Biotypic and genotypic diversity in Pasteurella canis isolated from host animals and humans: differences in trehalose fermentation and nucleotide sequences encoding trehalose-6-phosphate hydrolase (treC).

The biotypic and genotypic features of Pasteurella canis isolated from dogs, cats, and humans were clarified by repetitive sequence-based fingerprinting and nucleotide sequences encoding trehalose-6-phosphate hydrolase (treC). Thirty P. canis and 48 P. multocida isolates were collected from dogs, cats, and humans to perform biotyping. The genotyping of P. canis by fingerprinting was followed by dendrogram construction. The whole-genome sequences (WGSs) were searched for the enzyme-coding nucleotide sequences around the main and adjacent loci constituting the operon. Full-length nucleotide sequences encoding the enzyme were determined using polymerase chain reaction and direct sequencing. Biotypic results were compared to the dendrogram and nucleotide sequence data. We observed a difference in trehalose fermentation with a positivity rate of 46.7%. Two (A-1/A-2) and three (B-1/B-2/B-3) clades were located on the dendrograms generated based on two repetitive sequence-based fingerprinting techniques, showing no association between trehalose fermentation and the clades. Based on the WGSs, two variants of the gene, namely, a 1,641 bp gene treC and a pseudogene (1,335 bp) of treC with its first 306 nucleotides deleted, were observed. Trehalose-positive isolates harbored treC, whereas trehalose-negative isolates lacked treC with or without the pseudogene. Our observations suggest biotypic and genotypic diversity among the P. canis isolates from animal and human hosts, with respect to trehalose fermentation and treC nucleotide sequences. This is the first report on the diversity of treC nucleotide sequences among these isolates.

Virulence-associated Genome Sequences of Pasteurella canis and Unique Toxin Gene Prevalence of P. canis and Pasteurella multocida Isolated from Humans and Companion Animals.

Background: Comparative analysis of virulence factors (VFs) between Pasteurella canis and Pasteurella multocida are lacking, although both cause zoonotic infections. We determined the virulence-associated genome sequence characteristics of P. canis and assessed the toxin gene prevalence unique to P. canis among clinical isolates of P. canis and P. multocida. Methods: We selected 10 P. canis and 16 P. multocida whole-genome sequences (WGSs) from the National Center for Biotechnology database. The VFanalyzer tool was used to estimate P. canis-characteristic VFs. Amino acid sequences of VFs were compared with multiple-aligned sequences. The genome structure containing P. canis-characteristic and adjacent loci was compared to the corresponding P. multocida genome structure. After designing primer sequences and assessing their accuracy, we examined the gene prevalence of the P. canis-characteristic VFs using PCR among clinical isolates of P. multocida and P. canis. Results: Using VFanalyzer, we found virulence-associated cytolethal distending toxin (cdt)A-cdtB-cdtC loci common to all P. canis WGSs that were not found in P. multocida WGSs. Similarities in the multiple alignments of CdtA-CdtB-CdtC amino acid sequences were found among the 10 P. canis WGSs. Shared or similar loci around cdtA-cdtB-cdtC were identified between the P. canis and P. multocida genome structures. The PCR-based cdtA-cdtB-cdtC prevalence differed for P. canis and P. multocida clinical isolates. Conclusions: P. canis-specific cdtA-cdtB-cdtC prevalence was identified among clinical isolates. These three loci may be unique toxin genes and promising targets for the rapid identification of P. canis in clinical settings.

Isolation Rate of Neisseria meningitidis in Japanese Children with Respiratory Tract Infections.

Although invasive meningococcal disease is rare in Japan (0.028 cases per 100,000 population), its incidence is 10 times greater in many other countries. Colonization is a prerequisite for invasive meningococcal disease. However, no study in Japan has involved specifically analyzing the carriage rate of Neisseria meningitidis in children. During 5 months in 2015, the respiratory tract specimens of patients who presented to 3 hospitals with respiratory symptoms were cultured. The bacteria were identified in selective media using a meningococcal detection kit and the serogroup was identified using polymerase chain reaction analysis. In 389 patients aged ≤15 years with respiratory symptoms, the N. meningitidis isolation rate was 0.26% (1/389). The serogroup of the only child who tested positive was Y. In this study, we detected a low meningococcal isolation rate in pediatric patients. Due to increasing globalization, the risk of invasive meningococcal disease is likely increasing in Japan. Accordingly, invasive meningococcal diseases should be continuously monitored in Japan. Future large-scale studies should assess meningococcal isolation rates and corresponding serogroups.

[Antimicrobial Susceptibility of Streptococcus pneumoniae Isolated from 8 Hospitals in Chiba Prefecture Following the Introduction of 7-valent Pneumococcal Conjugate Vaccine].

We investigated the susceptibility of Streptococcus pneumoniae isolated from 8 hospitals in Chiba prefecture during 2012-2013. We further checked the serotype of S. pneumoniae derived from invasive pneumococcal disease (IPD). We tested for antimicrobial susceptibility in 256 clinical isolates (137 isolates from children, 119 isolates from adults) for 25 drugs. In MIC50 and MIC90, there were very little differences between children and adults, but there were 3 isolates from adults which were resistant to levofloxacin. The most major serotypes were 15A and 3 in IPD. Additionally there was no isolation of the type contained in the 7-valent pneumococcal conjugate vaccine in children, so it seems that the vaccination is very effective for children. Furthermore, in contrast with our preceding report, a decreasing was seen in PCG resistant proportion of S. pneumoniae. The maximum PCG-MIC was 2 µg/mL.

Gene expression profile in human leukocytes.

Leukocytes are classified as myelocytic or lymphocytic, and each class of leukocytes consists of several types of cells that have different phenotypes and different roles. To define the gene expression in these cells, we have performed serial analysis of gene expression (SAGE) using human leukocytes and have provided the gene database for these cells not only at the resting stage but also at the activated stage. A total of 709,990 tags from 17 libraries were analyzed for the manifestation of gene expression profiles in various types of human leukocytes. Types of leukocytes analyzed were as follows: peripheral blood monocytes, colony-stimulating factor-induced macrophages, monocyte-derived immature dendritic cells, mature/activated dendritic cells, granulocytes, natural killer (NK) cells, resting B cells, activated B cells, naive T cells, CCR4(-) memory T cells (resting T(H)1 cells), CCR4(+) memory T cells (resting T(H)2 cells), activated T(H)1 cells, and activated T(H)2 cells. Among 38,961 distinct tags that appeared more than once in the combined total libraries, 27,323 tags were found to represent unique genes in certain type(s) of leukocytes. Using probability (P) and hierarchical clustering analysis, we identified the genes selectively expressed in each type of leukocytes. Identification of the genes specifically expressed in different types of leukocytes provides not only a novel molecular signature to define different subsets of resting and activated cells but also contributes to further understanding of the biologic function of leukocytes in the host defense system.

Comprehensive gene expression analysis of human NK cells and CD8(+) T lymphocytes.

Cytotoxic lymphocytes, NK cells and CD8(+) T cells play a pivotal role in the host defense. To reveal the biological function of these cells through establishing a comprehensive gene expression profile, serial analysis of gene expression was performed in human peripheral blood NK cells and CD8(+) T cells. In total, 85,848 tags corresponding to >20,000 different transcripts were sequenced. The genes expressed abundantly in these libraries mostly consisted of genes encoding MHC class I and molecules related to protein synthesis. Among gene transcripts which related to cytotoxicity, granulysin, perforin, granzyme B and alpha-defensin 1 were highly expressed in NK cells. Resting CD8(+) T cells did not express the genes related to cytotoxicity, but expressed abundantly the genes encoding chemokines, tumor necrosis factor family. When CD8(+) T cells were sorted into naive, memory and effector subsets based on the expression of CD45RA and CD27, perforin and granzyme B were expressed in the CD45RA(+)CD27(-) effector subset. Alpha-defensin 1, one of the selectively expressed genes in NK cells, induced migration of naive CD8(+)CD45RA(+)CD27(+) T cells, but not memory CD8(+)CD45RA(-)CD27(+) or effector CD8(+)CD45RA(+)CD27(-) T cells. Furthermore, treatment with IL-15, a stimulator of NK cell development, differentiation, survival and cytotoxicity, rapidly enhanced the expression of alpha-defensin 1 in NK cells. The identification of the genes preferentially expressed in NK and CD8(+) T cell subsets may give important insights into the functions of these cells against virus infection and in tumor immunity.