Shigeki Misawa

Heat shock proteins and superantigenic properties of bacteria from the gastrointestinal tract of patients with Kawasaki disease.

We previously suggested that gut bacteria may be involved in the onset of Kawasaki disease (KD). In this study, we evaluated the production of heat shock proteins (hsps) and superantigens (sAgs) by microorganisms isolated from the jejunal mucosa of 19 children with KD in the acute phase and from 15 age‐matched control children. We identified 13 strains of Gram‐negative microbes from patients with KD; these microbes produced large amounts of hsp60 and induced pro‐inflammatory cytokine production by peripheral blood mononuclear cells. The Gram‐negative microbes also elicited endogenous hsp60 production, leading to the secretion of anti‐inflammatory intereukin‐10 (IL‐10). We also identified 18 strains of Gram‐positive cocci that had superantigenic properties and which induced the expansion of Vβ2 T cells in vitro. All bacteria identified in this study were antibiotic resistant. These data suggest that sAg and hsps produced by gut bacteria might be involved in KD.

Genetic Identification of Members of the Genus Corynebacterium at Genus and Species Levels with 16S rDNA-Targeted Probes

16S rRNA gene‐targeted probes were designed for the identification of corynebacteria at the genus and species levels. The genus‐specific probe hybridized all clinically important members of the genus Corynebacterium and could distinguish them from other coryneform bacteria and phylogenetically related high G + C% gram‐positive bacteria, including Actinomyces, Rhodococcus, Gordona, Nocardia, Streptomyces, Brevibacterium and Mycobacterium. The species‐specific probes for C. jeikeium and C. diphtheriae could differentiate these two species from other members of this genus. The probes were used to select corynebacteria among gram‐positive clinical isolates which had been tentatively identified as corynebacteria by biochemical tests. We screened 59 strains with the genus‐specific probe; 51 strains hybridized to the genus‐specific probe, 8 did not. Of the 51 strains that hybridized to the genus‐specific probe, 1 hybridized to the C. diphtheriae species probe and 13 hybridized to the C. jeikeium species probe. The 8 strains that did not hybridize to the genus probe were further characterized by analyzing cell wall diaminopimelic acid and partial 16S rRNA sequencing. The results indicated that these strains were distributed in the genera Arthrobacter and Brevibacterium.

Association between antimicrobial consumption and clinical isolates of methicillin-resistant Staphylococcus aureus: a 14-year study

The objective of this study was to determine the relationship between clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and antimicrobial consumption in hospitalized patients over a 14-year period. The study was retrospectively conducted between January 1995 and December 2008 at Juntendo University Hospital, Tokyo, Japan, a 1,020-bed tertiary-care teaching hospital. The incidence of MRSA isolates was examined using clinical specimens presented to the microbiology laboratory in the hospital. Antimicrobial consumption through intravenous injection was calculated in terms of the number of defined daily doses per 100 bed-days. The correlation between the incidence of MRSA isolates and antimicrobial consumption was determined employing a multiple stepwise regression analysis. A total of 109,946 bacterial isolates were consecutively collected over the 14-year period, and, of these, 13,872 (64% of S. aureus strains excluding coagulase-negative staphylococci) were MRSA strains. The longitudinal observation showed that the number and rate of MRSA isolates marginally decreased. The rate of MRSA isolates among S. aureus strains in 1995 was 68.5%, whereas that in 2008 was 53.8%. Consumption of cephalosporins decreased. Among carbapenems, the rate of imipenem (IPM) consumption decreased, whereas that of meropenem increased. A multiple stepwise regression analysis revealed that the antimicrobial consumption of cefmetazole, cefotiam, and IPM was positively correlated with the incidence of MRSA isolates. The use of β-lactam antimicrobials may contribute to the development of MRSA strains.

Analysis of Staphylococcal cassette chromosome mec in BD GeneOhm MRSA assay-negative strains.

ABSTRACT The BD GeneOhm MRSA assay could identify methicillin-resistant Staphylococcus aureus (MRSA) strains at a high ratio (97.8%). Analysis of 11 assay-negative MRSA strains suggested that insertion of non-mec staphylococcal cassette chromosome elements (SCCs) downstream of orfX, and carriage of SCCmecs with a left extremity that cannot be detected by the kit, might lead to their being given an incorrect negative status.

Multicenter evaluation of the Verigene Gram-negative blood culture nucleic acid test for rapid detection of bacteria and resistance determinants in positive blood cultures

The Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN) is a microarray-based assay that enables rapid detection of 9 common Gram-negative bacteria and 6 resistance determinants directly from positive blood cultures. We compared the performance of BC-GN with currently used automated systems, testing 141 clinical blood cultures and 205 spiked blood cultures. For identification of BC-GN target organisms in clinical and spiked blood cultures, the BC-GN assay showed 98.5% (130/132) and 98.9% (182/184) concordance, respectively. Of 140 resistance genes positively detected in clinical and spiked blood cultures with the BC-GN test, 139 (99.3%) were confirmed by PCR, and the detection results were consistent with the resistance phenotypes observed. The BC-GN assay, thus, can potentially improve care for sepsis patients by enabling timely detection and targeted antimicrobial therapy.

Comparative bactericidal activity of four fluoroquinolones against Pseudomonas aeruginosa isolated from chronic suppurative otitis media

BackgroundThe aim of the present study was to evaluate the bactericidal activity of four new fluoroquinolones against current isolates of Pseudomonas aeruginosa from the patients with chronic suppurative otitis media (CSOM).MethodsWe examined bactericidal activity of four types of fluoroquinolones, garenoxacin (GRNX), levofloxacin (LVFX), ciprofloxacin (CPFX) and sitafloxacin (STFX) against current isolates of P. aeruginosa (50 strains).ResultsSTFX exhibited the most potent activity of both MIC50 and MIC90, followed by CPFX, LVFX, and GRNX. The number of GRNX-resistant strains was significantly greater than those of LVFX, CPFX, and STFX (P < 0.05).ConclusionSTFX showed the most potent activity against P. aeruginosa for recent pathogens recovered from CSOM as compared with the others, suggesting that the clinical application of topical STFX would be useful to prevent the emergence of resistant mutants of P. aeruginosa.

Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called “the Eprobe leukemia assay,” for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

Efficacy of PCR-based open reading frame typing assay for outbreak investigation of metallo-β-lactamase-producing Pseudomonas aeruginosa in hematology unit

We investigated the efficacy of the PCR-based open reading frame typing (POT) assay for outbreak investigation of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa (MBL-PA). A total of 53 P. aeruginosa isolates were detected between January 2010 and December 2012 on a hematology ward, of which 6 were identified as MBL-PA with the blaIMP-1 gene. The POT assay revealed the same genotype (207-41) in 3 of 6 MBL-PA, suggesting an outbreak caused by a single strain. Environmental investigation of bathroom samples revealed the same POT genotype (207-41) as those of the clinical isolates and no other MBL-PA strains. Genetic relatedness of the MBL-PA isolates was confirmed by the DiversiLab repetitive-sequence-based PCR typing system, suggesting the POT type 207-41 as a genetically identical clone. The POT assay can be successfully applied to MBL-PA genotyping.

Molecular Epidemiology of Community-Associated and Hospital-associated Methicillin-resistant Staphylococcus aureus in a Japanese University Hospital

Abstract Background Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been reported in healthcare facilities worldwide. But details of CA-MRSA in Japanese healthcare facilities are rarely reported. The aims of this study are to know the distribution of CA-MRSA and healthcare-associated MRSA (HA-MRSA) with detailed molecular typing, and to assess the efficacy of infection control practice in a Japanese hospital. Methods From July to October in 2015, first MRSA isolate from each patient was collected in Juntendo University Hospital, Tokyo, Japan. MRSA strains were categorized as CA-MRSA and HA-MRSA according to the clinical definition of CDC. Detection of toxin genes and SCCmec typing were performed by PCR. Genetic relatedness among isolates was determined by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing was performed using whole genome sequencing data. Results A total of 102 MRSA strains were collected in the study period, and categorized as 34 CA-MRSA (33.3 %) and 68 HA-MRSA (66.7 %), respectively. Among the 73 strains isolated from hospitalized patients, 10 were CA-MRSA (13.7 %). Whereas among the 29 strains isolated in clinic, 5 were HA-MRSA (17.2 %). Three major types were as follows: ST8-SCCmec IV (n = 26, 25.5 %, CA: HA= 10: 16), ST5-SCCmec IIa (n = 17, 16.7 %, CA: HA= 5: 12), and ST1-SCCmec IVa (n = 13, 12.7 %, CA: HA= 6: 7). Among ST8-SCCmec IV strains, SCCmec IVl, originally reported in Japanese CA-MRSA, was found both in CA-MRSA and HA-MRSA (n = 11, 10.8 %, CA: HA= 5: 6). Only one ST772-SCCmec V strain carried Panton–Valentine leukocidin (PVL) gene. Two ST765-SCCmec IIa strains in CCU, 2 ST765-SCCmec IIa strains in a general ward, and 6 ST2764-SCCmec IVa strains in NICU showed genetic relatedness by PFGE, respectively. Especially, ST2764-SCCmec IVa was a clone originally reported as HA-MRSA in another Juntendo-affiliated hospital. Conclusion CA-MRSA and HA-MRSA were comparably found both in hospital and clinic. Unique Japanese clones were found in this study, but it seemed impossible to distinguish CA-MRSA and HA-MRSA simply by ST-SCCmec typing. In contrast, transmission of MRSA rarely happened in hospital. This heterogenous population structure of MRSA suggested that conventional HA-MRSA had lost its predominance by sufficient infection control, resulting in relative increase of CA-MRSA in hospital environment. Disclosures All authors: No reported disclosures.