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Biomedical subjects

Haruo Watanabe

Publications and source records attributed to Haruo Watanabe.

At least 91 records · Page 5Linked to original sources

Identification of a novel antigen of pathogenic Leptospira spp. that reacted with convalescent mice sera.

The virulence of leptospires isolated from human patients against C3H/HeJ mice was investigated. Infection with clinical isolates from patients with severe leptospirosis was lethal to C3H/HeJ mice, suggesting that C3H/HeJ mice are suitable as an acute lethal model of severe leptospirosis. Using this model, a novel antigen of pathogenic Leptospira spp. (named LAg42), which reacted with convalescent mice sera, was identified. LAg42 is a 42 kDa inner-membrane protein and its immunogenic region is located in the C-terminal region. The gene for LAg42 is conserved among pathogenic leptospires but not among non-pathogenic leptospires, which suggests its involvement in virulence.

Animals↗

Activation of hilA expression at low pH requires the signal sensor CpxA, but not the cognate response regulator CpxR, in Salmonella enterica serovar Typhimurium.

A two-component regulatory system, cpxR-cpxA, plays an important role in the pH-dependent regulation of virF, a global activator for virulence determinants including invasion genes, in Shigella sonnei. The authors examined whether the cpxR-cpxA homologues have some function in the expression of Salmonella enterica serovar Typhimurium invasion genes via the regulation of hilA, an activator for these genes. In a Salmonella cpxA mutant, the hilA expression level was reduced to less than 10 % of that in the parent strain at pH 6.0. This mutant strain also showed undetectable synthesis of an invasion gene product, SipC, at pH 6.0 and reduced cell invasion capacity - as low as 20 % of that of the parent. In this mutant, the reduction in hilA expression was much less marked at pH 8.0 than at pH 6.0 - no less than 50 % of that in the parent, and no significant reduction was observed in either SipC synthesis or cell invasion rate, compared to the parent. Unexpectedly, a Salmonella cpxR mutant strain and the parent showed no apparent difference in all three characteristics described above at either pH. These results indicate that in Salmonella, the sensor kinase CpxA activates hilA, and consequently, invasion genes and cell invasion capacity at pH 6.0. At pH 8.0, however, CpxA does not seem to have a large role in activation of these factors. Further, the results show that this CpxA-mediated activation does not require its putative cognate response regulator, CpxR. This suggests that CpxA may interact with regulator(s) other than CpxR to achieve activation at low pH.

Bacterial Proteins↗

Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism.

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.

Anti-Infective Agents↗

Probable laboratory contamination of clinical specimens with Leptospira meyeri.

Leptospira meyeri were isolated from the blood or the pleural effusion cultures of four patients at a commercial clinical laboratory. All isolates were identical in their 16S rDNA sequences and NotI or SfiI restriction profiles of the chromosome DNA on pulsed-field gel electrophoresis. However, intraperitoneal inoculation of the isolate (NIID1) failed to kill hamsters and none of the patients' sera reacted with L. meyeri strains isolated, indicating leptospiral contamination occurred during laboratory investigation.

Animals↗

[A case of leptospirosis diagnosed early by flaB-PCR].

We report a case of leptospirosis that was diagnosed in the early stage of infection by using flaB-PCR. The flaB gene of Leptospira was amplified from the DNA extract of the patient's blood at hospital day 1, while leptospiral antibody was negative by the method of microscopic agglutination test and micocapsule agglutination test at that time. Seroconversion occurred at hospital day 6 and Leptospira was isolated from the patient's blood at hospital day 16, demonstrating that flaB-PCR method was very useful in early diagnosis of leptospirosis.

Aged↗

Determination of the InvE binding site required for expression of IpaB of the Shigella sonnei virulence plasmid: involvement of a ParB boxA-like sequence.

The InvE protein positively regulates the expression of virulence genes ipaBCD in Shigella sonnei. The InvE has significant homology with ParB of plasmid P1, which is known as a plasmid partitioning factor with DNA binding ability. Although the DNA binding activity of InvE has been predicted, it is not known whether the DNA binding activity is necessary for type III secretion system-associated gene expression. In this study, we determined the transcription start site of the icsB-ipaBCD operon (ipa operon) and constructed a series of deletions of the icsB promoter region in the Escherichia coli K-12 background. The deletion study revealed that an 86-bp region upstream of the icsB transcription start site was essential for expression of the ipa operon, where the ParB binding motif (ParB BoxA-like sequence) was observed. Purified glutathione S-transferase-InvE fusion protein bound directly to the -93 to -54 region (designating the icsB transcription start site as nucleotide +1) containing the ParB BoxA-like sequence. These results indicated that InvE bound directly to the promoter region.

Bacterial Proteins↗

Genotypic analyses of Vibrio parahaemolyticus and development of a pandemic group-specific multiplex PCR assay.

A total of 54 Vibrio parahaemolyticus strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the "pandemic group") were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences, toxRS/new or orf8, that had been reported elsewhere to be specific for the pandemic group. Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone. In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin (tdh) gene also belonged to this cluster and possessed the toxRS/new sequence. However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the orf8 sequence. The evidence suggests that neither the toxRS/new nor the orf8 sequence is a reliable gene marker for definite identification of the pandemic group. We therefore developed a novel multiplex PCR assay specific for the pandemic group. The assay successfully distinguished pandemic group strains from other V. parahaemolyticus strains by yielding two distinct PCR products for tdh (263 bp) and the toxRS/new sequence (651 bp).

Bacterial Proteins↗

Life-threatening infantile diarrhea from fluoroquinolone-resistant Salmonella enterica typhimurium with mutations in both gyrA and parC.

Salmonella Typhimurium DT12, isolated from a 35-day-old infant with diarrhea, was highly resistant to ampicillin, tetracycline, chloramphenicol, streptomycin, gentamycin, sulfamethoxazole/trimethoprim, nalidixic acid, and fluoroquinolones. The patient responded to antibiotic therapy with fosfomycin. Multidrug-resistance may become prevalent in Salmonella infections in Japan, as shown in this first case of a patient infected with fluoroquinolone-resistant Salmonella.

Anti-Infective Agents↗

Multiplex polymerase chain reaction assay for selective detection of Salmonella enterica serovar typhimurium.

A multiplex polymerase chain reaction (PCR) assay was developed for the identification of Salmonella enterica serovar Typhimurium. Three sets of primers were designed for detecting O4, H:i, and H:1,2 antigen genes from the antigen-specific genes rfbJ, fliC, and fljB, respectively. These were evaluated in a multiplex PCR assay by using DNAs from S. enterica serovar Typhimurium, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens. Multiplex PCR proved to be capable of identifying S. enterica serovar Typhimurium specifically and differentiating it from other Salmonella serovars in addition to non-Salmonella enteric pathogens. Thus, this multiplex PCR assay can be practically applied to the identification of S. enterica serovar Typhimurium.

Polymerase Chain Reaction↗

Genomic diversity of enterohemorrhagic Escherichia coli O157 revealed by whole genome PCR scanning.

Enterohemorrhagic Escherichia coli O157 is one of the leading worldwide public health concerns, causing large outbreaks of hemorrhagic colitis as well as numerous small outbreaks and sporadic cases. The variability of restriction enzyme-digestion patterns of O157 genomes, which is widely used to distinguish strains in the molecular epidemiology of O157 infections, suggests the presence of some genomic diversity among the strains. Based on the complete genome sequence of O157 Sakai, we analyzed the whole genome structures of eight O157 strains displaying diverse XbaI-digestion patterns by a systematic PCR analysis that we have named whole genome PCR scanning. This analysis identified not only the O157-specific sequences that are highly conserved among the strains, but also revealed an unexpectedly high degree of genomic diversity. In particular, prophages, including Shiga toxin-transducing phages, exhibited extensive structural and positional diversity, implying that variation of bacteriophages is a major factor in generating genomic diversity among the O157 lineage.

Chromosomes, Bacterial↗

Critical role of myeloperoxidase and nicotinamide adenine dinucleotide phosphate-oxidase in high-burden systemic infection of mice with Candida albicans.

Oxygen metabolites generated by myeloperoxidase (MPO) and nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase contribute to microbial killing by phagocytes. To compare the importance of the 2 enzymes for host defense, MPO-deficient (MPO(-/-)) mice and NADPH-oxidase-deficient mice with chronic granulomatous disease (CGD mice) were intraperitoneally infected with 3 different doses of Candida albicans, and their infection severity was analyzed. CGD mice had increased mortality and exhibited increased tissue fungal burden in a dose-dependent manner, whereas normal mice showed no symptoms. Of interest, at the highest dose, the mortality of MPO(-/-) mice was comparable to that of CGD mice, but at the lowest dose, it was the same as that of normal mice. At the middle dose, the number of fungi disseminated into various organs of the MPO(-/-) mice was comparable to that of the CGD mice at day 6 of infection, but it was significantly lower at day 14. These results suggest that MPO and NADPH-oxidase are equally important for early host defense against a large inoculum of Candida.

Animals↗