Vibrio cholerae is an aetiological agent of cholera that inhabits marine and estuarine environments. It can survive harsh environments by entering the viable but non-culturable (VBNC) state, but the related changes in gene expression have not been described. Here, we experimentally induced the VBNC state in V. cholerae O1, by incubation in artificial seawater at 4°C. Bacterial cells that were incubated for 70 days retained their membrane integrity and were pathogenic, colonizing the gut of iron-dextran-treated mice, even though they formed no colonies on tryptic soy agar (TSA) or TSA amended with pyruvate. We therefore used this stage of cells as the VBNC bacteria. We compared the global transcription pattern of the VBNC cells with that of stationary-phase cells grown in rich medium. A total of 100 genes were induced by more than fivefold in the VBNC state, and the modulated genes were mostly those responsible for cellular processes. Furthermore, real-time RT-PCR analysis verified the changes in the expression levels, showing that the VC0230 [iron(III) ABC transporter], VC1212 (polB), VC2132 (fliG) and VC2187 (flaC) mRNAs were increased in the non-culturable state. Thus, these genes may be suitable markers for the detection of VBNC V. cholerae. To our knowledge, this is the first report of a comprehensive transcriptome analysis of V. cholerae in the VBNC state. The significance of this gene expression profile compared with those of in vivo isolates and non-stressed bacteria (culturable in vitro) is its potential to provide information about the public health risk from dormant bacteria.

A new subspecies, Staphylococcus schleiferi subsp. couguluns, was isolated from the external auditory meatus of dogs suffering from external ear otitis and is described on the basis of studies of 21 strains. Phenotypic studies showed that these strains are more closely related to Staphylococcus intermedius than to other staphylococci, but DNA hybridization studies indicated that they are closely related to Staphylococcus schleiferi NS50274T. On the basis of biochemical distinctiveness (positive test tube coagulase test and different carbohydrate reactions) and the etiological importance (frequent isolation from otitis specimens from dogs) of these strains, we propose to classify them as a subspecies of S. schleiferi. The strains of this new subspecies are coagulase tube test, P-hemolysin, and heat-stable nuclease positive but clumping factor negative. A simple scheme for the differentiation of S . schleiferi subsp. cougulans from the other coagulase-positive staphylococci is presented. The type strain is GA211 (=JCM 7470).The coagulase-positive species Staphylococcus intermedius is often isolated from veterinary clinical specimens from dogs (1-3,15) and is sometimes isolated from those from cats (2, 8). In the course of our taxonomic studies on staphylococci isolated from dogs and cats, a group of unidentified coagulase-positive strains was discovered. These strains were isolated from the external auditory meatus of dogs suffering from external ear otitis. Though strains from this group were phenotypically similar to strains of S . intermedius, we cannot identify these strains as S. intermedius, because acetoin is produced and acid is not generated from trehalose, unlike the reactions of S . intermedius. In the course of DNA-DNA hybridization studies, our unidentified group of strains revealed high levels of homology with S . schleiferi N850274T. S . schleiferi was reported by Freney et al. in 1988 as a new species isolated from human clinical specimens. It produced no coagulase in tube tests (9). Despite a relatively high level of DNA homology, phenotypic characteristics of S . schleiferi do not match those of our strains. MATERIALS AND METHODSBacterial strains. Twenty-one strains (GA11, GA64, GA89, GA116, GA124, GA199, GA211, GA222, GA227, GA238, GA247, GA288, GA337, GA347, GA390, GA400, GA412, GA416, GA456, GA483, and GA499) were isolated between 1982 and 1988 from discharges of the external ear of dogs diagnosed as suffering from external ear otitis. The isolation medium was Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% defibrinated horse blood. Cultures were maintained at -80°C in skim milk (Difco Laboratories, Detroit, Mich.). Unless noted otherwise, inocula for biochemical tests were prepared from overnight cultures on P agar plates (13), and incubation for tests was at 37°C. The type strains of various Staphylococcus species were obtained as previously described (11).Methods. Cell and colony characteristics, the production * Corresponding author.of catalase, oxida...

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Campylobacter jejuni is a microaerophilic bacterium that causes diarrhea in humans. The first step in establishing an infection is adherence to a host cell, which involves two major cell-binding proteins, Peb1A (CBF1) and Peb4 (CBF2). Because the functional role of Peb4 on the cell adhesion remains unclear compared with that of Peb1A, a C. jejuni peb4 deletion mutant was constructed and cell adherence and ability to colonize mouse intestine were studied. The result showed that adherence of the peb4 mutant strain to INT407 cells was 1-2% that of the wild-type strain. Mouse challenge experiments showed a reduced level and duration of intestinal colonization by the mutant compared with the wild-type strain. In addition, fewer peb4 mutant cells than wild-type cells responded to stress by forming a biofilm. Proteomic analysis revealed that the expression levels of proteins involved in various adhesion, transport, and motility functions, which are required for biofilm formation by the pathogen, were lower in the peb4 mutant than in the wild-type strain. A Peb4 homolog has prolyl cis/trans-isomerase activity, suggesting that the loss of this activity in the mutant strain may be responsible for the repression of these proteins.

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Lactobacillus casei, L. paracasei, and L. rhamnosus form a closely related taxonomic group (Lactobacillus casei group) within the facultatively heterofermentative lactobacilli. Here, we report the complete genome sequences of L. paracasei JCM 8130 and L. casei ATCC 393, and the draft genome sequence of L. paracasei COM0101, all of which were isolated from daily products. Furthermore, we re-annotated the genome of L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG), which we have previously reported. We confirmed that ATCC 393 is distinct from other strains previously described as L. paracasei. The core genome of 10 completely sequenced strains of the L. casei group comprised 1,682 protein-coding genes. Although extensive genome-wide synteny was found among the L. casei group, the genomes of ATCC 53103, JCM 8130, and ATCC 393 contained genomic islands compared with L. paracasei ATCC 334. Several genomic islands, including carbohydrate utilization gene clusters, were found at the same loci in the chromosomes of the L. casei group. The spaCBA pilus gene cluster, which was first identified in GG, was also found in other strains of the L. casei group, but several L. paracasei strains including COM0101 contained truncated spaC gene. ATCC 53103 encoded a higher number of proteins involved in carbohydrate utilization compared with intestinal lactobacilli, and extracellular adhesion proteins, several of which are absent in other strains of the L. casei group. In addition to previously fully sequenced L. rhamnosus and L. paracasei strains, the complete genome sequences of L. casei will provide valuable insights into the evolution of the L. casei group.

Lactobacillus rhamnosus is a facultatively heterofermentative lactic acid bacterium and is frequently isolated from human gastrointestinal mucosa of healthy individuals. L. rhamnosus ATCC 53103, isolated from a healthy human intestinal flora, is one of the most widely used and well-documented probiotics. Here, we report the finished and annotated genome sequence of this organism.The complete genome sequence of Lactobacillus rhamnosus ATCC 53103 was determined by a whole-genome shotgun strategy with the Sanger method. Genomic libraries containing 2-kb inserts were constructed and sequenced, and 39,936 sequences were generated, giving 8.6-fold coverage from both ends of the genomic clones. Sequence reads were assembled with the Phred-Phrap-Consed program (2). Remaining gaps between contigs were closed by direct sequencing of clones. Prediction and annotation of protein-coding genes were performed as described previously (5).The genome of L. rhamnosus ATCC 53103 consists of a circular 3,005,051-bp chromosome containing 2,834 predicted protein-coding genes and has no plasmid. Of all predicted protein-coding genes, we could assign 1,939 (68%) to known functions, 610 (22%) as conserved hypothetical genes, and 285 (10%) as novel hypothetical genes. This strain has a relatively high number of proteins involved in carbohydrate and amino acid metabolism and transport and defense mechanisms, compared with other sequenced intestinal lactobacilli. The genome encodes 28 complete phosphoenolpyruvate-carbohydrate phosphotransferase-type transporter systems (PTSs) and 25 putative glycosyl hydrolases, which are classified into 12 different carbohydrate-active enzyme families (http://www.cazy .org/). Of the 12 families, alpha-L-fucosidase (GH29) and alpha-mannosidase (GH38) are not found in other sequenced intestinal lactobacilli. Of the 28 PTSs, 12 are encoded by genes adjacent to glycosyl hydrolase genes and transcriptional regulator genes, allowing localized transcriptional control. This organism carries 22 multidrug ABC transporters, eight antimicrobial peptide ABC transporters, and seven beta-lactamases, suggesting its broad range of antibiotic resistance. The genome contains 17 complete two-component regulatory systems, which are most abundant among sequenced lactobacilli.Of the 17 sensor-responder pairs, 1 appears to be potentially associated with bacteriocin production, and 7 are located adjacent to genes for multidrug ABC transporters. Furthermore, the genome contains Ͼ90 putative transcriptional regulators.As expected, extensive similarity at the sequence level is observed between L. rhamnosus ATCC 53103 and its closely related strain Lactobacillus casei ATCC 334 (4), with overall genome synteny. However, a reciprocal BLASTP search reveals 755 (27%) protein-coding genes that are present in L. rhamnosus ATCC 53103 but absent in L. casei ATCC 334. These include six carbohydrate utilization gene clusters, which contain the genes for PTSs, glycoside hydrolases, transcriptional regulators, and other carbohydrate-related prote...

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide. This study aimed at a better understanding of the genetic diversity of this pathogen disseminated in Japan. We performed multilocus sequence typing (MLST) of Campylobacter jejuni isolated from different sources (100 human, 61 poultry, and 51 cattle isolates) in Japan between 2005 and 2006. This approach identified 62 sequence types (STs) and 19 clonal complexes (CCs), including 11 novel STs. These 62 STs were phylogenetically divided into 6 clusters, partially exhibiting host association. We identified a novel ST (ST-4526) that has never been reported in other countries; a phylogenetic analysis showed that ST-4526 and related STs showed distant lineage from the founder ST, ST-21 within CC-21. Comparative genome analysis was performed to investigate which properties could be responsible for the successful dissemination of ST-4526 in Japan. Results revealed that three representative ST-4526 isolates contained a putative island comprising the region from Cj0737 to Cj0744, which differed between the ST-4526 isolates and the reference strain NCTC11168 (ST-43/CC-21). Amino acid sequence alignment analyses showed that two of three ST-4526 isolates expressed 693aa- filamentous hemagglutination domain protein (FHA), while most of other C. jejuni strains whose genome were sequenced exhibited its truncation. Correspondingly, host cell binding of FHA-positive C. jejuni was greater than that of FHA-truncated strains, and exogenous administration of rFHA protein reduced cell adhesion of FHA-positive bacteria. Biochemical assays showed that this putative protein exhibited a dose-dependent binding affinity to heparan sulfate, indicating its adhesin activity. Moreover, ST-4526 showed increased antibiotic-resistance (nalidixic acid and fluoroquinolones) and a reduced ability for DNA uptake. Taken together, our data suggested that these combined features contributed to the clonal thriving of ST-4526 in Japan.