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Enterohemorrhagic Escherichia coli O157 (O157) strains can be subdivided into clades based on their single-nucleotide polymorphisms, but such analysis using conventional methods requires intense effort by laboratories. Although multi-locus variable-number tandem repeat analysis (MLVA), which can be performed with low laboratory burden, has been used as a molecular epidemiological tool, it has not been evaluated whether MLVA can be used the clade subdivision of O157 strains like it can for that of other pathogenic bacteria. This study aimed to establish a method for subdividing O157 strains into clades using MLVA data. The standardized index of association, ISA, for O157 strains isolated in Chiba prefecture, Japan (Chiba isolates) revealed the presence of unique tandem repeat patterns in each major clade (clades 2, 3, 7, 8, and 12). A likelihood database of tandem repeats for these clades was then constructed using the Chiba isolates, and a formula for maximum a posteriori (MAP) estimation was constructed. The ratio of the number of O157 strains putatively subdivided into a clade by MAP estimation from MLVA data relative to the number of O157 strains subdivided using single-nucleotide polymorphism analysis (designated as the concordance ratio [CR]) was calculated using the Chiba isolates and O157 strains isolated in Yamagata prefecture (Yamagata isolates). The CRs for the major Chiba and Yamagata isolate clades, other than clade 2, were 89%-100%. Although the CR for clade 2 Chiba isolates was >95%, that of the Yamagata isolates was only 78.9%. However, these clade 2 CRs were not significantly different from one another, indicating that clade 2 strains can be subdivided correctly by MAP estimation. In conclusion, this study expands the utility of MLVA, previously applied predominantly for molecular epidemiological analysis, into a low-laboratory-burden tool for subdividing O157 strains into phylogenetic groups., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hirai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Eighty strains of enterohemorrhagic Escherichia coli O157:H7/H- were analyzed by three single-nucleotide polymorphism (SNP) panels using whole-genome sequencing data. The partial concordance of SNP types among the different SNP panels was observed on minimum spanning trees reconstructed with SNP data. As for lineage I/II strains, some of the clade 7 strains belonged to one unique SNP type as determined by three panels, suggesting that clade 7 should be divided into at least two genotypes, namely, the unique type and the rest. In addition, clade 8 contained two unique genotypes, which was consistent with the previous prediction. Similarly, for lineage II, clade 12 should be divided into three genotype strains. In contrast, many strains of several clades belonging to lineage I were clustered into the same node on each minimum spanning tree upon testing with the three SNP panels. Previous studies reported that lineage I diverged more recently than lineages I/II and II. Such low diversity in lineage I in this study may have arisen because this lineage has not accumulated SNPs because of its relatively recent divergence. Based on the concordance observed in this study, some of the previously published O157 genotype distribution data were successfully interpreted to clarify the clade distribution, which was well supported by previous literature.

Introduction: 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., Methods: Nine hundred and ninety modern Beijing strains were studied using minimum spanning tree (MST) analysis and neighbor-net analysis of MLVA and WGS data., Results: 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., Conclusions: 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., (Copyright © 2022 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The prevalence of Shiga toxin-producing Escherichia coli O157 (STEC O157) strains in wild deer and boar in Japan was investigated. STEC O157 strains were isolated from 1.9% (9/474) of the wild deer and 0.7% (3/426) of the wild boar examined. Pulsed-field gel electrophoresis (PFGE) analysis classified the wild deer and boar strains into five and three PFGE patterns, respectively. The PFGE pattern of one wild boar strain was similar to that of a cattle strain that had been isolated from a farm in the same area the wild boar was caught, suggesting that a STEC O157 strain may have been transmitted between wild boar and cattle. Clade analysis indicated that, although most of the strains were classified in clade 12, two strains were classified in clade 7. Whole-genome sequence (WGS) analysis indicated that all the strains carried mdfA, a drug resistance gene for macrolide antibiotics, and also pathogenicity-related genes similar to those in the Sakai strain. In conclusion, our study emphasized the importance of food hygiene in processing meat from Japanese wild animals for human consumption.

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.

Enterohaemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Although stx1 and stx2 were found within the late operons of the Stx-encoding phages (Stx-phages), stx1 could mainly be transcribed from the stx1 promoter ( P Stx1 ), which represents the functional operator-binding site (Fur box) for the transcriptional regulator Fur (ferric uptake regulator), upstream of stx1 . In this study, we found that the production of Stx1 by EHEC was affected by oxygen concentration. Increased Stx1 production in the presence of oxygen is dependent on Fur, which is an Fe 2+ -responsive transcription factor. The intracellular Fe 2+ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe 2+ availability drove the formation of less Fe 2+ -Fur, less DNA binding to the P Stx1 region, and an increase in Stx1 production.

In Japan's Kanto region, the number of Salmonella enterica serovar Chester infections increased temporarily between 2014 and 2016. Concurrently with this temporal increase in the Kanto region, S . Chester isolates belonging to one clonal group were causing repetitive outbreaks in Europe. A recent study reported that the European outbreaks were associated with travelers who had been exposed to contaminated food in Morocco, possibly seafood. Because Japan imports a large amount of seafood from Morocco, we aimed to establish whether the temporal increase in S . Chester infections in the Kanto region was associated with imported Moroccan seafood. Short sequence reads from the whole-genome sequencing of 47 S . Chester isolates from people in the Kanto region (2014-2016), and the additional genome sequences from 58 isolates from the European outbreaks, were analyzed. The reads were compared with the complete genome sequence from a S . Chester reference strain, and 347 single nucleotide polymorphisms (SNPs) were identified. These SNPs were used in this study. Cluster and Bayesian cluster analyses showed that the Japanese and European isolates fell into two different clusters. Therefore, Φ PT and I A S values were calculated to evaluate genetic differences between these clusters. The results revealed that the Japanese and European isolates were genetically distinct populations. Our root-to-tip analysis showed that the Japanese isolates originating from one clone had accumulated mutations, suggesting that an emergence of this organism occurred. A minimum spanning tree analysis demonstrated no correlation between genetic and geographical distances in the Japanese isolates, suggesting that the emergence of the serovar in the Kanto region did not involve person-to-person contact; rather, it occurred through food consumption. The d N / d S ratio indicated that the Japanese strain has evolved under positive selection pressure. Generally, a population of bacterial clones in a reservoir faces negative selection pressure. Therefore, the Japanese strain must have existed outside of any reservoir during its emergence. In conclusion, S . Chester isolates originating from one clone probably emerged in the Kanto region via the consumption of contaminated foods other than imported Moroccan seafood. The emerging strain may have not established a reservoir for survival in the food supply chain resulting in its disappearance after 2017., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ando, Sekizuka, Yokoyama, Aihara, Konishi, Matsumoto, Ishida, Nagasawa, Jourdan-Da Silva, Suzuki, Kimura, Le Hello, Murakami, Kuroda, Hirai and Fukaya.)

This study investigated the genetic and pathogenic variation of the subgroups of clade 2 strains of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157. A total of 111 strains of STEC O157 isolated in Shimane prefecture, Japan, were classified in clade 2 (n = 39), clade 3 (n = 16), clade 4/5 (n = 3), clade 7 (n = 14), clade 8 (n = 17), and clade 12 (n = 22) by single-nucleotide polymorphism analysis and lineage-specific polymorphism assay-6. These results showed a distinct difference from our previous study in which clade 3 strains were the most prevalent strains in three other prefectures in Japan, indicating that the clade distribution of O157 strains was different in different geographic areas in Japan. Phylogenetic analysis using insertion sequence (IS) 629 distribution data showed that clade 2 strains formed two clusters, designated 2a and 2b. Stx2 production by cluster 2b strains was significantly higher than by cluster 2a strains (P < 0.01). In addition, population genetic analysis of the clade 2 strains showed significant linkage disequilibrium in the IS629 distribution of the strains in clusters 2a and 2b (P < 0.05). The Φ PT values calculated using the IS629 distribution data indicated that strains in clusters 2a and 2b were genetically different (P < 0.001). Cluster 2b strains are a highly pathogenic phylogenetic group and their geographic spread may be a serious public health concern.

In Japan, only one outbreak of Salmonella enterica subsp. enterica serovar Chester (S. Chester) has been confirmed in 1999. We performed a single-center retrospective case review of S. Chester infections that occurred in a rural area of Japan in 2016 (n=8). Case 5 and 6 occurred in twin infants who had contact with a pet dog. The dog's stool culture was positive for S. Chester. Pulsed-field gel electrophoresis and cluster analysis of S. Chester strains revealed that all the isolates appeared to be derived from the same genetic clone. Emergence of Salmonella infection can be overlooked if cases are not reported to health authorities; therefore, core hospitals should play a role to alert the occurrence of public health issue.

Our previous studies found that a dominant serovar of Salmonella enterica isolates from three farms raising broilers in 2014 and 2015 was serovar Agona and the number of Infantis isolates decreased (the serovar shift). In this study, 52 S. Agona strains which isolated between 1993 and 2008, were compared to the serovar shift clone by molecular epidemiology and phylogenetic analyses, using pulsed field gel electrophoresis and whole genome sequence analyses. Of the 52 strains, one strain isolated from a human case in 1995 was genetically identical to the serovar shift clone, even though it was isolated prior to the serovar shift. These results suggested that the S. Agona serovar shift clone had existed in a source other than chicken penetrated chicken population.

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., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The name "Actinomyces suis" was applied to each actinomycete isolate from swine actinomycosis by Grässer in 1962 and Franke in 1973. Nevertheless, this specific species was not included in the "Approved List of Bacterial Name" due to absence of the type cultures. Therefore, "Actinomyces suis" based on the description of Franke 1973 has been considered as "species incertae sedis". We isolated a number of Actinomyces strains from swine. The representative strains of them was designated as Chiba 101 that was closely similar to the description in "Actinomyces suis" reported by Franke in 1973. Interestingly, it was found that the biological characteristics of these strains were also very similar to those of Actinomyces denticolens. Furthermore, the average nucleotide identity (ANI) value between strain Chiba 101 and the type-strain of Actinomyces denticolens (=DSM 20671 T ) was found to be 99.95%. Sequences of the housekeeping genes and 16S rRNA gene showed 100% homology. These results strongly suggested that "Actinomyces suis" Franke 1973 is the same species as Actinomyces denticolens. Since actinomycosis caused by Actinomyces denticolens have been demonstrated in horses recently, it is necessary to recognize that Actinomyces denticolens is the pathogenic actinomycetes in broader range of animals.

[This corrects the article DOI: 10.1371/journal.pone.0191834.].

Enterohemorrhagic Escherichia coli O157 (O157) strains can be classified into clades (one of several phylogenetic groups) by single nucleotide polymorphisms (SNPs): these are clade 1, clade 2, clade 3, descendant and ancestral clades 4/5, clade 6, clade 7, clade 8, clade 9, and clade 12. Some recent studies showed that some O157 strains in clade 8 produced a larger amount of Shiga toxin (Stx) 2 than other strains. In this study, 1121 epidemiologically unlinked strains of O157 isolated in Chiba Prefecture, Japan were classified into clades during 1996-2014. Clade 8 strains were further classified into subclade 8a (67 strains) and subclade 8b (48 strains) using SNP analysis. In the absence of mitomycin C (MMC), subclade 8a strains in this study produced significantly greater amounts of Stx2 than subclade 8b strains. However, in the presence of MMC, the levels of Stx2 production in subclade 8b strains were significantly greater than subclade 8a strains. On the other hand, a recent study reported that the Stx2 production level in O157 strains was determined mainly by the subtypes of Stx2a phage (ϕStx2&#95;α, β, γ, δ, ε, and ζ). Using O157 strains in this study, the Stx2a phages were classified into these subtypes. In this study, all strains of subclades 8a and 8b carried ϕStx2a&#95;γ and ϕStx2a&#95;δ, respectively. Some strains in clade 6 also carried ϕStx2a&#95;δ. In the presence of MMC, subclade 8b strains produced significantly greater amounts of Stx2 than clade 6 strains carrying ϕStx2&#95;δ. In this study, we propose that Stx2 production in subclade 8b strains in the presence of MMC might be enhanced due to genetic factors other than ϕStx2&#95;δ.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Nitric oxide (NO), which acts as an antimicrobial defense molecule, was found to enhance the production of Stx1 and Stx2 in EHEC under anaerobic conditions. Although EHEC O157 has two types of anaerobic NO reductase genes, an intact norV and a deleted norV, in the deleted norV-type EHEC, a high concentration of NO (12-29 μmol/L, maximum steady-state concentration) is required for enhanced Stx1 production and a low concentration of NO (~12 μmol/L, maximum steady-state concentration) is sufficient for enhanced Stx2 production under anaerobic conditions. These results suggested that different concentration thresholds of NO elicit a discrete set of Stx1 and Stx2 production pathways. Moreover, the enhancement of Shiga toxin production in the intact norV-type EHEC required treatment with a higher concentration of NO than was required for enhancement of Shiga toxin production in the deleted norV-type EHEC, suggesting that the specific NorV type plays an important role in the level of enhancement of Shiga toxin production in response to NO. Finally, Fur derepression and RecA activation in EHEC were shown to participate in the NO-enhanced Stx1 and Stx2 production, respectively., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

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.

Actinomyces sp. strain Chiba101, isolated from an arthritic leg joint of a pig raised in Japan, is a bacterium closely related to Actinomyces denticolens Here, we deciphered the complete genome sequence of Actinomyces sp. Chiba101 and the high-quality draft genome sequence of A. denticolens DSM 20671 T ., (Copyright © 2017 Kanesaki et al.)

Nine Shiga toxin-producing Escherichia coli O157:H7/H- (O157) strains were serially cultured three times on LB agar plates. After each sub-culture, five colonies were picked for DNA isolation and whole genome sequence (WGS) analysis. After exclusion of possible recombination-related SNPs, 11, 9, and 34 single-nucleotide polymorphisms (SNPs) were detected in genes in the backbone, O-island, and mobile elements gene categories. This suggested that those SNPs due to cultivation could influence the threshold value set for molecular epidemiological studies of O157. Significant differences were observed by the Kruskal-Wallis test (P < 0.01) when the number of the SNPs in a strain was compared to that in other strains. This indicated that a specific number of strains could be used for setting the threshold value in molecular epidemiological studies. Due to cultivation, the SNPs were also detected in genes in a few core genome or core gene sets, suggesting that those SNPs could affect studies of phylogeny as well as molecular epidemiology. To improve the accuracy of phylogenetic and molecular epidemiological studies, genes in which the SNPs have arisen due to cultivation should be excluded from WGS data.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We have studied 167 epidemiologically unlinked strains of enterohemorrhagic Escherichia coli O157 (O157) isolated from patients with hemorrhagic colitis (HC), 87 strains from patients not with HC and 35 asymptomatic carriers (the not HC group), and differentiated these strains into clades using high resolution melting analysis. In addition, lineage analysis was carried out using lineage-specific polymorphism assay-6 and analysis of the distribution of IS629 insertion sites was carried out using IS-printing. Most strains were correctly clustered by minimum spanning tree analysis, and strains in the major clades showed linkage disequilibrium, confirming the clade differentiation in this study. The number of O157 strains in the different clades isolated from HC patients and the not HC group was significantly different (Chi square test, P<0.05), indicating that strains in different clades had different pathogenicities for hemorrhagic colitis. Pairwise comparison of the number of strains in different clades isolated from HC patients indicated that clade 12 strains were weakly pathogenic for HC. Stx2 titers and the number of strains carrying an stx2 gene were significantly different for different clades (Kruscal-Wallis test and Chi square test, P<0.05). Pairwise comparison of the Stx2 titer and the number of strains with an stx2 gene in different clades showed that the weak HC pathogenicity of clade 12 strains would be related to the low number of clade 12 strains with an stx2 gene and the low Stx2 production in those strains. Interestingly, the Stx2 titer and the prevalence of strains with an stx2 gene were significantly higher among clade 6 and 8 strains compared to clade 7 strains, although clades 6, 7, and 8 were all in lineage I/II. These results were discordant with the O157 evolutionary model, suggesting that insertion of an stx2 gene in lineage I/II strains after divergence of each clade., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Variable number of tandem repeats (VNTR) analysis is one of the methods for molecular epidemiological studies of Mycobacterium tuberculosis. VNTR analysis is a method based on PCR, provides rapid highly reproducible results and higher strain discrimination power than the restriction fragment length polymorphism (RFLP) analysis widely used in molecular epidemiological studies of Mycobacterium tuberculosis. Genetic lineage compositions of Mycobacterium tuberculosis clinical isolates differ among the regions from where they are isolated, and allelic diversity at each locus also differs among the genetic lineages of Mycobacterium tuberculosis. Therefore, the combination of VNTR loci that can provide high discrimination capacity for analysis is not common in every region. The Japan Anti-Tuberculosis Association (JATA) 12 (15) reported a standard combination of VNTR loci for analysis in Japan, and the combination with hypervariable (HV) loci added to JATA12 (15), which has very high discrimination capacity, was also reported. From these reports, it is thought that data sharing between institutions and construction of a nationwide database will progress from now on. Using database construction of VNTR profiles, VNTR analysis has become an effective tool to trace the route of tuberculosis infection, and also helps in decision-making in the treatment course. However, in order to utilize the results of VNTR analysis effectively, it is important that each related organization cooperates closely, and analysis should be appropriately applied in the system in which accurate control and private information protection are ensured.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A micro-organism resembling members of the genus Dermatophilus, strain W254(T), which was isolated from the submandibular lymph node of a pig, and an additional 16 strains isolated from swine tonsils, were studied to establish their taxonomic status. Although all 17 strains were isolated anaerobically under an atmosphere of 100 % CO2, all of them were aerotolerant anaerobes. The micro-organisms showed at least five cellular morphologies: (i) a radially protrusive thallus, which proliferated into tuber-like cells; (ii) segmentation in both tubers and thallus followed by multilocule formation, (iii) development of coccoid forms in the locules; (iv) a change from the coccoid forms to zoospores; (v) resting cells, which were able to develop into protrusive thalli again. The micro-organisms were positive for nitrate reduction, but negative for catalase, indole production, hydrolysis of urea and gelatin liquefaction. Milk was not decomposed and none of the strains was haemolytic. A total of 16 compounds, including glucose, were utilized as sole carbon sources and seven compounds, including l-arabinose, were not utilized. Three out of the 17 strains were subjected to further studies. The micro-organisms had meso-diaminopimelic acid in their peptidoglycan and galactose, glucose, madurose and a trace of mannose in their whole-cell sugar patterns. The major phospholipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol.Cellular fatty acids were C15 : 0 (35.7-23.1 %), C16 : 0 (5.9-2.4 %) C17 : 0 (62.9-39.5 %), C17 : 1 (24.4-0 %) and C18 : 0 (3-1.6 %). The predominant menaquinone was MK-8 (H4). The G+C content of the DNA was 69.6-71.8 mol%. Analysis of the 16S rRNA gene sequences showed that the strains clustered with the type strains of members of the family Dermatophilaceae. Based on the polyphasic taxonomic characterization carried out, all 17 strains are considered to belong to a novel species in a new genus, for which the name Tonsilliphilus suis gen. nov., sp. nov. is proposed. The type strain of the type species is W254(T) ( = ATCC 35846(T) = CCM 3774(T) = DSM 21880(T) = JCM 15727(T)).

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 © 2012 Elsevier B.V. All rights reserved.)

The sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects.

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 (B(SUB)) B1-B5 and T2 by minimum spanning tree (MST) analysis. The Φ(PT) values among the B(SUB), a measure of their molecular variance, were significantly different from zero with 999 permutations, indicating the validity of B(SUB) 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 B(SUB) 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 B(SUB). Haploid genetic diversity and standardized index of association values were different in each B(SUB), indicating that the diversity of each B(SUB) was different. Although the differences in B(SUB) diversity were mostly in accordance with the relative divergence order of the B(SUB) 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 B(SUB) indicated increased diversity of Beijing lineage strains, perhaps contributing to the survival and dissemination of these strains., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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 stx(2) 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.

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 © 2010 Elsevier B.V. All rights reserved.)

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 2010 Elsevier B.V. All rights reserved.)

Objectives: To compare the efficacy and safety of silodosin and tamsulosin in patients with lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) by a randomized crossover method., Methods: BPH patients with the complaint of LUTS were included in this study, and were randomly divided into two groups: a silodosin-preceding group (4 weeks of twice-daily administration of silodosin at 4 mg, followed by 4 weeks of once-daily administration of tamsulosin at 0.2 mg) or a tamsulosin-preceding group (4 weeks' administration of tamsulosin, followed by 4 weeks' administration of silodosin). No drug withdrawal period was provided when switching the drug., Results: In the first treatment period, both drugs significantly improved the International Prostate Symptom Score total score, but the improvement by silodosin was significantly superior to that by tamsulosin. After crossover treatment, significant improvement was observed only with silodosin treatment. Moreover, intergroup comparison of changes revealed that silodosin showed significant improvement of straining and nocturia with first and crossover treatments, respectively, compared with tamsulosin. Silodosin also significantly improved quality of life (QOL) score in both treatment periods, while tamsulosin significantly improved QOL score only in the first treatment period. The most frequent adverse drug reaction was ejaculatory disorder with silodosin; however, the incidence of dizziness with silodosin was similar to that with tamsulosin., Conclusions: In BPH/LUTS patients, silodosin exhibits excellent efficacy in improving subjective symptoms in both initial and crossover treatment, and it appears to improve the QOL of patients., (© 2010 The Japanese Urological Association.)

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 primers 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 better 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.

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.

Purpose: Androgen deprivation therapy (ADT) in patients with prostate cancer is associated with bone loss. We investigated the effectiveness of risedronate about a decreasing bone mineral density in patients with prostate cancer on ADT., Material and Method: A prospective study was conducted in Kitasato University Hospital from April 2004 to October 2006. A total of 69 men with prostate cancer were assigned to receive either oral risedronate or none during ADT (hormone naïve). The treatment group was 58 men and taking 2.5 mg risedronate per day. The control group was 11 men. At baseline, we assessed BMD (bone mineral density) by DEXA and urinary NTX, and measured for these changes every 6 months., Result: At baseline, each BMDs had no significant difference at the lumber and total hip. At the first 6-month stage, the change in BMD percentage between the 2 groups was statistically significantly different at lumber (p = 0.002) and total hip (p = 0.038). At the 12-month stage, the change in the BMD percentage between the 2 groups was statistically significantly different at the lumber (p = 0.038). And each difference made out that the risedronate group was preserving BMD. In urinary NTX, bone turn over was statistically significantly decreased with the risedronate group compared with the control group at the 12-month stage (p = 0.017)., Conclusion: We assure the beginning of bone loss at an early date (6 months) with ADT. Daily oral risedronate in patients with receiving ADT reduces bone mineral loss and maintain BMD.

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.

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.

In an outbreak of gastroenteritis in elementary school students and their families in Chiba Prefecture, Japan, Campylobacter jejuni was isolated from the stools of 14 patients who developed diarrheal illness after a one-day bus trip. C. jejuni was also isolated from the stools of 3 patients not going on the bus trip. Pulsed-field gel electrophoresis (PFGE) analysis was done on 17 isolates of C. jejuni to study genetic relationships among them. PFGE profiles of isolates treated with restriction enzymes Sma I, Ksp I and Kpn I were separated into 9, 10, and 10 types, but the relationship between PFGE profiles and epidemiological profiles was unclear. Dendrograms of PFGE of isolates double-digested with both Sma I and Ksp I were typed into D1, D2, D3 and D4, and profiles compared to profiles of serotyping and flagellin typing of isolates and epidemiological profiles to evaluate genetical and epidemiological relationships. Thirteen isolates of PFGE type D1 possessed serotype G and flagellin type Al and were isolated from patients going on the bus trip. Type D2 isolated from a student going on the bus trip and type D3 isolates from two students not going on the bus trip had serotype B and flagellin type A2. C. jejuni of PFGE type D4, serotype UT, and flagellin type A3 was also isolated from a student not going on the trip. Our results show that at least two outbreaks of C. jejuni occurred simultaneously in people related to the school. Restriction enzyme double-digestion PFGE was thus useful in the molecular epidemiological analysis of the C. jejuni outbreak.

Entomopathogens and other econutritional fungi belonging to Clavicipitaceae were phylogenetically analyzed on the basis of the 18S rRNA gene and mating-type genes (MAT1-1-1 and MAT1-2-1). The phylogenies of the mating-type genes yielded better resolutions than that of 18S rRNA gene. Entomopathogens (Cordyceps bassiana, Cordyceps brongniartii, Cordyceps militaris, Cordyceps sinclairii, Cordyceps takaomontana, Isaria cateniannulata, Isaria farinosa, Isaria fumosorosea, Isaria javanica, Lecanicillium muscarium and Torrubiella flava) were considered as a phylogenetically defined group, and were closely related to mycopathogens (Lecanicillium psalliotae and Verticillium fungicola). They located at more descendant positions in the mating-type trees than other fungi, and lacked the mating-type gene MAT1-1-3. The deletion of MAT1-1-3 was supposed to have occurred once in Clavicipitaceae, and a good indication for the evolution of Clavicipitaceae. Other entomopathogens (Cordyceps cylindrica, Cordyceps subsessilis, Metarhizium anisopliae and Nomuraea rileyi) and pathogens of plants, nematodes and slime molds, were relatively related to each other, and possessed MAT1-1-3, but were supposed to be heterogeneous. Root-associated fungi did not form any clade with other species.

An inhibitor of the prophenoloxidase activation using extract from a silkworm pupa was isolated from a culture filtrate of Cordyceps militaris and identified as dipicolinic acid (DPA). The production of DPA in Clavicipitaceae fungi was examined. Entomogenous fungi that produce DPA were integrated into one group by a phylogenetic analysis based on 18S rDNA. It is suggested that the group acquired an ability to produce DPA during its evolution from plant pathogenic fungi to entomogenous fungi.

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.

A trypsin-like protease, P-1-1, was purified from the culture supernatant of the fungus Cordyceps militaris by (NH(4))(2)SO(4) precipitation, chromatography on DEAE Bio-Gel Agarose, TSKgel CM-5PW, and gel-filtration with HiLoad 26/60 Superdex 75 pg, and its properties were examined. Purified P-1-1 showed a single band by SDS-PAGE and was estimated to have a molecular mass of 23,405 by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The optimum pH of the enzyme was between 8.5 and 12.0. It was inhibited strongly by leupeptin and diisopropyl fluorophosphate (DFP), and definitely did by N(alpha)-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), phenylmethanesulfonyl fluoride (PMSF) and chymostatin. The carbonyl group sides of Arg and Lys were confirmed as the sites of cleavage by the enzyme toward cecropin B. These results indicate that P-1-1 is a trypsin-type serine protease. The N-terminal amino acid sequence of P-1-1 showed a high homology with those of trypsins or chymotrypsin derived from Diptera insects.

Methods for cluster analysis of IS6110 based restriction fragment length polymorphism (RFLP) patterns of Mycobacterium tuberculosis isolates were studied for an epidemiological investigation in Chiba prefecture. To normalize patterns, external size markers were adopted instead of typical internal size markers used in the standard method. RFLP patterns were run on 1.4% agarose gels and external markers were applied to outside and middle lanes on each gel for precise comparison. The resulting RFLP patterns of 74 isolates were clustered by similarity. Similarity was calculated with the Dice coefficient using parameter settings at 0.8% tolerance and 0.5% optimization. Patterns of 19 isolates from 8 outbreaks showed high similarity within each outbreak. Cluster analysis, as described here, provides insights into epidemiological tracing of tuberculosis in Chiba prefecture.

Perithecium formation of an entomopathogenic fungus Cordyceps takaomontana was promoted by treating the mycelia with cell wall-degrading enzymes and PEG 4000. Perithecia were formed in the mixed culture of both mating-type strains MAT1 and MAT2, and not in the culture of MAT1 or MAT2 alone. The MAT1 strains did not possess a mating-type gene MAT1-1-3, but could produce perithecia. These results strongly suggested that C. takaomontana is heterothallic, and does not need MAT1-1-3 for the perithecium formation. MAT1-1-3 was also not found in another entomopathogenic fungus Cordyceps militaris. On the other hand, phytopathogenic fungi Balansia sp., Claviceps purpurea and Epichloë typhina possessed MAT1-1-3. The structures of mating-type locus MAT1-1 of these phytopathogenic fungi in the family Clavicipitaceae were similar to that of a phytopathogenic fungus Gibberella fujikuroi in the family Nectriaceae, which is closely related to Clavicipitaceae. These results suggested that phytopathogen might be more ancestral group than entomopathogen in Clavicipitaceae, and that MAT1-1-3 might be lost in the course of the host shift from plants to insects.

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 48 h 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.