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Abstract Salmonella enterica serovar Agona strains isolated from human cases were compared to strains that were derived from a clone caused a serovar shift in broilers. Pulsed field gel electrophoresis (PFGE) analysis with XbaI or BlnI digestion showed that three of seven strains from human case strains and most of the 81 strains from broilers were clustered in single complex in a minimum spanning tree (MST) reconstructed from the PFGE data. All the strains from human cases and 22 randomly selected strains from broilers were also analyzed by whole genome sequencing (WGS). Analysis of single nucleotide polymorphism (SNP) in the S. Agona core genes showed that four strains from human cases and all the strains from broilers were clustered in a maximum likelihood phylogenetic tree (ML tree) and an MST. These results indicated that the strains derived from the clone caused the serovar shift had already spread to humans. PFGE analysis with XbaI showed that four strains from broilers did not cluster with the other strains in an MST, though all those strains clustered in an ML tree and an MST reconstructed from SNP data. Moreover, three strains from broilers did not cluster in an MST reconstructed from PFGE with BlnI digestion, though those strains clustered in an ML tree and an MST reconstructed from SNP data. Therefore, it was suggested that S. Agona strains derived from a particular clone could not be traced by PFGE analysis but can be investigated by WGS analysis. [ABSTRACT FROM AUTHOR]

Seventeen clusters of Shiga toxin-producing Escherichia coli O157:H7/- (O157) strains, determined by cluster analysis of pulsed-field gel electrophoresis patterns, were analyzed using whole genome sequence (WGS) data to investigate this pathogen's molecular epidemiology. The 17 clusters included 136 strains containing strains from nine outbreaks, with each outbreak caused by a single source contaminated with the organism, as shown by epidemiological contact surveys. WGS data of these strains were used to identify single nucleotide polymorphisms (SNPs) by two methods: short read data were directly mapped to a reference genome (mapping derived SNPs) and common SNPs between the mapping derived SNPs and SNPs in assembled data of short read data (common SNPs). Among both SNPs, those that were detected in genes with a gap were excluded to remove ambiguous SNPs from further analysis. The effectiveness of both SNPs was investigated among all the concatenated SNPs that were detected (whole SNP set); SNPs were divided into three categories based on the genes in which they were located (i.e., backbone SNP set, O-island SNP set, and mobile element SNP set); and SNPs in non-coding regions (intergenic region SNP set). When SNPs from strains isolated from the nine single source derived outbreaks were analyzed using an unweighted pair group method with arithmetic mean tree (UPGMA) and a minimum spanning tree (MST), the maximum pair-wise distances of the backbone SNP set of the mapping derived SNPs were significantly smaller than those of the whole and intergenic region SNP set on both UPGMAs and MSTs. This significant difference was also observed when the backbone SNP set of the common SNPs were examined (Steel-Dwass test, P ≤ 0.01). When the maximum pair-wise distances were compared between the mapping derived and common SNPs, significant differences were observed in those of the whole, mobile element, and intergenic region SNP set (Wilcoxon signed rank test, P ≤ 0.01). When all the strains included in one complex on an MST or one cluster on a UPGMA were designated as the same genotype, the values of the Hunter-Gaston Discriminatory Power Index for the backbone SNP set of the mapping derived and common SNPs were higher than those of other SNP sets. In contrast, the mobile element SNP set could not robustly subdivide lineage I strains of tested O157 strains using both the mapping derived and common SNPs. These results suggested that the backbone SNP set were the most effective for analysis of WGS data for O157 in enabling an appropriation of its molecular epidemiology. [ABSTRACT FROM AUTHOR]

A quantitative and qualitative comparison was carried out of Mycobacterium tuberculosis Beijing strains isolated in three remote areas of Japan. A total of 452 strains from Chiba Prefecture, 75 from Yamagata Prefecture, and 315 from Kobe City were analyzed for 24 loci by variable number of tandem repeats typing (24 Beijing -VNTR). All strains were classified in six Beijing subgroups (B SUB ), B1 to B5 and T, based on a minimum spanning tree reconstructed using data of a standard set of 15 VNTR loci. No significant difference was found in the distribution of strains in the B SUB in the three areas, with one exception due to a B5 outbreak in Yamagata, indicating no significant quantitative difference in the B SUB in the three areas ( P < 0.01, Chi -square test). In addition, when strains in each B SUB isolated in the three areas were mixed and standardized index of association ( I A s ) and variance ( Φ PT ) values were calculated, no significant qualitative difference in the B SUB in the three areas was found. These results suggested that the B SUB diverged prior to the introduction of M. tuberculosis Beijing strains into Japan. Differences in the distribution of strains in each B SUB between Japan and continental Asian countries suggested there had been genetic drift in the continental Asian countries in which B4 had been dominant. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Minimum spanning tree analysis showed biased IS629 distribution in EHEC O157 strains in each clade. [•] Linkage disequilibrium was observed for the IS629 distribution in EHEC O157 strains in each clade. [•] Population genetic analyses showed that clade 7 strains should be split into two clades based on lineages. [•] This study showed that IS629-related events were not involved in the divergence of O157 clades. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Population genetic analysis using variable-number tandem repeat (VNTR) data of 23 loci (15 “optimized MIRU” loci and eight “Beijing option” loci) was done on Mycobacterium tuberculosis Beijing lineage strains isolated in Japan. These strains were divided into Beijing subgroups (BSUB) B1–B5 and T2 by minimum spanning tree (MST) analysis. The ΦPT values among the BSUB, a measure of their molecular variance, were significantly different from zero with 999 permutations, indicating the validity of BSUB classification using the 23 VNTR loci. Higher number of migrants (Nm) values were observed between B1 and T2, B4 and T2, B3 and T2, and B3 and B4 in a phylogenetic network model reconstructed from previously reported single-nucleotide polymorphism (SNP) data. These BSUB combinations, except B3 and B4, shared SNP types; i.e., ST19 was in B1 and T2 and in B4 and T2, and STK was in B3 and T2. These results taken together suggested that shared SNP types were not due to homoplasy, but to strong genetic relatedness between those BSUB. Haploid genetic diversity and standardized index of association values were different in each BSUB, indicating that the diversity of each BSUB was different. Although the differences in BSUB diversity were mostly in accordance with the relative divergence order of the BSUB in a phylogenetic network model, the diversity of B4 was biased by a significant increase in the number of strains in this study from patients born after 1964 (Fisher’s exact test P <0.01). The different diversity of each BSUB indicated increased diversity of Beijing lineage strains, perhaps contributing to the survival and dissemination of these strains. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses were compared to determine whether VNTR analysis was effective for population genetic analysis of Mycobacterium tuberculosis strains. A total of 682 strains, 510 Beijing genotype and 172 non-Beijing genotype strains, were studied. The number of repeats was investigated for 24 VNTR loci: the 15 loci of “optimized miru”, the 8 loci of “Beijing option”, and 1 locus for “JATA12”. Six loci (miru31, Mtub4, QUB4156c, QUB3232, VNTR3820, and VNTR4120) showed significantly different median numbers of repeats in strains belonging to different lineages defined by LSP (P <0.01, Mann–Whitney U test). When a minimum-spanning tree (MST) was reconstructed using these 6 loci, most strains clustered in the expected branches in the MST branches. However, topology of the MST was not congruent with the evolutional hypothesis of M. tuberculosis, indicating that MST analysis using VNTR data should not use for phylogeny of the organism. When the standardized index of association (sI A) was calculated using data for the 6 VNTR loci, the value of sI A was significantly different from zero (Monte Carlo simulation with 10,000 resamplings) in every lineage, indicating the linkage disequilibrium in different lineage strains of M. tuberculosis. These results were consistent with the hypothesis that clonal evolution of lineages of the organism has occurred. Therefore, the 6 loci identified in this study would be effective for M. tuberculosis population genetic analysis due to their significantly different median numbers of repeat and linkage disequilibrium though VNTR data was not effective for phylogeny of the organism. [Copyright &y& Elsevier]

A novel gene amplification method, loop-mediated isothermal amplification (LAMP), has been recently developed as a rapid, specific diagnostic method for various infectious diseases. We have investigated whether LAMP can be used to detect small numbers of enteroaggregative Escherichia coli (EAEC) cells contaminated in food samples. Primers for LAMP reaction were designed with EAEC aggR gene sequences (available in GenBank). LAMP specificity with these primer', was the same as that of PCR in a study of 37 EAEC and 42 non-EAEC bacterial strains. The sensitivity of the LAMP method was belier than that of PCR in a study of serially diluted EAEC cells. The LAMP method was significantly more effective than was PCR in detecting EAEC-contaminated food samples (Fisher's exact test, P < 0.05). Therefore, the LAMP method described here should be useful for detecting small numbers of EAEC cells in food samples. [ABSTRACT FROM AUTHOR]

Ninety-five enterohemorrhagic Escherichia coli serovar O157 strains, including 30 strains isolated from 13 intrafamily outbreaks and 14 strains isolated from 3 mass outbreaks, were studied by pulsed-field gel electrophoresis (PFGE) and variable number of tandem repeats (VNTR) typing, and the resulting data were subjected to cluster analysis. Cluster analysis of the VNTR typing data revealed that 57 (60.0%) of 95 strains, including all epidemiologically linked strains, formed clusters with at least 95% similarity. Cluster analysis of the PFGE patterns revealed that 67 (70.5%) of 95 strains, including all but 1 of the epidemiologically linked strains, formed clusters with 90% similarity. The number of epidemiologically unlinked strains forming clusters was significantly less by VNTR cluster analysis than by PFGE cluster analysis. The congruence value between PFGE and VNTR cluster analysis was low and did not show an obvious correlation. With two-step cluster analysis, the number of clustered epidemiologically unlinked strains by PFGE cluster analysis that were divided by subsequent VNTR cluster analysis was significantly higher than the number by VNTR cluster analysis that were divided by subsequent PFGE cluster analysis. These results indicate that VNTR cluster analysis is more efficient than PFGE cluster analysis as an epidemiological tool to trace the transmission of enterohemorrhagic E. coli O157. [ABSTRACT FROM AUTHOR]

Abstract: Variable numbers of tandem repeat (VNTR) typing of Mycobacterium tuberculosis was performed on 54 strains including 23 strains derived from 9 outbreaks. PCR amplicon sizes of 12 mycobacterial interspersed repetitive unit tandem repeat loci were measured using both agarose gel electrophoresis and capillary electrophoresis. Similarities using agarose gel electrophoresis of Euclidian distances among the 23 strains derived from the 9 outbreaks were significantly lower than that using capillary electrophoresis (Wilcoxon signed ranks test, P <0.01). By clustering analysis using unweighted pair group method using arithmetic averages, all of the 23 strains derived from the 9 outbreaks were each clustered with more than 90% similarities based on the distance using capillary electrophoresis. In contrast, differential clusters with more than 90% similarity were observed with only 7 strains derived from 3 outbreaks when analyzed by agarose gel electrophoresis. These results indicated that measurement of PCR amplicon size of tandem repeat loci should be carried out using capillary electrophoresis and that agarose gel electrophoresis is not suitable for clustering analysis of M. tuberculosis VNTR typing. [Copyright &y& Elsevier]

Abstract: The actual prevalence of Listeria monocytogenes from contents of swine cecum was investigated. The efficiency of Listeria enrichment broth (LEB) for isolation was examined by the recovery of artificially inoculated L. monocytogenes in contents of swine cecum. The numbers of organisms did not increase after 48h incubation, but increased when the rapid decrease in pH of the LEB was adjusted. Between 1991 and 1993, 250 contents of swine cecum were examined for the prevalence of L. monocytogenes using LEB enrichment, either with or without pH adjustment. L. monocytogenes was isolated from 74 samples in 1993 with pH adjustment, however, no organisms were isolated in 1991 and 1992. It was suggested that the marked rise of the L. monocytogenes isolation was due to the spread of the organism among swine. Furthermore, 67 out of the 74 isolates were identified as 1/2c by serotyping. The serovar 1/2c strains showed genetic diversity by random amplified polymorphic DNA. [Copyright &y& Elsevier]

Abstract: Inhibition of isolation of Listeria monocytogenes by bacteriocin-like substance (BLS)-producing Listeria innocua after enrichment culture was investigated. When 26 L. monocytogenes strains were examined in combination with eight L. innocua strains using the spot on lawn method, 52/208 (25.0%) combinations showed the growth inhibition of L. monocytogenes. When two Listeria species were cultured simultaneously in selective enrichment broth, inhibition of isolation of L. monocytogenes was observed in 12/52 of the combinations at 24h (23.1%), in 24/52 at 48h (46.2%) and in 30/52 (57.7%) after 7 days of incubation. The randomly amplified polymorphic DNA profiles showed no interstrain similarities between either strains of the BLS-producing L. innocua or the BLS-sensitive L. monocytogenes strains. Therefore inhibition by BLS-producing L. innocua of isolation of L. monocytogenes after enrichment culture is unlikely to be dependent upon a particular genetic profile. [Copyright &y& Elsevier]

Enterohemorrhagic Escherichia coli serovar O157 (O157) strains with highly similar pulsed-field gel electrophoresis (PFGE) patterns were isolated in Japan during 2007 and 2008. Several genetic features related to O157 evolution were investigated to indicate whether homoplasy might have contributed to the highly similar PFGE patterns in these strains. The O157 strains were classified in lineage I/II, as defined by a lineage-specific polymorphism assay-6 with an atypical allele in Z5935 (code: 231111). Analysis of the insertion sites of stx2 phage in these strains showed that the sites were "occupied" in yehV and "intact" in wrbA, indicating that the strains were derived from "Cluster 1" of "Subgroup C." When a specific single-nucleotide polymorphism in ECs2357 in clade 8 strains was investigated, all of the strains in the present study were confirmed to be clade 8 strains. These results indicated that the O157 strains in this study had common genetic features, suggesting that the highly similar PFGE patterns of these strains were not due to homoplasy. Because no common source of these strains could be identified in 2007 to 2008 in Japan, these strains may have emerged from a unique O157 clade 8 clone and then spread by dissemination in Japan. [ABSTRACT FROM AUTHOR]

A novel virulence gene, norV , that encodes nitric oxide (NO) reductase, was examined to investigate the emergence of enterohemorrhagic Escherichia coli (EHEC) O157 subgroup C clusters 2 and 3 from subgroup C cluster 1. Deletion of norV occurred at a point between cluster 1 and cluster 2 just after or at the same time that an stx2 bacteriophage, which retains Shiga toxin 2 gene, was inserted into wrbA , which encodes a novel multimeric flavodoxin-like protein, in EHEC O157. Sensitivity of NO to anaerobic growth was correlated with the deletion of norV in all EHEC O157 individuals tested. The C467A mutation of fimH , which encodes minor component of type 1 fimbriae, occurred within cluster 1, not as a transition from cluster 1 to cluster 2, indicating that there is a cluster 1 minority branch that leads to cluster 2. These data refine the evolutionary history of an emerging EHEC O157. [ABSTRACT FROM AUTHOR]

The prevalence of the phylogenetic groups of Mycobacterium tuberculosis Beijing genotype has been reported to be similar in different areas of Japan. However, recent reports from rural areas of Japan show a low prevalence of modern Beijing strains, suggesting that the distribution of modern Beijing strains may have changed recently. Therefore, multi-locus variable number of tandem repeats analysis (MLVA) and draft whole genome sequence (DWGS) analysis were carried out to investigate the prevalence of particular genotype strains. Nine hundred and ninety modern Beijing strains were studied using minimum spanning tree (MST) analysis and neighbor-net analysis of MLVA and WGS data. An MST of M. tuberculosis Beijing genotype strains reconstructed from 12 loci-MLVA data showed two large complexes with the J 12 -0006 MLVA pattern. In one of the complexes, strains with the pECT07 pattern produced by 24 loci-MLVA and its SLVs were most prevalent. DWGS analysis was carried out for pECT07 and its SLV strains. Neighbor-net and MST analyses of the DWGS data showed that pECT07 and its SLV strains were grouped in separate clusters. When all the combinations of two of the tested strains were analyzed, MST analysis showed that only 9 (1.7%) of the 528 pairs of tested strains had 5 or less SNPs. The results of this study suggested that pECT07 and its variants were prevalent among M. tuberculosis modern Beijing strains in Chiba Prefecture, but the prevalence of those strains may not have been due to an earlier large-scale latent outbreak. [ABSTRACT FROM AUTHOR]

Highlights: [•] High resolution melting analysis was effective for clade differentiation of O157. [•] Of all clades, clade 12 strains had the lowest pathogenicity for hemorrhagic colitis. [•] The Stx2 titers in clade 7 strains were significantly lower than in clade 6 and 8 strains. [•] There were significantly less strains with the stx2 gene in clade 7 than in clade 6 and 8. [•] These were contradicted with the O157 evolutionary model comparing to other lineage I/II. [ABSTRACT FROM AUTHOR]