Another advantage of multi-locus variable-number tandem repeat analysis that can putatively subdivide enterohemorrhagic Escherichia coli O157 strains into clades by maximum a posteriori estimation.

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.

Clarification of relationship between single-nucleotide polymorphism panels of Shiga toxin-producing Escherichia coli O157:H7/H- strains.

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.

Molecular epidemiological analysis of Mycobacterium tuberculosis modern Beijing genotype strains isolated in Chiba Prefecture over 10 years.

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 J12-0006 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.

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

Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.

Enhanced production of Shiga toxin 1 in enterohaemorrhagic Escherichia coli by oxygen.

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 (PStx1), 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 Fe2+-responsive transcription factor. The intracellular Fe2+ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe2+ availability drove the formation of less Fe2+-Fur, less DNA binding to the PStx1 region, and an increase in Stx1 production.

Whole-genome sequence analysis of Shiga toxin-producing Escherichia coli O157 strains isolated from wild deer and boar in Japan.

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.

Whole Genome Analysis Detects the Emergence of a Single Salmonella enterica Serovar Chester Clone in Japan's Kanto Region.

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.

Phylogeny, Prevalence, and Shiga Toxin (Stx) Production of Clinical Escherichia coli O157 Clade 2 Strains Isolated in Shimane Prefecture, Japan.

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 ΦPT 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.

Emergence of Salmonella enterica subsp. enterica serovar Chester in a rural area of Japan.

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.

Genetic characteristics of emerging Salmonella enterica serovar Agona strains isolated from humans in the prior period to occurrence of the serovar shift in broilers.

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.

Isolation of Salmonella enterica serovar Agona strains and their similarities to strains derived from a clone caused a serovar shift in broilers.

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.

Actinomyces denticolens as a causative agent of actinomycosis in animals.

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 20671T) 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.

Correction: Enterohemorrhagic Escherichia coli O157 subclade 8b strains in Chiba Prefecture, Japan, produced larger amounts of Shiga toxin 2 than strains in subclade 8a and other clades.

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

Enterohemorrhagic Escherichia coli O157 subclade 8b strains in Chiba Prefecture, Japan, produced larger amounts of Shiga toxin 2 than strains in subclade 8a and other clades.

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_α, ß, 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_γ and ϕStx2a_δ, respectively. Some strains in clade 6 also carried ϕStx2a_δ. In the presence of MMC, subclade 8b strains produced significantly greater amounts of Stx2 than clade 6 strains carrying ϕStx2_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_δ.

Application of whole genome sequence data in analyzing the molecular epidemiology of Shiga toxin-producing Escherichia coli O157:H7/H.

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.

Whole-Genome Sequences of Two Closely Related Bacteria, Actinomyces sp. Strain Chiba101 and Actinomyces denticolens DSM 20671T.

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 20671T.

Characterization of Shiga toxin-producing Escherichia coli from feces of sika deer (Cervus nippon) in Japan using PCR binary typing analysis to evaluate their potential human pathogenicity.

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.

Nitric oxide-enhanced Shiga toxin production was regulated by Fur and RecA in enterohemorrhagic Escherichia coli O157.

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.

Single-Nucleotide Polymorphisms in the Whole-Genome Sequence Data of Shiga Toxin-Producing Escherichia coli O157:H7/H- Strains by Cultivation.

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.