14) in a broiler farm in Japan.

Cefotaxime (CTX)-resistant and -susceptible Salmonella enterica serovar Infantis isolates obtained from broilers raised on a farm in January 2010 in Japan were characterized to establish their resistance determinants. The CTX-resistant isolates produced CTX-M-14 extended-spectrum ß-lactamase and harbored 2 distinct plasmid of approximately 140- and 95-kb, whereas the CTX-susceptible isolates harbored one 140-kb plasmid. The 95-kb plasmids were replicon typed as IncI1 carrying the bla(CTX-M-14) gene, while the 140-kb plasmids were IncP and harbored the aphA1, aadA1, tetA, and sul1 genes. Genetic fingerprinting by pulsed-field gel electrophoresis revealed similar macrorestriction profiles amongst CTX-resistant and susceptible isolates, suggesting a clonal relationship. The presence of CTX-resistant S. Infantis on a broiler farm has occurred through the acquisition of IncI1 resistance plasmid.

Effect of cooled and chlorinated chiller water on Campylobacter and coliform counts on broiler carcasses during chilling at a middle-size poultry processing plant.

To evaluate the effect of cooled and chlorinated chill water for Campylobacter and coliforms at a middle-size processing plant which was considered to be difficult for eliminate pathogenic bacteria on carcasses, following three conditions were examined; keeping temperature at < 20, < 10 and < 10°C, and chlorine concentration at < 50, < 50 and 50 to 70 ppm during processing in experiment 1, 2 and 3 respectively. Fifteen prechill and 15 postchill carcasses were examined in each experiment. In lower temperature of experiment 2, decreasing rate (%) of coliforms was significantly higher (P<0.01) than that in experiment 1. In higher chlorination of experiment 3, no Campylobacter was detected from all postchill carcasses.