Shinichiro Hirai

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

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

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

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

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

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

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

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

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.

An evolutionary analysis of nitric oxide reductase gene norV in enterohemorrhagic Escherichia coli O157

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.

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 pathogens 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.

Evolutionary model of the divergence of enterohemorrhagic Escherichia coli O157 lineage I/II clades reconstructed from high resolution melting and Shiga-like toxin 2 analyses.

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.

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.

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.