Shuji Fujimoto

High-frequency S-layer protein variation in Campylobacter fetus revealed by sapA mutagenesis.

Campylobacter fetus utilizes paracrystalline surface (S‐) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full‐length S‐layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full‐length S‐layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 × 10‐2. Immunoblots of survivors showed that phenotypic reversion involving high‐level production of full‐length (98, 127 or 149 kDa) S‐layer proteins had occurred. Revertants were serum resistant but caused approximately 10‐fold less bacteraemia in orally challenged mice than did the wild‐type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high‐frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.

Restriction Fragment Length Polymorphism Analysis and Random Amplified Polymorphic DNA Analysis of Campylobacter jejuni Strains Isolated from Patients with Guillain-Barré Syndrome

Campylobacter jejuni serotype O19 strains associated with the Guillain-Barré syndrome (GBS) and other strains were examined by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction products of the flaA genes and by random amplified polymorphic DNA (RAPD) analysis. RFLP analysis showed that regardless of LIO serotype, geographic origins, or association with GBS, the O19 isolates shared an identical digestion pattern by each of four restriction endonucleases, DdeI, MboI, MseI, and AluI. In contrast, among C. jejuni O1 or O2 strains, RFLP patterns were different even among strains of the same LIO serotype. The results of the RAPD analysis were consistent with the flaA RFLP data. These data indicate that all of the O19 strains that were tested were closely related to one another whether they were or were not associated with GBS.

Analysis of the expression of CagA and VacA and the vacuolating activity in 167 isolates from patients with either peptic ulcers or non-ulcer dyspepsia

Objectives:The goals of this study were: 1) to examine the prevalence of cytotoxin-associated protein (CagA), vacuolating cytotoxin (VacA), and the vacuolating cytotoxin activity (VCA) in vitro of infecting Helicobacter pylori isolates and 2) to clarify the relation between the expression of these virulence factors and the occurrence of peptic ulceration.Methods:One hundred sixty-seven clinical isolates of H. pylori from patients with peptic ulcer disease (gastric ulcer, 62 cases; duodenal ulcer, 48 cases) and nonulcer dyspepsia (57 cases) were studied regarding their genetic and phenotypic properties.Results:Type 1 bacteria, which had both CagA and VCA, and type 2 bacteria, which did not express either CagA or VCA, represented 62.9% and 7.8%, respectively; the remaining 29.4% had an intermediate phenotype, expressing either CagA independent of the presence of VCA (CagA+VCA−) or vice versa (CagA−VCA+). CagA+VCA− and CagA−VCA+ bacteria represented 17.4% and 12.0%, respectively, both of which were more numerous than the type 2 category. The proportion of the CagA-positive isolates was significantly higher in both the duodenal ulcer (97.9%) and gastric ulcer (83.9%) patients than in the non-ulcer dyspepsia patients (61.4%) (p < 0.01). On the other hand, the proportion of VacA/VCA-positive isolates was not significantly different between peptic ulcer disease and non-ulcer dyspepsia.Conclusions:The currently used classification of this bacterium based on the concomitant expression of CagA and VacA/VCA into the two major types is not adequate. The CagA-positive phenotype thus may be important as a virulence marker for peptic ulcer disease independent of the presence of VacA/VCA.

Spirochaete-like swimming mode of Campylobacter jejuni in a viscous environment

The swimming patterns of Campylobacter jejuni in environments of low and high viscosity were examined by a video tracking method. In media of low viscosity, C. jejuni swam with an average velocity of 39.3 microm/s with frequent changes in direction. The velocity of C. jejuni increased in a medium at a little higher viscosity than that of a low viscosity buffer. In addition to this, C. jejuni showed a second increase of velocity in media of a high viscosity of about 40 centipoise. The swimming patterns at these two velocity peaks were compared. In the second peak the wild-type C. jejuni exhibited repeated back and forth swimming patterns which were more like the swimming pattern of spirochaetes than that of monotrichous bacteria. Thus C. jejuni may presumably use a different swimming mode in media of high viscosity than the original swimming mode mediated by the propelling force of the flagella. The spiral shape of this bacterium like that of spirochaetes may strongly influence its swimming ability in media of high viscosity such as the mucous layer of the intestinal tract.

Protection against Campylobacter jejuni Infection in Suckling Mice by Anti-Flagellar Antibody

We obtained two monoclonal antibodies of IgM class and IgA class of immunoglobulin prepared from mouse spleen cells immunized with crude flagellar preparation, and a polyclonal antibody raised against purified flagellin monomer of Campylobacter jejuni in a rabbit. The specificity of the reaction of these antibodies for flagellar filament was confirmed by Western blotting and by immunoelectron microscopy. These antibodies caused agglutination of the bacteria and inhibited the motility of the bacteria. When a strain of C. jejuni was treated with IgM class monoclonal antibody before being inoculated into suckling mice, it reduced colonization of the intestinal tract by this bacteria. Inhibition of the colonization by IgA class monoclonal antibody was less effective than that of IgM class, and the polyclonal antibody consisting mostly of IgG class immunoglobulin was without effect.

Epidemiologic Application of Pulsed-Field Gel Electrophoresis to an Outbreak of Campylobacter fetus Meningitis in a Neonatal Intensive Care Unit

An outbreak of nosocomial Campylobacter fetus meningitis occurred in a neonatal intensive care unit (NICU). Eight C. fetus strains were isolated from 4 infants with meningitis, the mother of the index patient and 2 infants who were asymptomatic intestinal carriers. The pulsed-field gel electrophoresis (PFGE) pattern with the restriction endonucleases Smal and Sall were found to be identical for the nosocomial C. fetus isolates, but the patterns were different from those of sporadic strains. These nosocomial strains were strongly suspected to be a single strain. The finding revealed that the index patient was infected by the mother, and that the outbreak developed from this patient by cross-infection. This is the first confirmed nosocomial C. fetus meningitis outbreak spread by cross-infection in a NICU.

Increase in Resistance to Extended-Spectrum Cephalosporins in Salmonella Isolated from Retail Chicken Products in Japan

Extended-spectrum β-lactamase (ESBL)-producing Salmonella are one of the most important public health problems in developed countries. ESBL-producing Salmonella strains have been isolated from humans in Asian countries neighboring Japan, along with strains harboring the plasmid-mediated extended-spectrum cephalosporin (ESC)-resistance gene, ampC (pAmpC). However, only a few studies have investigated the prevalence of ESC-resistant Salmonella in chicken products in Japan, which are the main vehicle of Salmonella transmission. The aim of this study was to investigate the prevalence of ESBL-producing, pAmpC-harboring, or carbapenem-resistant Salmonella in chicken products in Japan. In total, 355 out of 779 (45.6%) chicken product samples collected from 1996–2010 contained Salmonella, resulting in 378 distinct isolates. Of these isolates, 373 were tested for resistance to ESCs, cephamycins, or carbapenems. Isolates that showed resistance to one or more of these antimicrobials were then examined by PCR and DNA sequence analysis for the presence of the bla CMY, bla CTX-M, bla TEM, and bla SHV resistance genes. Thirty-five resistant isolates were detected, including 26 isolates that contained pAmpC (bla CMY-2), and nine ESBL-producing isolates harboring bla CTX-M (n = 4, consisting of two bla CTX-M-2 and two bla CTX-M-15 genes), bla TEM (n = 4, consisting of one bla TEM-20 and three bla TEM-52 genes), and bla SHV (n = 1, bla SHV-12). All pAmpC-harboring and ESBL-producing Salmonella isolates were obtained from samples collected after 2005, and the percentage of resistant isolates increased significantly from 0% in 2004 to 27.9% in 2010 (P for trend = 0.006). This increase was caused in part by an increase in the number of Salmonella enterica subsp. enterica serovar Infantis strains harboring an approximately 280-kb plasmid containing bla CMY-2 in proximity to ISEcp1. The dissemination of ESC-resistant Salmonella containing plasmid-mediated bla CMY-2 in chicken products indicates the need for the development of continuous monitoring strategies in the interests of public health.

Reduced Tyk2 gene expression in β-cells due to natural mutation determines susceptibility to virus-induced diabetes

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

Southern blotting analyses of strains ofCampylobacter fetus using the conserved region ofsapA

Chromosomal DNA of 27 strains ofCampylobacter fetus was analyzed by Southern blotting with a probe of the conserved region ofsapA. The probe hybridized with 23 strains that produced type A lipopolysaccharide. These strains had more than sixsapA homologs. In Southern blots ofSalI-digested chromosomal DNA separated by pulsed-field gel electrophoresis, one fragment from 19 strains and two fragments from 4 strains hybridized. These data indicate that multiplesapA homologs are localized to a limited region on the chromosomal DNA ofC. fetus and thus suggest the possibility of developing a typing system using this method.

Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan.