記述言語：その他   掲載種別：研究論文（学術雑誌）  

Abstract

Background

Within the genus Escherichia, several monophyletic clades other than the traditionally defined species have been identified. Of these, cryptic clade I (C-I) appears to represent a subspecies of E. coli, but due to the difficulty in distinguishing it from E. coli sensu stricto, the population structure and virulence potential of C-I are unclear.

Results

We defined a set of true C-I strains (n = 465), including a Shiga toxin 2a (Stx2a)-producing isolate from a patient with bloody diarrhoea identified by the retrospective analyses using a C-I-specific detection system. Through genomic analysis of 804 isolates from the cryptic clades, including these C-I strains, we revealed their global population structures and the marked accumulation of virulence genes and antimicrobial resistance genes in C-I. In particular, half of the C-I strains contained hallmark virulence genes of Stx-producing E. coli (STEC) and/or enterotoxigenic E. coli (ETEC). We also found the host-specific distributions of virulence genes, which suggests bovines as the potential source of human infections caused by STEC- and STEC/ETEC hybrid-type C-I strains, as is known in STEC.

Conclusions

Our findings demonstrate the emergence of human intestinal pathogens in C-I lineage. To better understand the features of C-I strains and their infections, extensive surveillance and larger population studies of C-I strains are needed. The C-I-specific detection system developed in this study will be a powerful tool for screening and identifying C-I strains.

DOI： 10.1186/s12915-023-01584-4

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Pathogenic Escherichia coli strains are important causes of several swine diseases that result in significant economic losses worldwide. In Japan, the use of antimicrobials in swine is much higher than that in other farm animals every year. Antimicrobial resistance in pathogenic E. coli strains also heavily impacts the swine industry due to the limited treatment options and an increase in the potential risk of the One Health crisis. In 2016, we investigated 684 Japanese isolates of swine pathogenic E. coli belonging to four major serogroups and reported the emergence and increase in the highly multidrug-resistant serogroups O116 and OSB9 and the appearance of colistin-resistant strains. In the present study, by expanding our previous analysis, we determined the serotypes and antimicrobial resistance of 1,708 E. coli strains isolated from diseased swine between 1991 and 2019 in Japan and found recent increases in the prevalences of multidrug-resistant strains and minor serogroup strains. Among the antimicrobials examined in this study that have been approved for animal use, a third-generation cephalosporin was found to be effective against the most isolates (resistance rate: 1.2%) but not against highly multidrug-resistant strains. We also analyzed the susceptibilities of the 1,708 isolates to apramycin and bicozamycin, both which are available for treating swine in Japan, and found that the rates of resistance to apramycin and bicozamycin were low (6.7% and 5.8%, respectively), and both antimicrobials are more effective (resistance rates: 2.7% and 5.4%, respectively) than third-generation cephalosporins (resistance rate: 16.2%) against highly multidrug-resistant strains.

DOI： 10.3389/fmicb.2023.1107566

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Abstract

Shiga toxin (Stx) is the key virulence factor of Stx-producing Escherichia coli (STEC). All known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages). Although the genetic diversity of Stx phages has frequently been described, systematic analyses of Stx phages in a single STEC lineage are limited. In this study, focusing on the O26:H11 STEC sequence type 21 (ST21) lineage, where the stx1a gene is highly conserved, we analysed the Stx1a phages in 39 strains representative of the entire ST21 lineage and found a high level of variation in Stx1a phage genomes caused by various mechanisms, including replacement by a different Stx1a phage at the same or different locus. The evolutionary timescale of events changing Stx1a phages in ST21 was also determined. Furthermore, by using an Stx1 quantification system developed in this study, we found notable variations in the efficiency of Stx1 production upon prophage induction, which sharply contrasted with the conserved iron regulated Stx1 production. These variations were associated with the Stx1a phage alteration in some cases but not in other cases; thus, Stx1 production in this STEC lineage was determined by differences not only in Stx1 phages but also in host-encoded factors.

DOI： 10.1038/s41598-023-32111-8

記述言語：英語   掲載種別：研究論文（学術雑誌）  

<jats:p>Among Shiga toxin (Stx)-producing <jats:italic>
<jats:named-content content-type="species">
<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.3093" xlink:type="simple">Escherichia coli</jats:ext-link>
</jats:named-content>
</jats:italic> (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 <jats:italic>sensu stricto</jats:italic> (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.</jats:p>

DOI： 10.1099/mgen.0.000959

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Among the nine clades of Shiga toxin (Stx)-producing Escherichia coli O157:H7, clade 8 is thought to be highly pathogenic, as it causes severe disease more often than other clades. Two subclades have been proposed, but there are conflicting reports on intersubclade differences in Stx2 levels, although Stx2 production is a risk factor for severe disease development. The global population structure of clade 8 has also yet to be fully elucidated. Here, we present genome analyses of a global clade 8 strain set (n=510), including 147 Japanese strains sequenced in this study. The complete genome sequences of 18 of the 147 strains were determined to perform detailed clade-wide genome analyses together with 17 publicly available closed genomes. Intraclade variations in Stx2 production level and disease severity were also re-evaluated within the phylogenetic context. Based on phylogenomic analysis, clade 8 was divided into four lineages corresponding to the previously proposed SNP genotypes (SGs): SG8_30, SG8_31A, SG8_31B and SG8_32. SG8_30 and the common ancestor of the other SGs were first separated, with SG8_31A and SG8_31B emerging from the latter and SG8_32 emerging from SG8_31B. Comparison of 35 closed genomes revealed the overall structure of chromosomes and pO157 virulence plasmids and the prophage contents to be well conserved. However, Stx2a phages exhibit notable genomic diversity, even though all are integrated into the argW locus, indicating that subtype changes in Stx2a phage occurred from the γ subtype to its variant (γ_v1) in SG8_31A and from γ to δ in SG8_31B and SG8_32 via replacement of parts or almost entire phage genomes, respectively. We further show that SG8_30 strains (all carrying γ Stx2a phages) produce significantly higher levels of Stx2 and cause severe disease more frequently than SG8_32 strains (all carrying δ Stx2a phages). Clear conclusions on SG8_31A and SG8_31B cannot be made due to the small number of strains available, but as SG8_31A (carrying γ_v1 Stx2a phages) contains strains that produce much more Stx2 than SG8_30 strains, attention should also be paid to this SG.

DOI： 10.1099/mgen.0.000935

記述言語：その他   掲載種別：研究論文（学術雑誌）  

The origin of human and calf infections by Shigatoxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli O80:H2 is still unknown. The aim of this study was to identify E. coli O80 in healthy cattle with an emphasis on melibiose non-fermenting E. coli O80:H2. Faecal materials collected from 149 bulls at 1 slaughterhouse and 194 cows on 9 farms were tested with O80 antigen-encoding gene PCR after overnight growth in enrichment broths. The 53 O80 PCR-positive broths were streaked on different (semi-)selective agar plates. Five E. coli colonies from 3 bulls and 11 from 2 cows tested positive with the O80 PCR, but no melibiose non-fermenting E. coli was isolated. However, these 16 E. coli O80 were negative with PCR targeting the fliCH2, eae, stx1, stx2 and hlyF genes and were identified by WGS to serotypes and sequence types O80:H6/ST8619 and O80:H45/ST4175. They were phylogenetically related to E. coli O80:H6 and O80:H45 isolated from different animal species in different countries, respectively, but neither to STEC and EPEC O80:H2/ST301, nor to other serotypes of the clonal complex 165. As a conclusion, healthy adult cattle were not identified as a source of contamination of humans and calves by STEC or EPEC O80:H2.

DOI： 10.3390/microorganisms11020230

記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Irritable bowel syndrome (IBS) is a disorder of gut-brain interaction, including dysregulation of the hypothalamic-pituitary-adrenal axis with salivary cortisol changes. However, the role of gastrointestinal microbiota during IBS symptom exacerbation remains unclear. We tested the hypothesis that the microbial species, gene transcripts, and chemical composition of fecal and oral samples are altered during the exacerbation of IBS symptoms. METHODS: Fecal, salivary, and dental plaque samples were collected at baseline from 43 men with IBS with diarrhea (IBS-D) and 40 healthy control (HC) men. Samples in the IBS-D patients were also collected during symptom exacerbation. The composition of the fecal microbiota was determined by analyzing the 16S rRNA gene, RNA-based metatranscriptome, and metabolites in samples from HC and IBS patients with and without symptom exacerbation. Oral samples were also analyzed using omics approaches. RESULTS: The fecal microbiota during IBS symptom exacerbation exhibited significant differences in the phylogenic pattern and short-chain fatty acid compared with fecal samples during defecation when symptoms were not exacerbated. Although there were no significant differences in the phylogenic pattern of fecal microbiota abundance between HCs and IBS-D patients, significant differences were detected in the expression patterns of bacterial transcriptomes related to butyrate production and neuroendocrine hormones, including tryptophan-serotonin-melatonin synthesis and glutamine/GABA. The composition of plaque microbiota was different between HC and IBS-D patients during normal defecation. CONCLUSIONS: Our findings suggest that colonic host-microbial interactions are altered in IBS-D patients during exacerbation of symptoms. There were no overlaps between feces and oral microbiomes.

DOI： 10.1007/s00535-022-01888-2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Insertion sequences (ISs) can modulate gene expression by gene inactivation or activation. While phenotypic changes due to IS insertion/transposition are frequently observed, gene reactivation by precise or simple IS excision rarely occurs.

DOI： 10.1128/aem.00760-22

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Helicobacter cinaedi is an enterohepatic Helicobacter that causes bacteremia and other diseases in humans. While H. cinaedi-like strains are isolated from animals, including dog isolates belonging to a recently proposed H. canicola, little is known about the genetic differences between H. cinaedi and these animal isolates. Here, we sequenced 43 H. cinaedi- or H. canicola-like strains isolated from humans, hamsters, rats and dogs and collected 81 genome sequences of H. cinaedi, H. canicola and other enterohepatic Helicobacter strains from public databases. Genomic comparison of these strains identified four distinct clades (clades I-IV) in H. cinaedi/canicola/'magderbugensis' (HCCM) complex. Among these, clade I corresponds to H. cinaedi sensu stricto and represents a human-adapted lineage in the complex. We identified several genomic features unique to clade I. They include the accumulation of antimicrobial resistance-related mutations that reflects the human association of clade I and the larger genome size and the presence of a CRISPR-Cas system and multiple toxin-antitoxin and restriction-modification systems, both of which indicate the contribution of horizontal gene transfer to the evolution of clade I. In addition, nearly all clade I strains but only a few strains belonging to one minor clade contained a highly variable genomic region encoding a type VI secretion system (T6SS), which could play important roles in gut colonization by killing competitors or inhibiting their growth. We also developed a method to systematically search for H. cinaedi sequences in large metagenome data sets based on the results of genome comparison. Using this method, we successfully identified multiple HCCM complex-containing human faecal metagenome samples and obtained the sequence information covering almost the entire genome of each strain. Importantly, all were clade I strains, supporting our conclusion that H. cinaedi sensu stricto is a human-adapted lineage in the HCCM complex.

DOI： 10.1099/mgen.0.000830

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Serratia marcescens is an important nosocomial pathogen causing various opportunistic infections, such as urinary tract infections, bacteremia and sometimes even hospital outbreaks. The recent emergence and spread of multidrug-resistant (MDR) strains further pose serious threats to global public health. This bacterium is also ubiquitously found in natural environments, but the genomic differences between clinical and environmental isolates are not clear, including those between S. marcescens and its close relatives. In this study, we performed a large-scale genome analysis of S. marcescens and closely related species (referred to as the ‘S. marcescens complex’), including more than 200 clinical and environmental strains newly sequenced here. Our analysis revealed their phylogenetic relationships and complex global population structure, comprising 14 clades, which were defined based on whole-genome average nucleotide identity. Clades 10, 11, 12 and 13 corresponded to S. nematodiphila, S. marcescens sensu stricto, S. ureilytica and S. surfactantfaciens, respectively. Several clades exhibited distinct genome sizes and GC contents and a negative correlation of these genomic parameters was observed in each clade, which was associated with the acquisition of mobile genetic elements (MGEs), but different types of MGEs, plasmids or prophages (and other integrative elements), were found to contribute to the generation of these genomic variations. Importantly, clades 1 and 2 mostly comprised clinical or hospital environment isolates and accumulated a wide range of antimicrobial resistance genes, including various extended-spectrum β-lactamase and carbapenemase genes, and fluoroquinolone target site mutations, leading to a high proportion of MDR strains. This finding suggests that clades 1 and 2 represent hospital-adapted lineages in the S. marcescens complex although their potential virulence is currently unknown. These data provide an important genomic basis for reconsidering the classification of this group of bacteria and reveal novel insights into their evolution, biology and differential importance in clinical settings.

DOI： 10.1099/mgen.0.000793

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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.

DOI： 10.1099/mgen.0.000716

記述言語：その他   掲載種別：研究論文（学術雑誌）  

Shiga toxin-producing <italic>Escherichia coli</italic> (STEC) is a major intestinal pathogen and causes serious gastrointestinal illness, which includes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. The major virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which belong to the AB-type toxin family. Among several subtypes of Stx1 and Stx2, the production of Stx2a is thought to be a risk factor for severe STEC infections, but Stx2a production levels vary markedly between STEC strains, even strains with the same serotype. Therefore, quantitative analyses of Stx2 production by STEC strains are important to understand the virulence potential of specific lineages or sublineages. In this study, we developed a novel Stx2 quantification method by utilizing homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technology. To determine suitable “sandwich” assay conditions, we tested 6 combinations of fluorescence-labeled monoclonal antibodies (mAbs) specific to Stx2 and compared the HTRF signal intensities obtained at various incubation times. Through this analysis, we selected the most suitable mAb pair, one recognizing the A subunit and the other recognizing the B subunit, thus together detecting Stx holotoxins. The optimal incubation time was also determined (18 h). Then, we optimized the concentrations of the two mAbs based on the range for linearity. The established HTRF assay detected 0.5 ng/ml of the highly purified recombinant Stx2a and Stx2e proteins and the working range was 1–64 ng/ml for both Stx2a and Stx2e. Through the quantification analysis of Stx proteins in STEC cell lysates, we confirmed that other Stx2 subtypes (Stx2b, Stx2c, Stx2d and Stx2g) can also be quantified at a certain level of accuracy, while this assay system does not detect Stx2f, which is highly divergent in sequence from other Stx2 subtypes, and Stx1. As the HTRF protocol we established is simple, this assay system should prove useful for the quantitative analysis of Stx2 production levels of a large number of STEC strains.

DOI： 10.7717/peerj.11871

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, <italic>Escherichia coli</italic> strains such as Shiga toxin (Stx)-producing <italic>E</italic>. <italic>coli</italic> (STEC) and enteropathogenic <italic>E</italic>. <italic>coli</italic> (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (<italic>att</italic>) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified <italic>attB</italic> sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different <italic>attB</italic> sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of <italic>E</italic>. <italic>coli</italic> strains as well as other prophage-rich bacterial species.

DOI： 10.1371/journal.ppat.1009073

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/jam.14759

記述言語：英語   掲載種別：研究論文（学術雑誌）  

<title>ABSTRACT</title>
Dermonecrotic toxin (DNT) is one of the representative toxins produced by <named-content content-type="genus-species">Bordetella pertussis</named-content>, but its role in pertussis, <named-content content-type="genus-species">B. pertussis</named-content> infection, remains unknown. In this study, we identified the T-type voltage-gated Ca²⁺ channel Ca_V3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As Ca_V3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of <named-content content-type="genus-species">B. pertussis</named-content>. The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.


<bold>IMPORTANCE</bold> <named-content content-type="genus-species">Bordetella pertussis</named-content>, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca²⁺ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of <named-content content-type="genus-species">B. pertussis</named-content> infection.

DOI： 10.1128/mbio.03146-19

記述言語：英語   掲載種別：研究論文（学術雑誌）  

<title>Abstract</title>
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.

DOI： 10.1093/ofid/ofaa021

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1099/mgen.0.000323

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Escherichia albertii is a recently recognized human enteropathogen that is closely related to Escherichia coli. In many Gram-negative bacteria, including E. coli, O-antigen variation has long been used for the serotyping of strains. In E. albertii, while eight O-serotypes unique to this species have been identified, some strains have been shown to exhibit genetic or serological similarity to known E. coli/Shigella O-serotypes. However, the diversity of O-serotypes and O-antigen biosynthesis gene clusters (O-AGCs) of E. albertii remains to be systematically investigated. Here, we analysed the O-AGCs of 65 E. albertii strains and identified 40 E. albertii O-genotypes (EAOgs) (named EAOg1-EAOg40). Analyses of the 40 EAOgs revealed that as many as 20 EAOgs exhibited significant genetic and serological similarity to the O-AGCs of known E. coli/Shigella O-serotypes, and provided evidence for the inter-species horizontal gene transfer of O-AGCs between E. albertii and E. coli. Based on the sequence variation in the wzx gene among the 40 EAOgs, we developed a multiplex PCR-based O-genotyping system for E. albertii (EAO-genotyping PCR) and verified its usefulness by genotyping 278 E. albertii strains from various sources. Although 225 (80.9 &#37;) of the 278 strains could be genotyped, 51 were not assigned to any of the 40 EAOgs, indicating that further analyses are required to better understand the diversity of O-AGCs in E. albertii and improve the EAO-genotyping PCR method. A phylogenetic view of E. albertii strains sequenced so far is also presented with the distribution of the 40 EAOgs, which provided multiple examples for the intra-species horizontal transfer of O-AGCs in E. albertii.

DOI： 10.1099/mgen.0.000314

記述言語：英語   掲載種別：研究論文（学術雑誌）  

<title>Abstract</title>
In bacterial genome and metagenome sequencing, Illumina sequencers are most frequently used due to their high throughput capacity, and multiple library preparation kits have been developed for Illumina platforms. Here, we systematically analysed and compared the sequencing bias generated by currently available library preparation kits for Illumina sequencing. Our analyses revealed that a strong sequencing bias is introduced in low-GC regions by the Nextera XT kit. The level of bias introduced is dependent on the level of GC content; stronger bias is generated as the GC content decreases. Other analysed kits did not introduce this strong sequencing bias. The GC content-associated sequencing bias introduced by Nextera XT was more remarkable in metagenome sequencing of a mock bacterial community and seriously affected estimation of the relative abundance of low-GC species. The results of our analyses highlight the importance of selecting proper library preparation kits according to the purposes and targets of sequencing, particularly in metagenome sequencing, where a wide range of microbial species with various degrees of GC content is present. Our data also indicate that special attention should be paid to which library preparation kit was used when analysing and interpreting publicly available metagenomic data.

DOI： 10.1093/dnares/dsz017

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

BspR/BtrA, an Anti-σ factor, regulates the ability of Bordetella bronchiseptica to cause cough in rats
Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections, many of which are characterized by coughing of the infected hosts. The pathogenesis of the coughing remains to be analyzed, mainly because there were no convenient infection models of small animals that replicate coughing after Bordetella infection. Here, we present a coughing model of rats infected with B. bronchiseptica. Rats, which are one of natural hosts of B. bronchiseptica, were readily infected with the organisms and showed frequent coughing. B. pertussis also caused coughing in rats, which is consistent with previous reports, but the cough response was less apparent than the B. bronchiseptica-induced cough. By using the rat model, we demonstrated that adenylate cyclase toxin, dermonecrotic toxin, and the type III secretion system are not involved in cough production, but BspR/BtrA (different names for the same protein), an anti-σ factor, regulates the production of unknown factor(s) to cause coughing. Rat coughing was observed by inoculation of not only the living bacteria but also the bacterial lysates. Infection with bspR (btrA)-deficient strains caused significantly less frequent coughing than the wild type; however, intranasal inoculation of the lysates from a bspR (btrA)-deficient strain caused coughing similarly to the wild type, suggesting that BspR/BtrA regulates the production of the cough factor(s) only when the bacteria colonize host bodies. Moreover, the cough factor(s) was found to be heat labile and produced by B. bronchiseptica in the Bvg phase. We consider that our rat model provides insight into the pathogenesis of cough induced by the Bordetella infection. +

DOI： 10.1128/mSphere.00093-19

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Genomic Characterization of β-Glucuronidase-Positive Escherichia coli O157:H7 Producing Stx2a.
Among Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a β-glucuronidase-positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (β-glucuronidase-negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones.

DOI： 10.3201/eid2412.180404

記述言語：英語   掲載種別：研究論文（学術雑誌）  

A novel Gram-negative bacterium, designated 4G11^T, was isolated from the sea surface microlayer of a marine inlet. On the basis of 16S rRNA gene sequence analysis, the strain showed the closest similarity to Amylibacter ulvae KCTC 32465T (99.0 &#37;). However, DNA-DNA hybridization values showed low DNA relatedness between strain 4G11^Tand its close phylogenetic neighbours, Amylibacter marinus NBRC 110140T (8.0±0.4 &#37;) and Amylibacter ulvae KCTC 32465^T(52.9±0.9 &#37;). Strain 4G11T had C_{18: 1}, C_{16: 0}and C_{18: 2}as the major fatty acids. The only isoprenoid quinone detected for strain 4G11T was ubiquinone-10. The major polar lipids were phosphatidylglycerol, phosphatidylcholine, one unidentified polar lipid, one unidentified phospholipid and one unidentified aminolipid. The DNA G+C content of strain 4G11T was 50.0 mol&#37;. Based on phenotypic and chemotaxonomic characteristics and analysis of the 16S rRNA gene sequence, the novel strain should be assigned to a novel species, for which the name Amylibacter kogurei sp. nov. is proposed. The type strain of Amylibacter kogurei is 4G11^T(KY463497=KC^TC 52506^T=NBRC 112428^T).

DOI： 10.1099/ijsem.0.002911

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussis. B. bronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to B. pertussis CyaA. The hemolytic activity of B. bronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, B. bronchiseptica CyaA was phosphorylated at Ser ³⁷⁵ , which constitutes a motif (RSXpSXP [pS is phos-phoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of B. bronchiseptica CyaA based on its enzyme activity were markedly attenuated. B. parapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to B. bronchiseptica CyaA; however, B. pertussis CyaA has Phe ³⁷⁵ instead of Ser, and thus, was not affected by 14-3-3. In addition, B. pertussis CyaA impaired the barrier function of epithelial cells, whereas B. bronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/B. parapertussis and B. pertussis. IMPORTANCE Bordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella. The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins.

DOI： 10.1128/mBio.00628-18

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Here, we report the draft genome sequence of Saccharospirillum sp. strain MSK14-1, isolated from surface seawater collected at Aburatsubo Inlet in Japan. The genome sequence of strain MSK14-1 should contribute to our understanding of the characteristics of the genus Saccharospirillum.

DOI： 10.1128/genomeA.00469-18

記述言語：英語  

DOI： 10.1111/1348-0421.12552

記述言語：英語  

Characterization of the functional activity of botulinum neurotoxin subtype B6
Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A–G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C-terminal heavy chain (H_C) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously-characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. H_C/B1 and H_C/B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its H_c.domain.

DOI： 10.1111/1348-0421.12540

記述言語：英語  

Pseudomonas aeruginosa is one of the most common model bacterial species, and genomes of hundreds of strains of this species have been sequenced to date. However, currently there is only one available genome of an oceanic isolate. Here, we report two complete and six draft genome sequences of P. aeruginosa isolates from the open ocean.

DOI： 10.1128/genomeA.01255-17

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re-emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine.

DOI： 10.1111/1348-0421.12504

記述言語：英語  

Here, we report the draft genome sequences of Tersicoccus phoenicis DSM 30849T, isolated from a spacecraft assembly cleanroom at the National Aeronautics and Space Administration (NASA), and Tersicoccus sp. strain Bi-70, isolated from Lake Biwa, the largest lake in Japan. These genome sequences facilitate our understanding of the adaptation of these closely related strains to different habitats.

DOI： 10.1128/genomeA.00079-17

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bordetella species display phase modulation between Bvg⁺ and Bvg^- phases. Because expression of known virulence factors is up-regulated in the Bvg⁺ phase, bacteria in this phase are considered competent for infection. However, the Bvg^- phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg^- phase (bvg-repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (Bordetella RTX-family Adhesin), which is a typical bvg-repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan-type cadherin-like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca²⁺-dependent bacteria-to-substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg-repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis.

DOI： 10.1111/1348-0421.12356

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Analyses of bacterial genes expressed in response to the host environment provide clues to understanding the host-pathogen interactions that lead to the establishment of infection. In this study, a novel method named In Vivo Expressed-Tag ImmunoPrecipitation (IVET-PI) was developed for detecting genes expressed in bacteria that are recovered in a small numbers from host tissues. IVET-IP was designed to overcome some drawbacks of previous similar methods. We applied IVET-IP to Bordetella bronchiseptica colonizing rat trachea and identified 173 genes that were expressed in the bacteria over the entire course of an infection. These gene products included two transcriptional factors that are involved in the expression of filamentous hemagglutinin, adenylate cyclase toxin, and major virulence factors for the bordetellae. We consider that this method might provide novel insight into the course of Bordetella infection.

DOI： 10.1111/1348-0421.12247

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bordetella bronchiseptica is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of B. bronchiseptica S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs) at positions -129, -72, +22, and +38 in the region upstream of dnt encoding dermonecrotic toxin (DNT), when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and β-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of dnt and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for dnt. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases.

DOI： 10.1371/journal.pone.0116604

記述言語：日本語  

DOI： 10.1016/j.vetmic.2012.11.009

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.

DOI： 10.1111/j.1348-0421.2012.00490.x

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species
Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicleassociated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals. © 2012, American Society for Microbiology.

DOI： 10.1128/IAI.00302-12

記述言語：英語  

Clostridium botulinum produces large complex toxins, which include botulinum neurotoxin (BoNT) and auxiliary non-toxic proteins. We prepared monoclonal antibodies (mAbs) from mice that were immunized several times with BoNT/A after basal immunization with toxoid. We then examined the reactivities of these mAbs to BoNT and toxoid and showed that some mAbs reacted to only BoNT. This result indicates that the antigenicity of BoNT/A partially disappeared with formalin treatment. Some mAbs that specifically recognized either BoNT/A1 or BoNT/A2 were considered useful as detection antibodies specific for the BoNT/A subtype. Results of a neutralizing test with mAbs against either BoNT/A1 or BoNT/A2 showed that neutralizing antibody recognition sites were present in the light chain, heavy chain (N-terminal half), and heavy chain (C-terminal half) domains. Investigation of the different binding capabilities of the mAbs to BoNT and the complex toxin by immunoprecipitation suggested that the light chain of BoNT is exposed at the molecular surface of the complex toxin since there was no difference in the binding of light chain-specific mAb to BoNT and the complex toxin. The heavy chain is related to BoNT binding to non-toxic components, because the reactivity of the heavy chain to some mAbs was influenced by non-toxic components.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3′-sialyllactose at a resolution of 3.0 Å. In the structure, an electron density derived from the 3′-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

DOI： 10.1016/j.bbrc.2011.06.173

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Botulinum toxin (BoNT) from Clostridium botulinum OFD05, isolated from bovine botulism, is a D/C mosaic-type BoNT. BoNTs possess binding, translocation and catalytic domains. The BoNT/OFD05 binding domain exhibits significant sequence identity to BoNT/C, which requires a single ganglioside as a binding receptor on neuronal cells, while BoNT/A and BoNT/B require two receptors for specific binding. To determine the binding mechanism of BoNT/OFD05 and its ganglioside receptors on neuronal cells, recombinant BoNT/OFD05 receptor-binding domain has been expressed, purified and crystallized. Native and SeMet-derivative crystals showed X-ray diffraction to 2.8 and 3.1 Å resolution, respectively. The crystals belonged to space group P212121.

DOI： 10.1107/S1744309110012182

記述言語：日本語  

DOI： 10.1016/j.vetmic.2009.07.023

開催年月日： 2020年2月

記述言語：日本語  

国名：その他  

The mechanism responsible for the change in lactose hydrolysis during EHEC O121:19 cultivation

開催年月日： 2019年11月

記述言語：日本語  

国名：その他  

開催年月日： 2019年9月

記述言語：日本語  

国名：その他  

開催年月日： 2016年9月

記述言語：日本語  

開催地：宮崎市民プラザ・オルブライトホール   国名：日本国  

【目的】腸管出血性大腸菌 (enterohemorrhagic Escherichia coli, EHEC)は志賀毒素 (Shiga toxin, Stx1およびStx2)など、様々な病原因子を産生する。EHECの代表的なO血清群であるO157、O26、O111、O103、O121、O145、O45のうち、当研究室では前5者の全ゲノムを解読し、病原性進化機構の解明などを進めている。本研究ではO145の全ゲノム配列決定とO145菌株内でのゲノム多様性の実体解明を目的とした比較ゲノム解析を行った。 【方法】血便患者由来O145株(10942株)の全ゲノム配列を決定し、アノテーションや病原関連遺伝子の同定等を行った。また、ベルギー株を含む49株のドラフトゲノム配列を取得し、全ゲノム系統解析を実施するとともにStx2ファージ挿入部位を決定した。さらに、各菌株のStx2産生量をELISA法で定量した。 【結果】10942株の染色体は5,374 kbで、93 kbの病原プラスミドを保持していた。EHECの他の血清型と同様に、染色体上に多数のプロファージを有し、３型分泌系エフェクターなどの病原因子レパートリーも他のEHECと類似していた。ベルギー株も含めた全ゲノム系統解析から、O145には複数の亜系統が存在しており、いずれの亜系統にも国内株が含まれることが明らかとなった。また、Stx2ファージの挿入部位は亜系統によって異なり、Stx2産生量において各菌株間に明らかな違いが見られた。 【考察】O157などと同様に、O145も水平伝播により多数の病原遺伝子を獲得し、EHECへ進化したと推察された。また、O145における菌株間のゲノム多様性は比較的高く、日本国内にも複数の亜系統が分布していると考えられた。O145株に挿入されたStx2ファージのゲノム多型と各菌株のStx2産生量の違いの関連性については、詳細に解析する必要があるため、現在、全株においてStx2ファージ領域の配列決定を行っている。