Population structure of Escherichia coli O26 : H11 with recent and repeated stx2 acquisition in multiple lineages.

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.

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.

Evaluation of streptococcal toxic shock-like syndrome caused by group B streptococcus in adults in Japan between 2009 and 2013.

Infection with Streptococcus agalactiae has long been recognized in infants. In recent years, S. agalactiae is an important cause of morbidity and mortality among adults and among those with underlying medical condition. Several cases of GBS infection and more fulminant disease similar to streptococcal toxic shock syndrome have recently been reported. We report here that 19 S. agalactiae strains were isolated from streptococcal toxic shock-like syndrome cases involving adult patients in Japan between 2009 and 2013. The average age of the patients was 66.3 years. At least one underlying disease was present in 47.4% (9/19) of the patients. The most prevalent serotype among these strains was Ib. All serotype Ib strains belonged to clonal complex 10 and were ciprofloxacin resistant. In contrast, all strains were susceptible to penicillin G, ampicillin, cefazolin, cefotaxime, imipenem, panipenem, and linezolid. The characteristic type distributions of streptococcal toxic shock-like syndrome isolates differed between isolates obtained from vaginal swabs of women and infants with invasive infections.

Streptococcal toxic shock syndrome from necrotizing soft-tissue infection of the breast caused by a mucoid type strain.

Streptococcal toxic shock syndrome is a severe infectious disease. We report a Japanese case of Streptococcal toxic shock syndrome caused by a highly mucoid strain of Streptococcus pyogenes. A 31-year old female with shock vital sign presented at a tertiary medical center. Her left breast was necrotizing and S. pyogenes was detected by Immunochromatographic rapid diagnostic kits. Intensive care, including administration of antibiotics and skin debridement, was performed. After 53 days in our hospital, she was discharged. The blood cultures and skin swab cultures all grew S. pyogenes which displayed a highly mucoid morphology on culture media. In her course of the disease, the Streptococcus strain had infected two other family members. All of the strains possessed the T1 and M1 antigens, as well as the emm1.0 gene. As for fever genes, the strains were all positive for speA, speB, and speF, but negative for speC. All of the strains exhibited and the same pattern in PFGE with the SfiI restriction enzyme. The strain might have spread in the local area by the data from the Japanese Infectious Disease Surveillance Center. Immunochromatographic rapid diagnostic kits are very useful for detecting S. pyogenes. However, they can not be used to diagnose severe streptococcul disease by highly mucoid strain alone. Careful observation of patients and colony morphology are useful methods for diagnosing severe streptococcal disease by highly mucoid strain.

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.

Evaluation of the culture method NIHSJ-02 alternative to ISO 10272-1:2006 for the detection of campylobacter jejuni and campylobacter coli in chicken: collaborative study.

For the surveillance of the prevalence of Campylobacter jejuni and Campylobacter coli in raw chicken products in Japan, a qualitative method, National Institute of Health Sciences Japan (NIHSJ)-02, was developed as an alternative to International Organization for Standardization (ISO) 10272-1:2006. In the NIHSJ-02 culture method, the enrichment step is carried out in a reduced volume of Preston broth at 42 +/- 1 degrees C to reduce cost and space, and to prevent the overgrowth of background bacteria. To evaluate the performance of NIHSJ-02, a collaborative study was conducted, and the results obtained by NIHSJ-02 were compared with those obtained using the reference method, ISO 10272-1:2006. Fifteen laboratories participated; each examined 48 minced chicken samples consisting of test samples uninoculated, inoculated with C. jejuni at a low or high level, and inoculated with C. coli at a low level. The average probabilities of detection by NIHSJ-02 across laboratories were 0.033, 0.222, 0.678, and 0.267 in samples uninoculated, inoculated with C. jejuni at a low and high level, and with C. coli at a low level, respectively. Those by ISO 10272-1:2006 were 0.051, 0.128, 0.551, and 0.090. Significantly higher probabilities of detection were determined by NIHSJ-02 compared to ISO 10272-1:2006, except for uninoculated samples. On the other hand, significantly lower frequency of occurrence of background bacteria was observed by NIHSJ-02 (43.1%) compared with ISO 10272-1:2006 (92.6%). NIHSJ-02 showed better performance than ISO 10272-1:2006 with regard to the selective detection of C. jejuni and C. coli in chicken.

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.

Diversity of Melissococcus plutonius from honeybee larvae in Japan and experimental reproduction of European foulbrood with cultured atypical isolates.

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for ß-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research.

Surveillance of severe invasive group G streptococcal infections in Japan during 2002-2008.

Group G Streptococcus strains isolated from patients with severe invasive infections in the period 2002-2008 were surveyed and their prevalence compared with that observed in the period 1995-2001 in Japan. Strains with genotypes stg485, stg6792, stc36, stg6, and stg652 were isolated in both periods, whereas various new genotypes appeared in 2002-2008 and some genotypes found in 1995-2001 were not found subsequently, thus indicating a change in the prevalent genotyped strains causing severe invasive streptococcal infections.

Characterization of lysine decarboxylase-negative strains of Salmonella enterica serovar Enteritidis disseminated in Japan.

Salmonella enterica serovar Enteritidis is one of the leading causes of food-borne diseases in Japan. Typically, Salmonella spp. test positive for lysine-decarboxylase. However, the number of isolates of serovar Enteritidis without lysine-decarboxylase activity increased in Japan in 2003. Among 109 strains from distinct outbreaks, 10 lacked lysine-decarboxylase activity. Nine of the ten lysine-decarboxylase-negative strains showed quite similar pulsed-field gel electrophoresis profiles. Their lysine-decarboxylase phenotype was recovered by introduction of the cadBA locus from an lysine-decarboxylase-positive strain. Although the cad loci of the lysine-decarboxylase-negative strains seemed to be intact without any insertion sequences, cadC, a positive regulator of cadBA, had a single-base deletion at the same position, the 973rd base (cytosine), in all the nine lysine-decarboxylase-negative strains, whereas the wild-type cadC gene has a 1542 bp coding region (514 amino acids). This deletion was expected to produce a truncated (338 amino acids) form of CadC due to a frameshift. Because CadC senses environmental cues such as external pH and lysine through its putative C-terminal periplasmic domain, it is likely that the truncated CadC is not sensitive enough to external signaling to activate the cadBA operon, resulting in loss of the lysine-decarboxylase activity. Our results suggest that dissemination of these genetically closely related strains of serovar Enteritidis accounts for the unusual increase in the isolation of lysine-decarboxylase-negative strains.