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Kiyofumi Ohkusu

Publications and source records attributed to Kiyofumi Ohkusu.

16 recordsLinked to original sources
PubMed2007

The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species.

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.

Bacterial Typing Techniques↗
PubMed2007

dnaJ gene sequence-based assay for species identification and phylogenetic grouping in the genus Staphylococcus.

In the last few years, many attempts have been made to use conserved gene sequences for identification and for phylogenetic studies of Staphylococcus species. In an effort to identify a more reliable approach, a dnaJ gene sequence-based database was created. In this study, an approximately 883 bp portion of the dnaJ gene sequence from 45 staphylococcal type strains was compared with 16S rRNA and other conserved gene (hsp60, sodA and rpoB) sequences available in public databases. Nucleotide sequence comparisons revealed that the staphylococcal dnaJ gene showed higher discrimination (mean similarity 77.6 %) than the 16S rRNA (mean similarity 97.4 %), rpoB (mean similarity 86 %), hsp60 (mean similarity 82 %) and sodA (mean similarity 81.5 %) genes. Analysis of the dnaJ gene sequence from 20 Staphylococcus isolates representing two clinically important species showed <1 % sequence divergence. Phylogenetic data obtained from the dnaJ gene sequence were in general agreement with those of 16S rRNA gene sequence analysis and DNA-DNA reassociation studies. In conclusion, the dnaJ gene sequence-based assay is an effective alternative to currently used methods, including 16S rRNA gene sequencing, for identification and taxonomical analysis of Staphylococcus species.

Bacterial Typing Techniques↗
PubMed2007

System for simultaneous detection of 16 pathogens related to urethritis to diagnose mixed infection.

OBJECTIVE: Urethritis is not always caused by a single pathogen, and isolation of more than two pathogens from one patient is not uncommon. We developed a method to simultaneously detect 16 pathogens related to urethritis. METHODS: We designed specific primers used for amplification of urethritis pathogens in our 16-well microplate assay. Sixteen microliters of each reaction mixture containing template DNA was added to each well to amplify 16 pathogens simultaneously. RESULTS: After we evaluated the specificity and sensitivity of this microplate polymerase chain reaction method, we used it to detect pathogens in clinical samples. Of 163 clinical samples, 49.7% (81/163) were positive for specific pathogens, and 6.7% (11/163) showed mixed infection. A specific pathogen was not identified in 43.6% (71/163) of cases. CONCLUSIONS: We developed a 16-well microplate assay with 16 specific primers to identify pathogens associated with urethritis.

Humans↗
PubMed2006

Helicobacter cinaedi cellulitis and bacteremia in immunocompetent hosts after orthopedic surgery.

At various times after orthopedic operations (more than a few weeks, with an average of 29.9 days), 11 patients had a sudden onset of high temperature (average 38.9 degrees C) and local cellulitis at different sites on the operated sides. The wounds had completely healed, without complicated infections, when the cellulitis occurred. The clinical picture of cellulitis in all patients was atypical: diffuse salmon-pink skin color, local heat, swelling, spontaneous pain, and tenderness but no eruptions. No patient had any underlying immunocompromising conditions or had been given immunosuppressive agents. Gram-negative spiral bacteria were isolated from blood cultures and were identified as Helicobacter cinaedi on the basis of 16S rRNA gene sequencing and DNA-DNA hybridization using standard strains. By means of phylogenetic analysis, we divided these clinical isolates into two clones. The H. cinaedi strain isolated via fecal cultures from two patients without intestinal symptoms was the same clone as the blood isolate. All isolates were quite susceptible to various antibiotics, and clinical and inflammatory symptoms of bacteremia and cellulitis improved after treatment with penicillins and cephalosporins. A relatively high incidence of recurrence of the same disease was observed, however. Almost all patients responded immunologically to the infection, as evidenced by the production of serum antibody against H. cinaedi. We thus suggest that H. cinaedi should not be regarded as simply an opportunistic pathogen but that it may be a pathogen in immunocompetent hosts and may cause infections together with bacteremia and cellulitis.

Adult↗
PubMed2006

Emerging multiple mutations and high-level fluoroquinolone resistance in methicillin-resistant Staphylococcus aureus isolated from ocular infections.

We investigated the prevalence of mutations in the gyrA, gyrB, grlA, and grlB genes in 21 clinical isolates of methicillin-resistant Staphylococcus aureus recovered from ocular infections. These isolates were classified into 16 groups on the basis of pulsed-field gel electrophoresis (PFGE) patterns. Among these nonidentical strains, 9 different combinations of mutations were observed. We observed an unusually high frequency of mutations: 10 (62.5%) of the isolates had four or five mutations, 5 (31.3%) of the isolates had double mutations, and 1 isolate had a single mutation. Different mutations were detected in strains of the same PFGE type, and this is consistent with the independent acquisition of mutations. The minimun inhibitory concentrations values ranged from 0.5 to 512 microg/mL for levofloxacin and 0.125 to 128 microg/mL for gatifloxacin. Only 1 isolate was found to be susceptible to both agents. These results suggest that application of high concentration fluoroquinolone ophthalmic solutions selects for isolates presenting such mutations that may result in a high level of resistance to fluoroquinolones.

Drug Resistance, Bacterial↗
PubMed2006

dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus.

The dnaJ and gyrB nucleotide sequences were determined for members of the genus Streptococcus. The average similarity between the species tested was 76.4% (69.7-100%) for dnaJ and 75.9 (70.1-98.7%) for gyrB. These data indicated that the dnaJ and gyrB genes are more divergent and more discriminatory than the 16S rDNA gene. Furthermore, the variation in the dnaJ nucleotide sequences among the mitis group was greater than that of the gyrB nucleotide sequences, especially between Streptococcus pneumoniae and Streptococcus mitis. Subsequently, the high discrimination power of dnaJ within the mitis group was confirmed. Thus, we conclude that the dnaJ and gyrB genes are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains.

Bacterial Proteins↗
PubMed2006

Emergence of multidrug-resistant Corynebacterium striatum as a nosocomial pathogen in long-term hospitalized patients with underlying diseases.

During a 53-month period (March 1994 to August 1998), 48 Corynebacterium striatum isolates recovered from clinical specimens were characterized. The organisms were identified by both phenotypic characteristics and 16S rRNA gene sequence analysis. Thirty-six (75%) were isolated from sputum/bronchial aspirates, 10 (21%) from wound exudates/pus, 1 (2%) from vaginal discharge, and 1 (2%) from an otorrheic specimen. All 48 patients had been hospitalized for treatment of an underlying disease and had received antibiotics previously. The C. striatum isolates were considered pathogenic based on their abundance within polymorphonuclear neutrophils and their dominant growth in culture. Sensitivities of isolates to 11 antibiotics were determined by broth microdilution. MIC90 values of the isolates were 1 microg/mL for vancomycin, 16 microg/mL for penicillin and ampicillin, 32 microg/mL for minocycline, and > or = 32 microg/mL for cephalosporins, imipenem, ofloxacin, and macrolides. Restriction fragment-length polymorphism analysis with pulsed-field gel electrophoresis was used to determine the clonal identity. The pulse-field gel electrophoresis profiles revealed 14 distinct patterns with 20 subtypes. The isolates for the nosocomial outbreaks of C. striatum included 3 types (A, D, and E) with 4 subtypes (A1, A2, D2, and E). All 4 genotypes had broad-spectrum resistance to antimicrobial agents. Furthermore, type E strain isolated from 3 patients in the same ward was sensitive only to vancomycin. We conclude that C. striatum should be considered an emerging multidrug-resistant nosocomial pathogen in patients hospitalized for a prolonged period and/or in immunocompromised patients with such underlying conditions as cerebrovascular disease, pulmonary disease, diabetes, or malignancy.

Anti-Bacterial Agents↗
PubMed2006

A mixed bacterial infection of a bronchogenic lung cyst diagnosed by PCR.

An unusual paediatric case of a bronchogenic cyst infected with both Haemophilus influenzae type b and Streptococcus pneumoniae is described, which was detected not by culture of the purulent cyst fluid, but by real-time PCR amplification for several potential pathogens of DNA extracted from the fluid.

Base Sequence↗
PubMed2006

Virulence-defective strains of Salmonella enterica serovar Typhi as candidates for education at level 2 facilities.

The use of biosafety level 3 pathogens is an essential element of education and training at medical schools. We previously reported on invasion-defective strains of Salmonella enterica serovar Typhi, GTC 3P408 (DeltainvA, DeltasipB) and GTC 3P409 (DeltainvA, DeltasipB, and DeltaviaB), as candidates for use in educational programs. Vi negative strains of S. enterica serovar Typhi became extremely sensitive to complement attack but showed increased invasiveness. Therefore, this study was conducted to construct two virulencedefective strains, GTC 3P460 (DeltainvA, DeltasipB, and DeltarpoS) and GTC 3P461 (DeltainvA, DeltasipB, DeltaviaB, and DeltarpoS), of S. enterica serovar Typhi by deleting rpoS from the GTC 3P409 and GTC 3P408 strains. Stress tests demonstrated that GTC 3P460 and GTC 3P461 are sensitive to conditions of starvation, acid stress and oxidative stress. These results suggest that these virulence-defective strains have difficulty surviving in the gastric environment and in macrophages, characteristics that make them ideal candidates for education at level 2 facilities. Colony morphology and conventional biochemical features of these strains are identical to the parent strain S. enterica serovar Typhi GIFU 10007.

Antigens, Bacterial↗
PubMed2006

Mycobacterium kumamotonense Sp. Nov. recovered from clinical specimen and the first isolation report of Mycobacterium arupense in Japan: Novel slowly growing, nonchromogenic clinical isolates related to Mycobacterium terrae complex.

Three mycobacterium strains isolated from clinical specimens in Japan were provisionally assigned to the genus Mycobacterium based on their phenotypical characteristics. These isolates were further investigated to determine their specific taxonomic statuses. Mycolic acid analysis and 16S rRNA gene, rpoB, and hsp65 sequence data for the isolates showed that they are most similar to M. terrae complex. DNA-DNA hybridization studies indicated that the three strains were of two species and were distinguishable from M. terrae, M. nonchromogenicum, and M. hiberniae. Therefore, these strains represent two novel species within the genus Mycobacterium. However, one potential new species should have been considered as M. arupense with the 16S rRNA gene and hsp65 sequences similarities of 99.8% and 100% respectively; it was isolated from human specimens in the United States and was proposed in June 2006 as a new species. This report describes the first isolation of M. arupense in Japan, suggesting that the organism is clinically relevant. In addition, we propose the novel species designation Mycobacterium kumamotonense sp. nov. The type strain is CST 7247(T) (=GTC 2729(T), =JCM 13453(T), =CCUG 51961(T)).

Bacterial Proteins↗
PubMed2005

Quantitative microarray-based DNA-DNA hybridization assay for measuring genetic distances among bacterial species and its application to the identification of family Enterobacteriaceae.

Quantitative DNA-DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3-labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA-DNA hybridization for measuring chromosome relatedness among bacterial species.

DNA, Bacterial↗
PubMed2005

Corynebacterium resistens sp. nov., a new multidrug-resistant coryneform bacterium isolated from human infections.

Five strains of an unknown, multidrug-resistant coryneform, gram-positive rod were isolated from blood, bronchial aspirate, and abscess specimens. Four of the five strains isolated were highly resistant to antimicrobial agents, including beta-lactams, aminoglycosides, macrolides, quinolones, and tetracyclines, except for glycopeptides. In immunocompromised patients, bacteremia associated with this organism was rapidly fatal. This coryneform bacterium was nonmotile, lipophilic, and nonsaccharolytic. Lack of pyrazinamidase activity differentiated this organism from other lipophilic corynebacteria. Chemotaxonomic studies indicated that this multidrug-resistant coryneform bacterium belongs to the genus Corynebacterium. Comparative 16S rRNA gene sequencing and DNA-DNA hybridization analyses revealed that the five isolates were genetically identical and that they represent a new subline within the genus Corynebacterium, for which we propose the designation Corynebacterium resistens sp. nov. The type strain of Corynebacterium resistens is GTC 2026T (SICGH 158T, JCM 12819T, CCUG 50093T).

Corynebacterium↗
PubMed2004

Differential virulence of Mycobacterium avium strains isolated from HIV-infected patients with disseminated M. avium complex disease.

The role that colonization with Mycobacterium avium plays in the development of disseminated disease is unclear. In this study, we determined whether all M. avium strains isolated from the portals of M. avium infection are capable of crossing the mucosal border and causing infection. The patients in this study were enrolled in AIDS Clinical Trial Group protocol 341. The patients were divided into 3 groups; 2 groups differed in their immunological and clinical risk for M. avium disease. A third group (n=22 patients) had culture-documented disseminated M. avium complex disease at the time of entry in the study. Eight of 22 patients had M. avium isolated from both a colonized site and blood or bone marrow specimens. All 8 patients had distinct M. avium strains; 2 patients had a polyclonal infection. The virulence properties of 13 strains were determined, including invasion of gastrointestinal cells and replication in macrophages. There were significant differences in the virulence properties, and these differences may provide insight into the interplay between microbial pathogenesis and host defense.

AIDS-Related Opportunistic Infections↗
PubMed2004

[Rapid diagnostic system of bacterial infection].

Approach to develop genetic detection system for bacterial infection in blood is a demanding subject because number of bacteria in blood stream is very few and in most cases the number of bacteria is less than the detection limit of gene amplification methods. At the first stage of blood sampling, we only applied gene amplification method against fastidious organisms to culture and organisms causes severe infection. Organisms which are usually found very few in blood are subjected to blood culture. After monitoring system of blood culture gave positive signal of bacterial growth, DNA or RNA in culture media were extracted and amplified with universal primers for bacteria and fungi. Amplicons were applied to a rapid sequencer. However, in case of mixed infection, the sequencing is not successful. In such cases, the amplicons are analized with DNA microarray, immobilized 16S rDNA and 28S rDNA of 1,000 human pathogenic bacteria and fungi.

Bacterial Infections↗