Haruyuki Nakayama-Imaohji

The lifestyle of the segmented filamentous bacterium: A non-culturable gut-associated immunostimulating microbe inferred by whole-genome sequencing

Numerous microbes inhabit the mammalian intestinal track and strongly impact host physiology; however, our understanding of this ecosystem remains limited owing to the high complexity of the microbial community and the presence of numerous non-culturable microbes. Segmented filamentous bacteria (SFBs), which are clostridia-related Gram-positive bacteria, are among such non-culturable populations and are well known for their unique morphology and tight attachment to intestinal epithelial cells. Recent studies have revealed that SFBs play crucial roles in the post-natal maturation of gut immune function, especially the induction of Th17 lymphocytes. Here, we report the complete genome sequence of mouse SFBs. The genome, which comprises a single circular chromosome of 1 620 005 bp, lacks genes for the biosynthesis of almost all amino acids, vitamins/cofactors and nucleotides, but contains a full set of genes for sporulation/germination and, unexpectedly, for chemotaxis/flagella-based motility. These findings suggest a triphasic lifestyle of the SFB, which comprises two types of vegetative (swimming and epicellular parasitic) phases and a dormant (spore) phase. Furthermore, SFBs encode four types of flagellin, three of which are recognized by Toll-like receptor 5 and could elicit the innate immune response. Our results reveal the non-culturability, lifestyle and immunostimulation mechanisms of SFBs and provide a genetic basis for the future development of the SFB cultivation and gene-manipulation techniques.

High-Level Fluoroquinolone Resistance in Ophthalmic Clinical Isolates Belonging to the Species Corynebacterium macginleyi

ABSTRACT The clinical importance of nondiphtherial Corynebacterium, a ubiquitous member of the normal human microflora of the skin and mucous membrane, for ocular surface infections has been recognized recently. We performed an antimicrobial susceptibility test with Etest strips for three fluoroquinolones (ciprofloxacin, norfloxacin, and levofloxacin) and a taxonomic analysis on 21 isolates of Corynebacterium from ophthalmic samples. Of these, 16 isolates were identified as C. macginleyi at the species level on the basis of 16S rRNA gene sequence comparisons. The remaining five isolates were determined to be C. mastitidis (four) or C. accolens (one). Eleven of the C. macginleyi isolates showed high levels of resistance to all of the fluoroquinolones tested, and one isolate was resistant to norfloxacin alone. An analysis of the amplified quinolone-resistance-determining regions of the gyrA genes revealed that a single amino acid substitution in position 83 of the gyrA product was sufficient to generate the norfloxacin resistance phenotype, and double mutations leading to amino acid changes in positions 83 and 87 were necessary for high-level resistance against the other fluoroquinolones. We conducted the first example of multilocus sequence typing (MLST) analysis on C. macginleyi. The MLST analysis grouped the majority of C. macginleyi isolates into a single lineage, and another molecular strain typing by random amplified polymorphic DNA fragment patterns supported the finding, indicating that a particular lineage of C. macginleyi is dominant on the human ocular surface. This type of population might be particularly adaptable to the milieu on the human ocular surface.

Efficient Electrotransformation of Bacteroides fragilis

ABSTRACT This study describes refined electroporation parameters for efficient transformation of Bacteroides fragilis by plasmids prepared from laboratory strains of Escherichia coli. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four E. coli-Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable for B. fragilis transformation, and it generated 2- to 900-fold more transformants (about 104 transformants per μg pLYL05 DNA) than the other plasmids. For the 72-h cultivation period tested, B. fragilis cells harvested at 48 h yielded the highest numbers of transformants. The transformation efficiency of pLYL05 increased linearly with the electric field strength over a range from 5.0 to 12.5 kV/cm. At least 3 h of postpulse incubation was required to maximize the transformation efficiency. For deletion of B. fragilis genes by homologous recombination, competent cells grown to early exponential phase and 12 h of postpulse incubation were required for efficient integration of the pLYL05-based suicide vector into the target site. The expected integration was obtained in B. fragilis strain NCTC9343 only when a homologously prepared (i.e., in vivo methylated) suicide vector was used. Spontaneous resolution of the diploid successfully deleted the expected genetic region. Our simple and efficient plasmid transfer method enabled disruption of a B. fragilis gene using in vivo-methylated targeted vectors. Our optimized electroporation parameters provide a useful tool for genetic manipulation of Bacteroides species.

Identification of the site-specific DNA invertase responsible for the phase variation of SusC/SusD family outer membrane proteins in Bacteroides fragilis.

The human gut microbe Bacteroides fragilis can alter the expression of its surface molecules, such as capsular polysaccharides and SusC/SusD family outer membrane proteins, through reversible DNA inversions. We demonstrate here that DNA inversions at 12 invertible regions, including three gene clusters for SusC/SusD family proteins, were controlled by a single tyrosine site-specific recombinase (Tsr0667) encoded by BF0667 in B. fragilis strain YCH46. Genetic disruption of BF0667 diminished or attenuated shufflon-type DNA inversions at all three susC/susD genes clusters, as well as simple DNA inversions at nine other loci, most of which colocalized with susC/susD family genes. The inverted repeat sequences found within the Tsr0667-regulated invertible regions shared the consensus motif sequence AGTYYYN(4)GDACT. Tsr0667 specifically mediated the DNA inversions of 10 of the 12 regions, even under an Escherichia coli background when the invertible regions were exposed to BF0667 in E. coli cells. Thus, Tsr0667 is an additional globally acting DNA invertase in B. fragilis, which probably involves the selective expression of SusC/SusD family outer membrane proteins.

Corynebacterium lowii sp. nov. and Corynebacterium oculi sp. nov., derived from human clinical disease and an emended description of Corynebacterium mastitidis

Strains of members of the genus Corynebacterium derived from ophthalmologic patients in Japan, Belgium and Switzerland and found to be closely related to-, but distinguishable from Corynebacterium mastitidis by 16S rRNA gene sequencing, were characterized using biochemical, chemotaxonomic, MALDI-TOF mass spectrometry and antimicrobial susceptibility methods and DNA-DNA hybridization as well as by whole-genome sequencing (WGS). Based on this investigation, we describe Corynebacterium lowii sp. nov. and Corynebacterium oculi sp. nov., derived from human ocular specimens, as well as emend the description of Corynebacterium mastitidis. Type strains for these species are: C. lowii R-50085T (=LMG 28276T =CCUG 65815T) and C. oculi R-50187T (=LMG 28277T =CCUG 65816T). DNA G+C content was found to be 62.2 % (by HPLC) and 62.8 % (by WGS) for C. lowii R-50085T, 64.1 % (HPLC) and 64.8 % (WGS) for C. oculi R-50187T and 67.8 % (HPLC) for C. mastitidis LMG 19040T [=S-8T =CCUG 38654T =CECT 4843T =CIP 105509T =DSM 44356T =IFO (NBRC)16160T =JCM 12269T].

PhoB Regulates the Survival of Bacteroides fragilis in Peritoneal Abscesses

In response to phosphate limitation, bacteria employ the Pho regulon, a specific regulatory network for phosphate acquisition. The two-component signal transduction system of PhoRB plays a crucial role in the induction of Pho regulon genes, leading to the adaptation to phosphate starvation. Herein, we identified the PhoRB system in Bacteroides fragilis, a commensal gut bacterium, and evaluated its role in gut colonization and survival in peritoneal abscesses. BF1575 and BF1576 encoded PhoR (sensor histidine kinase) and PhoB (response regulator) in the sequenced B. fragilis strain YCH46, respectively. Transcriptome analysis revealed that deletion of phoB affected the expression of 585 genes (more than 4-fold change) in B. fragilis, which included genes for stress response (chaperons and heat shock proteins), virulence (capsular polysaccharide biosynthesis) and phosphate metabolism. Deletion of phoB reduced the ability of the bacterium to persist in peritoneal abscesses induced by an intra-abdominal challenge of B. fragilis. Furthermore, PhoB was necessary for survival of this anaerobe in peritoneal abscesses but not for in vitro growth in rich media or in intestinal colonization. These results indicate that PhoB plays an important role in the survival of B. fragilis under stressful extraintestinal conditions.

Mariner-based transposon mutagenesis for Bacteroides species

Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human‐Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/μg of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes.

Identification of a New Virulent Clade in Enterohemorrhagic Escherichia coli O26:H11/H- Sequence Type 29

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.

DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.

Microbicidal effects of weakly acidified chlorous acid water against feline calicivirus and Clostridium difficile spores under protein-rich conditions

Sanitation of environmental surfaces with chlorine based-disinfectants is a principal measure to control outbreaks of norovirus or Clostridium difficile. The microbicidal activity of chlorine-based disinfectants depends on the free available chlorine (FAC), but their oxidative potential is rapidly eliminated by organic matter. In this study, the microbicidal activities of weakly acidified chlorous acid water (WACAW) and sodium hypochlorite solution (NaClO) against feline calcivirus (FCV) and C. difficile spores were compared in protein-rich conditions. WACAW inactivated FCV and C. difficile spores better than NaClO under all experimental conditions used in this study. WACAW above 100 ppm FAC decreased FCV >4 log10 within 30 sec in the presence of 0.5% each of bovine serum albumin (BSA), polypeptone or meat extract. Even in the presence of 5% BSA, WACAW at 600 ppm FAC reduced FCV >4 log10 within 30 sec. Polypeptone inhibited the virucidal activity of WACAW against FCV more so than BSA or meat extract. WACAW at 200 ppm FAC decreased C. difficile spores >3 log10 within 1 min in the presence of 0.5% polypeptone. The microbicidal activity of NaClO was extensively diminished in the presence of organic matter. WACAW recovered its FAC to the initial level after partial neutralization by sodium thiosulfate, while no restoration of the FAC was observed in NaClO. These results indicate that WACAW is relatively stable under organic matter-rich conditions and therefore may be useful for treating environmental surfaces contaminated by human excretions.