Eiji Yokoyama

Production of bacteriocin-like-substance by Listeria innocua against Listeria monocytogenes.

Cultures and culture filtrates of 129 Listeria innocua strains were examined for inhibitory activity against 18 strains of Listeria monocytogenes. Of the strains examined, 114 (88.4%) cultures and 126 (97.7%) culture filtrates had an inhibitory activity against strains of L. monocytogenes and most filtrates were sensitive to trypsin treatment. The authors concluded that most L. innocua strains produce a trypsin sensitive bacteriocin-like substance against L. monocytogenes.

Biased distribution of IS629 among strains in different lineages of enterohemorrhagic Escherichia coli serovar O157.

The distribution of insertion sequence (IS) 629 among strains of enterohemorrhagic Escherichia coli serovar O157 (O157) was investigated and compared with the strain lineages defined by lineage specific polymorphism assay-6 (LSPA-6) to demonstrate the effectiveness of IS629 analysis for population genetics analysis. Using pulsed-field gel electrophoresis and variable-number tandem repeat typing, 140 strains producing both VT1 and VT2 and 98 strains producing only VT2 were selected from a total of 592 strains isolated from patients and asymptomatic carriers in Chiba Prefecture, Japan, during 2003-2008. By LSPA-6 analysis, six strains had atypical amplicon sizes in their Z5935 loci and five strains had atypical amplicon sizes in their arp-iclR intergenic regions. Sequence analyses of PCR amplified DNAs showed that five of the six loci used for LSPA-6 analysis had tandem repeats and the allele changes were due to changes in the number of tandem repeats. Subculturing and long-term incubation was found to have no detectable effect on the lineages defined by LSPA-6 analysis, demonstrating the robustness of LSPA-6 analysis. Minimum spanning tree analysis reconstruction revealed that strains in lineage I, I/II, and II clustered on separate branches, indicating that the distribution of IS629 was biased among O157 strains in different lineages. Strains with LSPA-6 codes 231111, 211113, and 211114 had atypical amplicon sizes and were clustered in lineage I/II branch, and strains with LSPA-6 codes 212114, 221123, 221223, 222123, 222224, 242123, 252123, and 242222 had atypical amplicon sizes and clustered in lineage II branches. Linkage disequilibrium was observed in strains in every lineage when the standardized index of association was calculated using IS629 distribution data. Therefore, the distribution analysis of IS629 may be effective for population genetics analysis of O157 due to the biased IS629 distribution among strains in the three O157 lineages.

Prevalence and Genetic Properties of Salmonella enterica Serovar Typhimurium Definitive Phage Type 104 Isolated from Rattus norvegicus and Rattus rattus House Rats in Yokohama City, Japan†

ABSTRACT Salmonella enterica serovar Typhimurium was isolated from the intestinal contents of Rattus rattus and Rattus norvegicus house rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carry Salmonella serovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 from R. rattus and R. norvegicus were similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0- and 1.2-kbp integrons, carrying genes pse1, pasppflo-like, aadA2, sulI, and tet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carrying pse1 and sulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.

Clade analysis of enterohemorrhagic Escherichia coli serotype O157:H7/H- strains and hierarchy of their phylogenetic relationships

Enterohemorrhagic Escherichia coli serotype O157:H7/H-(O157) strains isolated in Chiba prefecture, Japan, during 2002-2009 were studied by lineage, subgroup, cluster, and clade analysis. Lineage analysis of 470 O157 strains with no known epidemiological relationships using lineage specific polymorphism assay-6 showed that there were 242 lineage I strains, 160 lineage I/II strains, 67 lineage II strains, and 1 atypical strain. Clade analysis of these strains by single nucleotide polymorphism in eight loci showed that lineage I contained all the clade 1, clade 2, and clade 3 strains, and some of the clade 4/5 strains. In contrast, clade 7, clade 8, and the remaining clade 4/5 strains were divided between lineage I/II and II, and clade 6 was in lineage I/II, suggesting paraphyletic evolution of these lineages. Cluster and subgroup analysis of the stx phage insertion site showed that all lineage I strains were cluster 3 and all lineage I/II and II strains, with the exception of clade 9, were in cluster 1. Clade analysis also indicated that there were three phylogenetic groups of clade 4/5 strains: ancestral groups containing lineage I/IIand II strains and a descendant group containing lineages I. Analysis of stx2c gene distribution showed that stx2c was in ancestral clade 4/5 strains but not in descendant 4/5 strains, suggesting that the ancestral group may be clade 4 as reported by Manning et al. The results with the markers used in this study suggested that the hierarchy of O157 phylogenetic relationships was lineage as the upper level, followed by subgroup and then cluster, and clade as the lowest level. The need for refinement of clade definition and modification of the model of the O157 evolution have been discussed.

Linkage disequilibrium of the IS629 insertion among different clades of enterohemorrhagic Escherichia coli O157:H7/H-strains.

The distribution of insertion sequence (IS) 629 was investigated among enterohemorrhagic Escherichia coli O157:H7/H-(O157) strains in different clades. Minimum spanning tree analysis showed that most strains in each clade clustered in a separate branch, indicating biased distribution of the IS629 insertion in different clades. The standardized index of association of the IS629 distribution data showed linkage disequilibrium in the strains in every clade, indicating that IS629 distribution data could be used for population genetic analysis. For this population genetic analysis, the Φ(PT) value, an analogue of F(ST), was calculated and indicated that clade 7 strains could be split into two clades based on their lineages. The degree of pairwise linkage disequilibrium was quite different among these two groups. The clade 7 split was in agreement with the model of O157 paraphyletic evolution and a new designation of the lineage II clades was proposed. The prevalence of strains with an IS629 insertion in certain loci was significantly different in different clades. Some of these significant differences were in loci in strains in branches of clades that were close in the O157 phylogenetic model, suggesting that IS629 insertion/deletion was not directly related to the divergence of O157 clades.

Analysis of the population genetics of clades of enterohaemorrhagic Escherichia coli O157:H7/H- isolated in three areas in Japan

The genetic differences of enterohaemorrhagic Escherichia coli O157 (O157) strains isolated from humans in three widely‐separated areas in Japan were analysed to provide information on possible geographic aspects of O157 pathogenicity.

Mutational analysis of reduced telithromycin susceptibility of Streptococcus pneumoniae isolated clinically in Japan

A total of 132 Streptococcus pneumoniae isolates collected between 2005 and 2006 in Japan were examined for susceptibility to telithromycin (TEL) and macrolide. The overall resistance to macrolide was 80%. Among the isolates, 128 strains had low-level TEL susceptibility (minimal inhibitory concentrations [MICs] 0.03-1 microg mL(-1)), suggesting that pneumococci with reduced susceptibility to TEL have appeared without prior exposure to the drug, although none of the isolates were assigned as TEL-resistant (breakpoint, > or = 4 microg mL(-1)). Eight of these isolates (MIC 0.5-1 microg mL(-1)) were analyzed for macrolide resistance determinants and genetic relatedness. They all carried mefE-mel, which encodes the macrolide efflux genetic assembly, and three also harbored ermB, which encodes rRNA methylase. Allele replacement mutagenesis of the corresponding genes in the clinical isolates revealed that reduced TEL susceptibility (MIC 1 microg mL(-1)) in S. pneumoniae may be caused by acquisition of the mefE-mel element only and additionally conferred by the ermB determinant.

Phylogenetic and population genetic analysis of Salmonella enterica subsp. enterica serovar Infantis strains isolated in Japan using whole genome sequence data

Salmonella enterica subsp. enterica serovar Infantis has been reported to be carried asymptomatically in chickens and isolated from some human patients with diarrhea. The aim of this study was to investigate the phylogeny of S. Infantis strains isolated in Japan from chicken meat, chicken egg shells, environmental samples from a grading and packing center for chicken eggs (GP center), diarrhea patients, and asymptomatic carriers based on whole genome sequence data. The S. Infantis strains were in five clusters in a phylogenetic tree reconstructed by the maximum likelihood method. The five clusters were confirmed by neighbor-net and Bayesian cluster analyses. Two of the five clusters formed a group containing all of the strains isolated from chicken meat and some of the strains isolated from diarrhea patients and asymptomatic carriers. The median-joining network reconstructed in this study showed that strains in one of these two clusters diverged from one node with similar relatively short branches, suggesting clonal dissemination of these strains. The other three clusters formed a group containing all of the strains isolated from chicken egg shells and the GP center, and the remaining strains from diarrhea patients and asymptomatic carriers. Interestingly, strains isolated from patients did not cluster in only one group, indicating that none of the S. Infantis strains in this study had significantly higher human pathogenicity. The population genetic analyses in this study showed the separation of the five clusters into two groups was concordant with the sources where the strains in the clusters were isolated. These results suggested that evolutionary groups with higher hierarchy than the clusters identified in this study may be present, although such groups could not be determined by phylogenetic, neighbor-net, and Bayesian analyses in this study. Determination of higher level S. Infantis evolutionary groups should be investigated using other types of genetic markers.

A novel subpopulation of Salmonella enterica serovar Infantis strains isolated from broiler chicken organs other than the gastrointestinal tract.

Salmonella enterica subsp. enterica serovar Infantis strains were isolated from broiler chickens from six farms in Japan and the pathogenicity associated with the recently reported 280kbp mega plasmid was examined by possession of the plasmid and histopathology of tissues from these chickens. S. Infantis strains were isolated from 10 of 24 chickens. Phylogenetic, network and Bayesian cluster analyses were used to determine whether these strains were in the previously defined Clusters 1-5. Phylogenetic analysis classified the strains isolated in this study in two groups (Groups A and B). Both groups contained strains from gastrointestional contents, but only Group A also contained strains from spleen, liver, and lymphoid tissues. Histopathology showed suppurative splenitis in a spleen from which Group A strains were isolated. Although network and Bayesian cluster analyses were unable to differentiate Group A and B strains from the previously defined Clusters 1-5, population genetic analysis indicated that Group A was a different population from Cluster 5, indicating that Group A would be a subpopulation of Cluster 5. The irp2 gene, which is in the mega plasmid carried by a pathogenic S. Infantis strain recently isolated in Israel, was found in both Groups A and B strains and in the previously reported Clusters 4 and 5 strains. These results suggested that Group A would be a novel subpopulation of the previously defined Cluster 5, and presence of the mega plasmid may not be related whether S. Infantis strains can infect certain organs.

A Geographically Widespread Outbreak Investigation and Development of a Rapid Screening Method Using Whole Genome Sequences of Enterohemorrhagic Escherichia coli O121

From 2014 to 2015, we investigated a suspected nationwide outbreak of enterohemorrhagic Escherichia coli serogroup O121. However, similar pulsed field gel electrophoresis (PFGE) profiles and the lack of epidemiological links between the isolates made detection of the outbreak difficult. To elucidate a more precise genetic distance among the isolates, whole genome sequence (WGS) analyses were implemented in the investigation. The WGS-based single nucleotide polymorphism (SNP) analysis showed that 23 out of 44 isolates formed a distinct cluster (the number of intra-cluster SNPs was ≤8). Specific genomic regions in the clustered isolates were used to develop a specific PCR analysis. The PCR analysis detected all the clustered isolates and was suitable for rapid screening during the outbreak investigation. Our results showed that WGS analyses were useful for the detection of a geographically widespread outbreak, especially for isolates showing similar PFGE profiles and for the development of a rapid and cost-effective screening method.