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Enterohemorrhagic Escherichia coli (EHEC) causes severe human diseases worldwide. The type three secretion system and effector proteins are essential for EHEC infection, and are encoded by the locus of enterocyte effacement (LEE). RNA-binding protein Hfq is essential for small regulatory RNA (sRNA)-mediated regulation at a post-transcriptional level and full virulence of many pathogenic bacteria. Although two early studies indicated that Hfq represses LEE expression by post-transcriptionally controlling the expression of genes grlRA and/or ler, both of which encode LEE regulators mediating a positive regulatory loop, the detailed molecular mechanism and biological significance remain unclear. Herein, we show that LEE overexpression was caused by defective RNA-binding activity of the Hfq distal face, which post-transcriptionally represses grlA and ler expression. In vitro analyses revealed that the Hfq distal face directly binds near the translational initiation site of grlA and ler mRNAs, and inhibits their translation. Taken together, we conclude that Hfq inhibits grlA and ler translation by binding their mRNAs through the distal face in an sRNA-independent manner. Additionally, we show that Hfq-mediated repression of LEE is critical for normal EHEC growth because all suppressor mutations that restored the growth defect in the hfq mutant abolished hfq deletion-induced overexpression of LEE.

We report a fatal case of hemolytic uremic syndrome with urinary tract infection in Japan caused by Shiga toxin–producing Escherichia coli. We genotypically identified the isolate as OX18:H2. Whole-genome sequencing revealed 3 potentially pathogenic lineages (OX18:H2, H19, and H34) that have been continuously isolated in Japan.

In June 2020, a large-scale food poisoning outbreak involving about 3000 elementary and junior high school students occurred in Yashio, Saitama, Japan. A school lunch was the only food stuff ingested by all of the patients. Escherichia coli serotype O7:H4 carrying the astA gene for enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1) was detected in faecal specimens from the patients, and sample inspection revealed its presence in a seaweed salad and red seaweed (Gigartina tenella) as one of the raw materials. Analysis of the antibiotic sensitivity of the isolates revealed resistance to ampicillin and cefotaxime. All isolates were confirmed to be of the same origin by pulsed-field gel electrophoresis after digestion with the restriction enzyme XbaI, and single nucleotide polymorphism analysis using whole genome sequencing. To our knowledge, this is the first report of a large-scale food poisoning caused by E. coli O7:H4, which lacks well-characterized virulence genes other than astA.

Japan has had a substantial increase in syphilis cases since 2013. However, research on the genomic features of the Treponema pallidum subspecies pallidum (TPA) strains from these cases has been limited. Here, we elucidated the genetic variations and relationships between TPA strains in Japan (detected between 2014 and 2018) and other countries by whole-genome sequencing and phylogenetic analyses, including syphilis epidemiological surveillance data and information on patient sexual orientation. Seventeen of the 20 strains in Japan were SS14- and the remaining 3 were Nichols-lineage. Sixteen of the 17 SS14-lineage strains were classified into previously reported Sub-lineage 1B. Sub-lineage 1B strains in Japan have formed distinct sub-clusters of strains from heterosexuals and strains from men who have sex with men. These strains were closely related to reported TPA strains in China, forming an East-Asian cluster. However, those strains in these countries evolved independently after diverging from their most recent common ancestor and expanded their genetic diversity during the time of syphilis outbreak in each country. The genetic difference between the TPA strains in these countries was characterized by single-nucleotide-polymorphism analyses of their penicillin binding protein genes. Taken together, our results elucidated the detailed phylogenetic features and transmission networks of syphilis.

We identified and characterized the first 2 Neisseria gonorrhoeae strains with high-level azithromycin resistance isolated in Japan. These were in the clade of ceftriaxone- and azithromycin-resistant strains isolated in Australia and the United Kingdom. The multilocus sequence typing, N. gonorrhoeae multiantigen sequence typing, and N. gonorrhoeae sequence typing for antimicrobial resistance types of these strains were found in gonococci from eastern Asia.

Since the Shiga toxin-producing enteroaggregative Escherichia coli (Stx-EAEC) O104:H4 strain caused a massive outbreak across Europe in 2011, the importance of Stx-EAEC has attracted attention from a public health perspective. Two Stx-EAEC O86 isolates were obtained from patients with severe symptoms in Japan in 1999 and 2015. To characterize the phylogeny and pathogenic potential of these Stx-EAEC O86 isolates, whole-genome sequence analyses were performed by short-and long-read sequencing. Among genetically diverse E. coli O86, the Stx-EAEC O86 isolates were clustered with the EAEC O86:H27 ST3570 subgroup. Strikingly, there were only two loci with single nucleotide polymorphisms (SNPs) between the Stx2a phage of a Japanese O86:H27 isolate and that of the European epidemic-related Stx-EAEC O104:H4 isolate. These results provide evidence of global distribution of epidemic-related Stx2a phages among various lineages of E. coli with few mutations.

Phages and plasmids play important roles in bacterial evolution and diversification. Although many draft genomes have been generated, phage and plasmid genomes are usually fragmented, limiting our understanding of their dynamics. Here, we performed a systematic analysis of 239 draft genomes and 7 complete genomes of Shiga toxin (Stx)-producing Escherichia coli O145:H28, the major virulence factors of which are encoded by prophages (PPs) or plasmids. The results indicated that PPs are more stably maintained than plasmids. A set of ancestrally acquired PPs was well conserved, while various PPs, including Stx phages, were acquired by multiple sublineages. In contrast, gains and losses of a wide range of plasmids have frequently occurred across the O145:H28 lineage, and only the virulence plasmid was well conserved. The different dynamics of PPs and plasmids have differentially impacted the pangenome of O145:H28, with high proportions of PP- and plasmid-associated genes in the variably present and rare gene fractions, respectively. The dynamics of PPs and plasmids have also strongly impacted virulence gene repertoires, such as the highly variable distribution of stx genes and the high conservation of a set of type III secretion effectors, which probably represents the core effectors of O145:H28 and the genes on the virulence plasmid in the entire O145:H28 population. These results provide detailed insights into the dynamics of PPs and plasmids, and show the application of genomic analyses using a large set of draft genomes and appropriately selected complete genomes.

SUMMARYA nationwide study of Shiga toxin-producingEscherichia coli(STEC) was performed to determine the prevalence, characteristics and risk factors for fecal shedding of STEC among cattle in Japan. Information on rearing practices was also collected to identify risk factors for fecal shedding of STEC. STEC was isolated from 24·1% of samples (133/551) collected from 59·1% of farms (65/110). Bayesian clustering using the virulence marker profiles of the isolates subdivided the isolates into four genetically distinct groups, two of which corresponded toeae- orsaa-positive STEC, which can cause severe disease in human. Both STEC groups exhibited characteristic phylogeny and virulence marker profiles. It is noteworthy that the tellurite resistance gene was not detected in allsaa-positive STEC isolates, suggesting that the standard isolation method using tellurite might lead to an underestimation of the prevalence ofsaa-positive STEC. A multivariate logistic regression model using epidemiological information revealed a significantly (P< 0·01) high odds ratio on STEC fecal shedding in tie-stall housing and a low odds ratio in flat feed box and mechanical ventilation. Information on isolate characteristics of the two major pathotypes and risk factors in rearing practices will facilitate the development of preventative measures for STEC fecal shedding from cattle.

We present the complete genome sequence of an enterohemorrhagic Escherichia coli O111:H8 strain. This strain was isolated from a hemolytic-uremic syndrome patient and was responsible for a large outbreak associated with the consumption of raw beef in 2011.

Enterohaemorrhagic Escherichia coli (EHEC) is an important human pathogen worldwide. Although serotype O157 is currently the most dominant and important EHEC strain, serotypes O26, O111, O91, O103 and O121 are also recognized as serious pathogens that affect public health. EHEC outbreaks often occur in nurseries and elderly care facilities. In 2012, a nursery outbreak of EHEC O121 occurred during which the bacterium acquired a plasmid-borne extended-spectrum β-lactamase (ESBL) gene. ESBL-producing E. coli O86 was concurrently isolated from one of the EHEC patients. Therefore, we investigated the isolates by whole-genome sequence (WGS) analysis to elucidate the transmission dynamics of the EHEC strains and the ESBL plasmid. According to WGS-based phylogeny, all 17 EHEC O121 isolates were clonal, while E. coli O86 was genetically distant from the EHEC O121 isolates. The complete sequence of an ESBL plasmid encoding the CTX-M-55 β-lactamase was determined using S1-PFGE bands, and subsequent mapping of the WGS reads confirmed that the plasmid sequences from EHEC O121 and E. coli O86 were identical. Furthermore, conjugation experiments showed that the plasmid was capable of conjugative transfer. These results support the hypothesis that EHEC O121 acquired an ESBL-producing plasmid from E. coli O86 during the outbreak. This report demonstrates the importance of implementing preventive measures during EHEC outbreaks to control both secondary infection and the spread of antimicrobial resistance factors.

Streptococcus pneumoniae serotype 12F rarely colonizes the nasopharynx but commonly causes invasive pneumococcal disease. Here, we report the complete genome sequence of a sequence type 4846 (ST4846) S. pneumoniae serotype 12F strain isolated from a cluster of invasive pneumococcal disease patients in Japan.

To determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The bla NDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: bla NDM-1 in 2 isolates, bla NDM-5 in 7 isolates, and bla NDM-7 in 5 isolates. The bla CTX-M gene was detected in 112 isolates (66.3%): bla CTX-M-15 in 108 isolates and bla CTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the bla NDM -positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

In Japan, hot springs and public baths are the major sources of legionellosis. In 2015, an outbreak of Legionnaires' disease occurred among 7 patients who had visited a spa house. Laboratory investigation indicated that L. pneumophila serogroup 1 and 13 strains caused the outbreak and that these strains were genetically related.

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.

Mosaic penA alleles have caused most of the cephalosporin resistance in Neisseria gonorrhoeae , but their evolution is mostly unknown. The penA gene from Neisseria cinerea strain AM1601 (ceftriaxone MIC, 1.0 μg/ml) caused ceftriaxone resistance (MIC, 1 μg/ml) in a ceftriaxone-susceptible gonococcal strain. The 3′-terminal half of AM1601 penA was almost identical to that of the ceftriaxone-resistant gonococcal GU140106 and FC428 strains. N. cinerea can serve as a reservoir of ceftriaxone resistance-mediating penA sequences that can be transferred to gonococci.

A key virulence factor of enterohaemorrhagic Escherichia coli (EHEC) is the bacteriophage-encoded Shiga toxin (Stx). Stxs are classified into two types, Stx1 and Stx2, and Stx2-producing strains are thought to cause more severe infections than strains producing only Stx1. Although O26 : H11 is the second most prevalent EHEC following O157 : H7, the majority of O26 : H11 strains produce Stx1 alone. However, Stx2-producing O26 strains have increasingly been detected worldwide. Through a large-scale genome analysis, we present a global phylogenetic overview and evolutionary timescale for E. coli O26 : H11. The origin of O26 has been estimated to be 415 years ago. Sequence type 21C1 (ST21C1), one of the two sublineages of ST21, the most predominant O26 : H11 lineage worldwide, emerged 213 years ago from one of the three ST29 sublineages (ST29C2). The other ST21 lineage (ST21C2) emerged 95 years ago from ST21C1. Increases in population size occurred in the late 20th century for all of the O26 lineages,but most remarkably for ST21C2. Analysis of the distribution of stx2-positive strains revealed the recent and repeated acquisition of the stx2 gene in multiple lineages of O26, both in ST21 and ST29. Other major EHEC virulence genes, such as type III secretion system effector genes and plasmid-encoded virulence genes, were well conserved in ST21 compared to ST29. In addition, more antimicrobial-resistance genes have accumulated in the ST21C1 lineage. Although current attention is focused on several highly virulent ST29 clones that have acquired the stx2 gene, there is also a considerable risk that the ST21 lineage could yield highly virulent clones. KEYWORDS: Shiga toxin; antimicrobial-resistance gene; enterohaemorrhagic Escherichia coli; genome evolution; population structure PMID: 29208163 PMCID: PMC5729918 DOI: 10.1099/mgen.0.000141, A key virulence factor of enterohaemorrhagic Escherichia coli (EHEC) is the bacteriophage-encoded Shiga toxin (Stx). Stxs are classified into two types, Stx1 and Stx2, and Stx2-producing strains are thought to cause more severe infections than strains producing only Stx1. Although O26 : H11 is the second most prevalent EHEC following O157 : H7, the majority of O26 : H11 strains produce Stx1 alone. However, Stx2-producing O26 strains have increasingly been detected worldwide. Through a large-scale genome analysis, we present a global phylogenetic overview and evolutionary timescale for E. coli O26 : H11. The origin of O26 has been estimated to be 415 years ago. Sequence type 21C1 (ST21C1), one of the two sublineages of ST21, the most predominant O26 : H11 lineage worldwide, emerged 213 years ago from one of the three ST29 sublineages (ST29C2). The other ST21 lineage (ST21C2) emerged 95 years ago from ST21C1. Increases in population size occurred in the late 20th century for all of the O26 lineages, but most remarkably for ST21C2. Analysis of the distribution of stx2-positive strains revealed the recent and repeated acquisition of the stx2 gene in multiple lineages of O26, both in ST21 and ST29. Other major EHEC virulence genes, such as type III secretion system effector genes and plasmid-encoded virulence genes, were well conserved in ST21 compared to ST29. In addition, more antimicrobial-resistance genes have accumulated in the ST21C1 lineage. Although current attention is focused on several highly virulent ST29 clones that have acquired the stx2 gene, there is also a considerable risk that the ST21 lineage could yield highly virulent clones.

Streptococcus pneumoniae colonizes the nasopharyngeal mucus in healthy individuals and can cause otitis media, pneumonia, and invasive pneumococcal diseases. In this study, we analyzed S. pneumoniae strains that caused 19 pneumonia episodes in long-term inpatients with severe underlying disease in a hospital during a period of 14 months (from January 2014 to February 2015). Serotyping and whole-genome sequencing analyses revealed that 18 of the 19 pneumonia cases were caused by S. pneumoniae strains belonging to 3 genetically distinct groups: clonal complex 9999 (CC9999), sequence type 282 (ST282), and ST166. The CC9999 and ST282 strains appeared to have emerged separately by a capsule switch from the pandemic PMEN 1 strain (Spain 23F -ST81). After all the long-term inpatients were inoculated with the 23-valent pneumococcal polysaccharide vaccine, no other nosocomial pneumonia infections occurred until March 2016.

Enterohemorrhagic Escherichia coli (EHEC) O26 infections cause severe human diseases such as hemolytic uremic syndrome and encephalopathy, and is the predominant serogroup among non-O157 EHEC in many countries. Shiga toxin (Stx), which consists of two distinct types (Stx1 and Stx2), plays a central role in EHEC pathogenesis. The major stx gene type in EHEC O26 strains is stx1, although isolates with only stx2 have emerged in Japan since 2012 and have been reported in Europe. In this study, we selected 27 EHEC O26 strains isolated in Japan and identified a distinct genetic clade within sequence type (ST) 29, designated ST29C1, that carried only stx2 and had the plasmid gene profile ehxA+/katP−/espP+/etpD−. We showed that ST29C1 strains produced higher Stx2a levels, and greater virulence in Vero cells and in germ-free mice than other lineages. We also showed that ST29C1 was a distinct phylogenetic clade by SNP analysis using whole genome sequences and clearly differed from the major European EHEC O26 virulent clone, which was designated ST29C2 in this study. The combination of toxin production analysis, virulence analysis in Vero cells and germ-free mice, and phylogenetic analysis identified a newly emerging virulent EHEC clade.

Background Little is known about the evolutionary process and emergence time of resistance mutations to fluoroquinolone in Salmonella enterica serovar Typhi. Methods We analyzed S. Typhi isolates collected from returned travelers between 2001 and 2016. Based on ciprofloxacin susceptibility, isolates were categorized as highly resistant (minimum inhibitory concentration [MIC] ≥ 4 μg/mL [CIPHR]), resistant (MIC = 1–2 μg/mL [CIPR]), intermediate susceptible (MIC = 0.12–0.5 μg/mL [CIPI]), and susceptible (MIC ≤ 0.06 μg/mL [CIPS]). Results A total of 107 isolates (33 CIPHR, 14 CIPR, 30 CIPI, and 30 CIPS) were analyzed by whole-genome sequencing; 2461 single nucleotide polymorphisms (SNPs) were identified. CIPS had no mutations in the gyrA or parC genes, while each CIPI had 1 of 3 single mutations in gyrA (encoding Ser83Phe [63.3%], Ser83Tyr [33.3%], or Asp87Asn [3.3%]). CIPHR had the same 3 mutations: 2 SNPs in gyrA (encoding Ser83Phe and Asp87Asn) and a third in parC (encoding Ser80Ile). CIPHR shared a common ancestor with CIPR and CIPI isolates harboring a single mutation in gyrA encoding Ser83Phe, suggesting that CIPHR emerged 16 to 23 years ago. Conclusions Three SNPs—2 in gyrA and 1 in parC—are present in S. Typhi strains highly resistant to fluoroquinolone, which were found to have evolved in 1993–2000, approximately 10 years after the beginning of the ciprofloxacin era. Highly resistant strains with survival advantages arose from strains harboring a single mutation in gyrA encoding Ser83Phe. Judicious use of fluoroquinolones is warranted to prevent acceleration of such resistance mechanisms in the future.

To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin-producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food-related fungi including fermentation starters. Spread distances of STEC O157 varied depending on the co-cultured fungal species, and the motile bacterial strain spread for longer distances than the non-motile strain. The population of STEC O157 increased when co-cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus. Confocal scanning microscopy visualization of green fluorescent protein-tagged STEC O157 on fungal hyphae revealed that the bacterium colonized in the water film that existed on and between hyphae. To investigate the physiological changes in STEC O157 caused by co-culturing with fungi, the bacterium was harvested after 7 days of co-culturing and tested for acid resistance. After co-culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi. Our results indicate that fungal hyphae can spread the contamination of STEC O157 and can also enhance the stress resistance of the bacteria.

The effects of secondary starter molds of common mold-ripened cheeses on the Shiga toxin-producing Escherichia coli (STEC) O157 were assessed in 3 model systems. In the 1st model, 8 STEC O157 strains were incubated in the spent culture of Penicillium camemberti or Penicillium roqueforti under mild acidic conditions at 25 °C. In the spent cultures of the mold at pH 4.8 to 5.0, the lag times of STEC O157 growth were significantly shorter than those observed in fresh medium. Analyses of the spent culture of P. camemberti showed that the causative agents of the growth enhancement were produced by the mold in response to an acidic environment and were not fully inactivated in heat treatment. In the 2nd model, P. camemberti and STEC O157 were cocultured in acidified milk at 25 °C. The population of STEC O157 reached 108 CFU/mL in the presence of the mold, whereas the population steadily declined in the absence of the mold. Although this growth enhancement was partially attributable to alkalization by the mold, it was observed even when the pH of this model was stabilized. In the 3rd model, 2 STEC O157 strains were incubated in the spent cultures of molds at pH 4.5 at 10 °C. In the spent culture, proportions of injured cells were significantly lower and D values were significantly higher than those in control, except one STEC O157 strain in the spent culture of P. camemberti. These results showed that the molds could enhance the growth and survival of STEC O157 by changing the environment. Practical Application: This study demonstrated that molds in foods can improve the growth and survival of the Shiga toxin-producing Escherichia coli O157. Because microbial interactions are ubiquitous in food, our results provide an important insight for understanding the behavior of microorganisms in food.

A comprehensive and quantitative analysis of the mycoflora on the surface of commercial fruit was performed. Nine kinds of fruits grown in Japan were tested. Overall fungal counts on the fruits ranged from 3.1 to 6.5 log CFU/g. The mean percentages of the total yeast counts were higher than those of molds in samples of apples, Japanese pears, and strawberries, ranging from 58.5 to 67.0%, and were lower than those of molds in samples of the other six fruits, ranging from 9.8 to 48.3%. Cladosporium was the most frequent fungus and was found in samples of all nine types of fruits, followed by Penicillium found in eight types of fruits. The fungi with the highest total counts in samples of the various fruits were Acremonium in cantaloupe melons (47.6% of the total fungal count), Aspergillus in grapes (32.2%), Aureobasidium in apples (21.3%), blueberries (63.6%), and peaches (33.6%), Cladosporium in strawberries (38.4%), Cryptococcus in Japanese pears (37.6%), Penicillium in mandarins (22.3%), and Sporobolomyces in lemons (26.9%). These results demonstrated that the mycoflora on the surfaces of these fruits mainly consists of common pre- and postharvest inhabitants of the plants or in the environment; fungi that produce mycotoxins or cause market diseases were not prominent in the mycoflora of healthy fruits. These findings suggest fruits should be handled carefully with consideration given to fungal contaminants, including nonpathogenic fungi, to control the quality of fruits and processed fruit products.

Between April 2001 and 2007, 18 Yersinia pseudotuberculosis outbreaks occurred in breeding monkeys at 12 zoological gardens in Japan, and 28 monkeys of 8 species died. A total of 18 Y. pseudotuberculosis strains from the dead monkeys, comprising one strain per outbreak, were examined for serotype and the presence of the virulence genes virF, inv, ypm (ypmA, ypmB and ypmC) and irp2. Of the 18 Y. pseudotuberculosis strains, 7 (38.9%) were serotype 4b, 7 (38.9%) were serotype 1b, and there was one each of serotypes 2b, 3, 6 and 7. All the 18 strains examined harbored virF and inv. Sixteen (88.9%) strains, including the strain of serotype 7, harbored ypmA. However, no strain harbored ypmB, ypmC and irp2. This study demonstrated that among other pathogenic factors, almost all the Y. pseudotuberculosis isolated from the outbreaks had the ypm gene encoding the superantigenic toxin, YPM. As most of the monkeys who died in those outbreaks originated from South America and other regions, where the presence of the ypm gene have not been reported, YPM might be the cause, or at least the most important factor for, the high mortality of the breeding monkeys infected by Y. pseudotuberculosis in Japan. This is also the first report of a fatal case due to Y. pseudotuberculosis serotype 7 infection in the world.

Salmonella enterica serovar Typhimurium pulsed-field gel electrophoresis cluster VII has been isolated from cattle populations in Japan since the mid-2000s. Some cluster VII isolates exhibited extended-spectrum cephalosporin resistance defined by the bla CMY-2 gene located in a chromosomal genomic island, GI-VII-6. We determined the whole-genome sequence of strain L-3553 as the reference strain.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- (S. 4,[5]12:i:-) is believed to be a monophasic variant of S. enterica serovar Typhimurium (S. Typhimurium). This study was conducted to corroborate this hypothesis and to identify the molecular and phenotypic characteristics of the S. 4,[5]12:i:- isolates in Japan. A total of 51 S. 4,[5]12:i:- isolates derived from humans, cattle, swine, chickens, birds, meat (pork), and river water in 15 prefectures in Japan between 2000 and 2010 were analyzed. All the S. 4,[5],12:i:- isolates were identified as S. Typhimurium by two different polymerase chain reactions (PCR) for identification of S. Typhimurium. Of the 51 S. 4,[5],12:i:- isolates, 39 (76.5%) harbored a 94-kb virulence plasmid, which is known to be specific for S. Typhimurium. These data suggest that the S. 4,[5],12:i:- isolates are monophasic variants of S. Typhimurium. The flagellar phase variation is induced by three adjacent genes (fljA, fljB, and hin) in the chromosome. The results of PCR mapping of this region and comparative genomic hybridization analysis suggested that the deletion of the fljAB operon and its flanking region was the major genetic basis of the monophasic phenotype of S. 4,[5],12:i:-. The fljAB operon and hin gene were detectable in eight of the S. 4,[5],12:i:- isolates with common amino acid substitutions of A46T in FljA and R140L in Hin. The introduction of these mutations into S. Typhimurium isolates led to the loss of selectability of isolates expressing the phase 2 H antigen. These data suggested that a point mutation was the genetic basis, at least in part, of the S. 4,[5],12:i:- isolates. The results of phenotypic analysis suggested that the S. 4,[5],12:i:- isolates in Japan consist of multiple distinct clones. This is the first detailed characterization of the S. 4,[5],12:i:- isolates derived from various sources across Japan.

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) is a serious public health concern, causing severe diarrhea and hemolytic-uremic syndrome. Patient symptoms are varied among STEC strains, possibly implying the presence of markers for STEC virulence other than Stx. To reveal the genotypic traits responsible for STEC virulence, we investigated 282 strains of various serogroups for the presence of 17 major virulence genes, i.e., stx 1 , stx 2a , stx 2c , stx 2d , stx 2e , stx 2f , eae , tir , espB , espD , iha , saa , subA , ehxA , espP , katP , and stcE . Next, we examined the prevalence of virulence genes according to the seropathotypes in which serotypes were classified (seropathotypes A through E) based on the reported frequencies in human illness, as well as known associations with outbreaks and with severe disease. Our results demonstrate that the presence of both katP and stcE in STEC, in addition to the genes located in the locus of enterocyte effacement (LEE), including eae , tir , espB , and espD , may indicate the most pathogenic genotype of STEC. A population structure analysis of the profiles of virulence genes statistically supported the pathogenic genotype and, furthermore, revealed that there are serogroups with potentially higher pathogenicity than previously thought. Some strains in serogroups O26, O145, and O165 may have high virulence equivalent to that of serogroup O157. Several serogroups, including O14, O16, O45, O63, O74, 119, O128, and O untypeable, also may be potentially pathogenic, although rarely in humans.

To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx 1 stx 2 genotype showed greater resistance to heat than strains of the stx 1 stx 2c genotype; moreover, strains of the stx 1 stx 2 genotype showed greater resistance to starvation than strains of the stx 2 or stx 2c genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx 1 stx 2 genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx 1 stx 2c genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx 1 stx 2 genotype, have greater multiple stress resistance than do strains of other genotypes.

To establish rapid methods to detect Shiga toxin (Stx)-producing Escherichia coli (STEC) in ground beef samples by using an immunochromatography kit, results of 8-h enrichment in various types of broth with shaking were compared. In pure culture, Stx was detected in the culture of trypticase soy broth (TSB) at 42°C and modified EC broth (mEC) at 36°C from all or most serogroups of O26, O111, O128, O157 and OUT. Ground beef samples inoculated with each serogroup were enriched in TSB at 42°C, mEC at 36°C and mEC with novobiocin (NmEC) at 42°C. Although all conditions led to the successful recovery of each serogroup by the plating method, enrichment in NmEC was relatively superior to the other conditions in the detection of Stx by an immunochromatography kit. These results indicated that the growth of STEC and the release of Stx from cells were different in pure cultures and in culture with ground beef. In addition, polymyxin B treatment for 10 min at 37°C and homogenizing with glass beads enhanced the detection of Stx. From the results, it was suggested that an immunochromatography kit in a combination with enrichment in NmEC at 42°C for 8 h, and treatment with polymyxin B or homogenizing would be a rapid method to detect STEC contamination in ground beef.

Genotypes of Shiga toxin-producing Escherichia coli (STEC) O157 isolated from humans and cattle were analyzed by uni- and multivariable logistic regression, and population structure methods, to gain insight into transmission and the nature of human infection. Eleven genotyping assays, including PCR typing of five virulence factors ( stx 1 , stx 2 , stx 2c , eae , and ehxA ) and a lineage-specific polymorphism assay using six markers (LSPA6), were considered in the analyses. The prevalence of the stx 1 , stx 2 , and stx 2c virulence factors was significantly different between human and cattle isolates. However, multivariable regression revealed that the presence of only the stx 2 gene was significantly associated with human isolates after controlling for confounding effects. LSPA6 typing demonstrated an apparent difference in the distribution of LSPA6 lineages between human and cattle isolates and a strong association between stx genotypes and LSPA6 genotypes. Population genetics tools identified three genetically distinct clusters of STEC O157. Each cluster was characterized by stx genotypes and LSPA6 genotypes. The human isolates typically comprised LSPA6 lineage I with stx 1 stx 2 strains and LSPA6 lineage I/II with stx 2 or stx 2 stx 2c strains. In contrast, the cattle isolates comprised LSPA6 lineage II strains with stx 2c or stx 2 stx 2c strains in addition to the clusters identified for the human isolates. Our analyses provide new evidence that the stx 2 gene is the most distinctive feature in human isolates compared to cattle isolates in Japan, and only a subset of the genetically diverse population isolated from cattle is involved in human illnesses. Our results may contribute to international comparisons and risk assessments of STEC O157.

To identify a rapid method for extracting a large amount of DNA from fungi associated with food hygiene, extraction methods were compared using fungal pellets formed rapidly in liquid media. Combinations of physical and chemical methods or commercial kits were evaluated with 3 species of yeast, 10 species of ascomycetous molds, and 4 species of zygomycetous molds. Bead grinding was the physical method, followed by chemical methods involving sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), and benzyl chloride and two commercial kits. Quantity was calculated by UV absorbance at 260 nm, quality was determined by the ratio of UV absorbance at 260 and 280 nm, and gene amplifications and electrophoresis profiles of whole genomes were analyzed. Bead grinding with the SDS method was the most effective for DNA extraction for yeasts and ascomycetous molds, and bead grinding with the CTAB method was most effective with zygomycetous molds. For both groups of molds, bead grinding with the CTAB method was the best approach for DNA extraction. Because this combination also is relatively effective for yeasts, it can be used to extract a large amount of DNA from a wide range of fungi. The DNA extraction methods are useful for developing gene indexes to identify fungi with molecular techniques, such as DNA fingerprinting.

Aim: To develop a novel multiplex polymerase chain reaction (PCR) assay with six primer pairs for Salmonella subspecies identification. Methods and Results: Five primer pairs were chosen to detect the genes (fljB, mdcA, gatD, stn and STM4057) responsible for several phenotypic traits or encoding (sub) species-specific regions. A primer pair for invA was added to simultaneously detect Salmonella. The combination of these primer pairs was expected to give unique results to all subspecies, including Salmonella bongori. The multiplex PCR assay was optimized and evaluated with 53 Salmonella strains representing all S. enterica subspecies, S. bongori and five non-Salmonella strains. The multiplex PCR assay revealed that the genotypes were well correlated with the phenotypes in the Salmonella strains tested. The unique band patterns to their subspecies were generated from 94·3% (50/53) of the Salmonella strains, and no product from other strains by the multiplex PCR assay. Conclusions: The multiplex PCR assay we developed was found to be a rapid, specific and easy to perform method compared with traditional biochemical tests for Salmonella subspecies identification, especially for rapid screening of large numbers of samples. Significance and Impact of the Study: The assay will be useful for characterizing Salmonella isolates from reptiles, which belong to various subspecies, and therefore add to the scientific understanding of reptile-associated Salmonellosis.

Pathogenic Yersinia enterocolitica serotypes O:3, O:5,27, O:8 and O:9 were inoculated into sliced and ground pork, and the samples were stored under vacuum or aerobic conditions at 2 and degrees C. All serotypes survived for 5 weeks in pork with or without vacuum packing without any discernable increases in their population. In sterilized pork with or without vacuum packing, there was no evident growth of Y. enterocolitica. In pork broth in which the pH had been artificially adjusted to 6.8, the growth of Y. enterocolitica was faster than that at 5.7. It is suggested that the growth of Y. enterocolitica in pork with or without vacuum packing may be inhibited by pH but not by the microflora or lactic acid bacteria in pork.