Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

Distribution of Severe Fever with Thrombocytopenia Syndrome Virus and Antiviral Antibodies in Wild and Domestic Animals in Oita Prefecture, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging life-threatening infectious disease caused by the tickborne SFTS virus (SFTSV), first identified in China in 2009 and then in Japan in 2013. Human SFTS cases were reported to be concentrated in western Japan, but the epidemiological conditions of SFTSV infection in a specific region are still obscure. We performed an epidemiological study of SFTSV in Oita Prefecture on the island of Kyushu, located in western Japan. For our research, we collected sera from wild and domestic animals (deer, wild boars, raccoons, cats, and dogs) and ticks from January 2010 to November 2020 in Oita. The anti-SFTSV antibody positivity rate of deer in 2014 was significantly higher than that in 2011 (65% versus 27%, P < 0.001). The anti-SFTSV antibody positivity rates of deer, wild boars, raccoons, wild dogs, domestic dogs, and domestic cats were 55%, 12%, 27%, 1.8%, 0.53%, and 1.4%, respectively. Moreover, RT-PCR could not detect SFTSV in any tick sample. Of the six areas of Oita Prefecture, only the Eastern area showed no incidence or possibility of SFTSV infection among wild and domestic animals, ticks, and human beings. Further investigation is required to assess whether local seroepidemiology in animals will help assess the risk of SFTSV infections in inhabitants.

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.

Escherichia coli O8:H8 Carrying a Novel Variant of the Heat-Labile Enterotoxin LT2 Gene Caused Outbreaks of Diarrhea.

No outbreaks caused by Escherichia coli-producing heat-labile enterotoxin LT2 have been reported to date. Here, we revealed that the E. coli O8:H8 strains isolated from patients in 2 independent diarrhea outbreaks were negative for any known virulence determinants in routine microbiological tests, were very closely related, and carried a prophage-encoded gene for a novel LT2 variant (LT2d) and the genes for colonization factor antigen III. We also showed that LT2d has a cytotonic activity similar to LT1. These data indicate the importance of E. coli strains producing LT2d as a human pathogen.

Differential dynamics and impacts of prophages and plasmids on the pangenome and virulence factor repertoires of Shiga toxin-producing Escherichia coli O145:H28.

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.

Epidemiological Aspects of Escherichia albertii Outbreaks in Japan and Genetic Characteristics of the Causative Pathogen.

Zoonotic pathogen Escherichia albertii has been identified as the cause of several human disease outbreaks; however, factors such as the general symptoms and incubation period of E. albertii infection have yet to be defined. Therefore, we aimed to determine the unique aspects of E. albertii outbreaks in Japan and to examine the genetic characteristics of the causative pathogen. We studied all known E. albertii outbreaks that occurred in Japan up until 2015, which consisted of five confirmed outbreaks and one putative outbreak (Outbreaks 1-6). Outbreaks were re-examined based on personal communications between researchers in prefectural and municipal public health institutes, and through examination of any published study conducted at the time. Draft genome sequences of outbreak-associated E. albertii isolates were also generated. The most common symptom displayed by patients across the six episodes was watery diarrhea (>80%), followed by abdominal pain (50-84%) and fever (37.0-39.5°C) (26-44%). The estimated average incubation period of E. albertii infection was 12-24 h. We assumed that most of the outbreaks were foodborne or waterborne, with restaurant foods, restaurant water, and boxed lunches being the suspected transmission vehicles. Three of the six outbreak-associated E. albertii isolates possessed intact ETT2 regions, while the remaining isolates contained disrupted ETT2-encoding genes. Virulence gene screening revealed that more than half (44/70) of the tested genes were present in all 5 strains examined, and that each of the strains contained more than 1 gene from 14 out of the 21 groups of virulence genes examined in this study. The five E. albertii strains were classified into four of the five known phylogroups. Therefore, we determined that multiple E. albertii genotypes in Japan have the potential to cause outbreaks of diarrhea, abdominal pain, and/or fever following infection of a human host.

[Molecular epidemiological study of community-acquired methicillin-resistant staphylococcus aureus (CA-MRSA) - an examination of commercially distributed meat as a possible vehicle for CA-MRSA].

Staphylococcus aureus has occupied an important position in public health as a cause of food poisoning and hospital-acquired MRSA (HA-MRSA) infections. The spread of community-acquired MRSA (CA-MRSA) infections has also recently become a concern. However, the sources of this infection remain unclear, and there are few reports of epidemiology information. In order to understand MRSA spread in the community, we investigated the distribution of MRSA strains in commercially distributed raw meat samples (n=305) and stool samples from outpatients with diarrhea (n=1,543) from the same meat distribution region in Oita Prefecture, Japan. 301 Staphylococcus aureus strains were isolated and 18 of them were MRSA (2 from chicken meat, 1 from duck meat, 1 from pork meat, and 14 from patients with diarrhea). All 18 MRSA strains were negative for Panton-Valentine leucocidin gene. In this study conducting a comparison of properties and a molecular epidemiological analysis of MRSA isolated from commercially distributed meat and diarrhea patient stools, the results suggest that commercially distributed meat could play a role in the prevalence of CA-MRSA in the community.

Association between aggregative adherence fimbriae types including putative new variants and virulence-related genes and clump formation among aggR-positive Escherichia coli strains isolated in Thailand and Japan.

Enteroaggregative Escherichia coli (EAggEC) are an important cause of diarrhea. Four types of AAF have been identified; however, their prevalence and association with virulence properties remain unclear. E. coli strains carrying the aggR gene as EAggEC that were isolated in Japan and Thailand (n = 90) were examined for AAF subunit genes, two toxin genes (pet/astA), and clump formation. The most prevalent AAF gene was hdaA (28%), followed by aafA (20%), aggA (12%), and agg3A (4%), as well as a putative new AAF sequence (25.6%). Retention status of the toxin genes and intensities of clump formation appeared to vary according to the AAF type.

Isolation and characteristics of Shiga toxin 2f-producing Escherichia coli among pigeons in Kyushu, Japan.

An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx 2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx 2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA O26, irp 2, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan.

Commercially distributed meat as a potential vehicle for community-acquired methicillin-resistant Staphylococcus aureus.

The incidence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection has been increasing; however, the sources of infection remain unclear. Therefore, we investigated the involvement of meat as a possible mediator of CA-MRSA infection. We examined the distribution of MRSA strains in commercially distributed raw meat samples (n = 197) and diarrheal stool samples of outpatients (n = 1,287) that were collected in Oita Prefecture, Japan, between 2003 and 2009 for routine legal inspections. Fourteen MRSA strains were isolated from three meat and 11 stool samples. Among these, seven isolates from three meat and four stool samples exhibited the same epidemiological marker profiles [coagulase type III, staphylococcal enterotoxin C, staphylococcal chromosomal cassette mec (SCCmec) type IV, ST8, spa type 606 (t1767), and toxic shock syndrome toxin-1 (TSST-1) producing type]. Furthermore, of the seven strains, three isolates from two meat samples and one stool sample collected in 2007 exhibited completely identical characteristics with respect to phage open reading frame (ORF) typing, pulsed-field gel electrophoresis, and drug susceptibility profiles. The results suggest that commercially distributed meat could play a role in the prevalence of CA-MRSA in the community.

Intracellular lipid content of liver and skeletal muscle in patients with adult growth hormone deficiency without diabetes mellitus.

BACKGROUND: Insulin resistance (IR) and visceral obesity are often observed in adult growth hormone deficiency patients (AGHDs). However, there is little information regarding the intrahepatic lipid (IHL) or the intramyocellular lipid (IMCL) content and their association with IR in AGHDs. The aim of this study was to directly assess IHL and IMCL in AGHDs by proton magnetic resonance spectroscopy and to evaluate the association of lipid levels with IR. METHODS: Appropriate hormone replacement therapy (RT) other than GH and estrogen was prescribed before evaluation. Ten AGHDs (aged 23-75 years) without diabetes or elevation of aminotransferases were examined the percent body fat, visceral fat area (VFA), IHL, IMCL, adipokines and glucose metabolism. In two AGHDs, changes of these parameters were evaluated after GHRT. RESULTS: Visceral obesity and metabolic syndrome was found in 100% and in 80% of the patients, respectively. IHL was significantly higher than that in non-obese healthy controls (12.5 ± 4.6 vs. 0.69 ± 0.46%, M ± SE, p = 0.0330), while IMCL did not differ between AGHDs and controls (528.8 ± 137.2 vs. 378 ± 51.1 mM, p = 0.2728). Homeostasis model assessment of IR was significantly correlated with IHL (r = 0.896, p = 0.0001) and IMCL (r = 0.749, p = 0.0102), but not with the VFA or percent truncal fat mass. A decrease of IHL and improvement of glucose tolerance were observed in the two patients after 6 M GHRT. CONCLUSION: These results demonstrated that IHL, but not IMCL, may increase in AGHDs, and that IHL may associate with IR. GHRT may decrease IHL along with amelioration of IR.

Distribution of non-locus of enterocyte effacement pathogenic island-related genes in Escherichia coli carrying eae from patients with diarrhea and healthy individuals in Japan.

The relationship to diarrhea of genes located on the pathogenicity islands (PAI) other than the locus of enterocyte effacement (LEE) was investigated. Enteropathogenic Escherichia coli (EPEC), the retention of espC on the EspC PAI, the OI-122 genes (efa1/lifA, nleB), the phylogenetic marker gene yjaA, and the bundle-forming pilus gene bfpA on the EPEC adherence factor (EAF) plasmid were studied. E. coli strains carrying the intimin gene (eae) without the Shiga toxin gene, isolated from patients with diarrhea (n = 83) and healthy individuals (n = 38) in Japan, were evaluated using PCR. The genotypes of eae and espC were identified by heteroduplex mobility assay (HMA). The proportions of strains isolated from individuals with and without diarrhea that carried these genes were as follows: bfpA, 13.3 and 7.9%, respectively; espC, 25.3 and 36.8%; efa1/lifA, 32.5 and 13.2%; nleB, 63.9 and 60.5%; yjaA, 42.2 and 55.3%. Statistical significance (P < 0.05) was achieved only for efa1/lifA. The proportion of strains lacking espC and carrying efa1/lifA was higher for patient-derived strains (30.1%) than for strains from healthy individuals (13.2%), but the difference was not significant. Strains carrying both espC and efa1/lifA were rare (2 strains from patients). Statistical analyses revealed significant relationships between espC and yjaA and between efa1/lifA and nleB, as well as significant inverse relationships between espC and efa1/lifA and between efa1/lifA and yjaA. espC was found in eae HMA types a1, a2, and c2, whereas efa1/lifA was found in types b1, b2, and c1. In addition, 6 polymorphisms of espC were found. The espC, yjaA, efa1/lifA, and nleB genes were mutually dependent, and their distributions were related to eae type, findings that should be considered in future epidemiological studies.

Classification of perA sequences and their correlation with autoaggregation in typical enteropathogenic Escherichia coli isolates collected in Japan and Thailand.

Enteropathogenic Escherichia coli (EPEC) strains produce a bundle-forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae- and bfpA-positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n=27) and Thailand (n=26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation. The degree of autoaggregation was well correlated with adherence to HEp-2 cells, contact hemolysis and BFP expression. Our results showed that functional deficiency due to frameshift mutation and subsequent nonsense mutation in perA reduced BFP expression in typical EPEC strains isolated in Japan.

The relationship between O-antigens and pathogenic genes of diarrhea-associated Escherichia coli.

To evaluate the serogrouping-based diagnosis of diarrheagenic Escherichia coli, a total of 1,130 strains of E. coli isolated in several countries were studied. The strains were regarded as enterovirulent on the basis of their O-antigens determined using a commercially available kit containing 43 antisera, and the presence of diarrhea-associated genes (eae, stx, aggR, est, elt, ipaH) was evaluated by PCR. Two hundred sixty-three strains of 1,130 (23.3%) were identified as diarrheagenic based on the presence of at least one pathogenic gene. The probability that E. coli identified as diarrheagenic on the basis of serogrouping actually possessed some pathogenic gene was highest for serogroup O119 (78.4%); other serogroups with a positive rate for pathogenic genes higher than 60% were O111 and O126. No target genes were detected among the strains belonging to serogroups O1, O29, O112ac, O143, O158 and O168. Our results suggest that, in practice, serogrouping is useful for the identification of diarrheagenic E. coli in a very limited number of serogroups.

[Hazard evaluation of livestock-derived, verotoxin-producing Escherichia coli by enterohemolysin production assay and eaeA gene detection].

We examined enterohemolysin (Ehly) production, and detected the hlyA gene and the eaeA gene for the intestinal mucosal adherence factor intimin in 131 strains of human-derived verotoxin-producing Escherichia coli (VTEC) and 140 strains of livestock (cattle and swine) -derived VTEC to evaluate their hazards to humans. The hlyA gene was confirmed in 98.5% of human-derived, in 50.5% of cattle-derived, and in 10.3% of swine-derived VTEC strains. Ehly-positive rates were 96.2-97.7%, 45.9-55.0%, and 10.3-20.7% in human-, cattle-, and swine-derived VTEC strains, respectively. Thus, the positive rates differed among strains of different species origins. However, all 12 cattle-derived O157VTEC strains had hlyA, and were Ehly-positive. Although 97.7% of human-derived strains and all cattle-derived O157VTEC strains had eaeA, only 8.1% of cattle derived strains of serotypes other than O157 and 3.4% of swine-derived strains had eaeA. In human- and cattle-derived strains, the presence of eaeA was associated with Ehly: all eaeA-carrying strains had hlyA, and almost all of them were Ehly-positive. Cattle-derived eaeA-carrying strains accounted for 29.5-35.3% of Ehly-positive strains, compared to 100% in human-derived strains. Only 3-4% of Ehly-negative strains had eaeA, and none of the non-hlyA-carrying strains had eaeA. These findings suggest that 2 factors, eaeA and Ehly, serve as useful indicators for the evaluation of hazard to humans, and that Ehly is a useful indicator because cattle-derived Ehly-positive strains may have eaeA.