Ryoichi Saito

Analysis of plasmid-mediated multidrug resistance in Escherichia coli and Klebsiella oxytoca isolates from clinical specimens in Japan

This study investigated the relationship of plasmid-mediated quinolone resistance (PMQR) and aminoglycoside resistance among oxyimino-cephalosporin-resistant Escherichia coli (n=46) and Klebsiella oxytoca (n=28) clinical isolates in Japan. Seventy-three isolates appeared to produce an extended-spectrum beta-lactamase (ESBL) and one K. oxytoca isolate produced IMP-1 metallo-beta-lactamase (MBL). Polymerase chain reaction (PCR) and sequencing confirmed that eight CTX-M-9/SHV-12-producing isolates, one IMP-1-producing K. oxytoca isolate, and six ESBL-positive E. coli isolates respectively possessed PMQR genes qnrA1, qnrB6, and aac(6)-Ib-cr. All qnr-positive isolates also carried either aac(6)-Ib or aac(6)-IIc aminoglycoside acetyltransferase genes. Resistance determinants to beta-lactams, quinolones and aminoglycosides were co-transferred with a plasmid of ca. 140 kb. The qnrA1 gene was located downstream of insertion sequence ISCR1 in complex class 1 integrons. A novel qnrA1-carrying class 1 integron with the cassette arrangement aac(6)-IIc-aadA2 as well as a unique class 1 integron with bla(IMP-1)-aac(6)-IIc cassettes on the plasmid carrying qnrB6 were found in K. oxytoca isolates. We describe the identification of qnrB6 and aac(6)-Ib-cr and the close association of qnr with aac(6)-Ib and aac(6)-IIc for the first time in clinical isolates producing ESBL or MBL in Japan.

Evaluation of a chromogenic agar medium for the detection of extended-spectrum β-lactamase-producing Enterobacteriaceae

Aim:u2002 To compare the performance of a new chromogenic agar medium CHROMagar ESBL (KC‐ESBL) to chromID ESBL (SB‐ESBL) for the detection and presumptive identification of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae directly from clinical specimens.

Antimicrobial susceptibility and mechanism of quinolone resistance in Campylobacter jejuni strains isolated from diarrheal patients in a hospital in Tokyo

We determined the minimum inhibitory concentrations of six types of antimicrobial agents for 523 strains of Campylobacter jejuni that were isolated from diarrheal patients in a general hospital in Tokyo during the period between 2003 and 2005. It was revealed that 20.2%, 22.9%, 6.7%, and 0.6% of all the C. jejuni strains tested were resistant to ciprofloxacin (CPFX), nalidixic acid, ampicillin, and fosfomycin, respectively. All the strains were susceptible to clarithromycin and erythromycin. To elucidate the mechanism of quinolone resistance, in a total of 55 strains selected randomly, we carried out sequence determination and analysis of the quinolone-resistance determining regions (QRDRs) of their gyrA and gyrB genes. Amino-acid substitution at codon 86 (Thr → IIe) of GyrA was found in all the 37 CPFX-resistant strains. There was no amino-acid substitution in the QRDR of the gyrB gene. All of the genomic DNAs of these 55 strains showed distinct pulsed-field gel electrophoresis patterns. Taken together, these results suggested that the quinolone resistance of C. jejuni was attributable mainly to the mutation at codon 86 (Thr → IIe) in the QRDR of GyrA, and that this particular mutation and other silent mutations could be found not only in a certain clone of C. jejuni but also universally in a wide variety of strains.

Molecular characterizations of carbapenem and ciprofloxacin resistance in clinical isolates of Pseudomonas putida

To analyze the genetic mechanisms of carbapenem and ciprofloxacin resistance in clinical isolates of Pseudomonas putida, 27 clinical isolates (comprising 11 carbapenem- and ciprofloxacin-resistant strains, 13 carbapenem-resistant and ciprofloxacin-susceptible strains, and 3 carbapenem- and ciprofloxacin-susceptible strains) were collected from different patients. Carbapenem resistance was examined by polymerase chain reaction (PCR) and DNA sequencing for metallo-β-lactamase (MBL) and integrase genes (IntI-1 and IntI-3), and by reverse transcriptase-PCR (RT-PCR) for expression of the porin gene (oprD). Ciprofloxacin resistance was characterized by PCR and DNA sequencing for mutations in the quinoloneresistance determining regions of the gyrA and parC genes. The blaIMP-1 MBL and intI-1 and/or intI-3 genes were detected in all carbapenem-resistant strains, and decreased expression of the oprD gene as compared to carbapenemsusceptible strains was observed in several strains. All the 11 strains with ciprofloxacin minimal inhibitory concentrations (MICs) of ≥64 mg/l had substitution in GyrA (Thr83Ile), and one (ciprofloxacin MIC of 512 mg/l) of these strains also had substitution in ParC (Ser87Leu). Overproduction of the efflux pump was observed in 10 of the 11 ciprofloxacin-resistant strains. We concluded that the production of IMP-1 type MBL was the most critical factor in developing high-level resistance to carbapenems, and mutations in the target proteins and overproduction of the efflux pump synergistically contribute to the acquisition of high-level resistance to ciprofloxacin in clinical isolates of P. putida.

Performance of an automated system for quantitation of hepatitis C virus core antigen.

The performance of an automated system for the HCV core antigen assay using the Lumispot LS-2000 automated analyzer was evaluated against that of the COBAS AMPLICOR HCV MONITOR Test, version 2.0 (COBAS HCM-2) for the testing of sera from 155 chronic hepatitis C patients. The within-run coefficient of variations (CVs) and the between-day CVs were <9.6 and <8.4%, respectively. The analytical detection limit of the HCV core antigen assay was 5.0 fmol/l and it was linear up to at least 45000 fmol/l. No blood elements interfered with the assay. HCV core antigen levels were significantly correlated with HCV RNA levels in both serogroup 1 and serogroup 2 (r=0.829, P<0.001). It is estimated that 100 fmol/l of HCV core antigen level was equivalent to approximately 30000 IU/ml of HCV RNA level. Three sera had HCV core antigen levels below the detection limit of the assay and their HCV RNA levels as determined by COBAS HCM-2 assay were very low at 1000, 1100 and 1700 IU/ml, respectively. In six IFN responders among seven patients, HCV core antigen levels were in parallel with HCV RNA levels. In conclusion, since this assay demonstrated good reproducibility, a favorable dynamic range and adequate sensitivity, it may be useful as an alternative direct marker of viral level monitoring in patients with hepatitis C.

A 5 Year Longitudinal Study of Water Quality for Final Rinsing in the Single Chamber Washer-Disinfector with a Reverse Osmosis Plant

This report deals with the construction and management of the reverse osmosis (RO) water system for final rinsing of surgical instruments in the washer-disinfector. Numerous operational challenges were encountered in our RO water system and these were analyzed utilizing the Ishikawa Fishbone diagram. The aim was to find potential problems and promote preventive system management for RO water. It was found that the measures that existed were inappropriate for preventing contamination in the heat-labile RO water system. The storage tank was found to be significantly contaminated and had to be replaced with a new one equipped with a sampling port and water drainage system. Additional filters and an UV treatment lamp were installed. The whole system disinfection started 1.5 years later using a peracetic acid–based compound after confirming the material compatibility. Operator errors were found when a new water engineer took over the duty from his predecessor. It was also found that there were some deficiencies in the standard operating procedures (SOPs), and that on-the-job training was not enough. The water engineer failed to disinfect the sampling port and water drainage system. The RO membrane had been used for 4 years, even though the SOP standard specified changing it as every 3 years. Various bacteria, such as Rothia mucilaginosa, were cultured from the RO water sampled from the equipment. Because Rothia mucilaginosa is a resident in the oral cavity and upper respiratory tract, it is believed that the bacteria were introduced into the system by the maintenance personnel or working environment. Therefore, the presence of R. mucilaginosa implied the failure of sanitary maintenance procedures. This study suggests that water systems should be designed based on the plans for profound system maintenance. It also suggests that SOP and on-the job training are essential to avoid any operator errors. These results must be carefully considered when either constructing new RO systems or performing maintenance and periodical examination of the equipment. LAY ABSTRACT: Reverse osmosis (RO) water is used for final rinsing in our washer-disinfector. The authors used the Ishikawa Fishbone diagram to clarify the critical points for optimizing RO water quality. There existed no measures to prevent contamination in the heat-labile RO water system. The storage tank was significantly contaminated and had to be replaced with a new one equipped with a sampling port and water drainage system. Additional filters and an UV treatment lamp were installed. The whole system disinfection started 1.5 years later using a peracetic acid–based compound after confirming the material compatibility. Operator errors occurred when a new water engineer took over the duty from his predecessor. There were neither standard operating procedures (SOPs) nor on-the-job training. The new water engineer had failed to disinfect the sampling port and water drainage system. Rothia mucilaginosa was cultured from the RO water. It is a resident in the oral cavity and upper respiratory tract. This implied the possible failure of sanitary procedures in the system maintenance. The Ishikawa Fishbone diagram was useful for this study. It suggests that water systems should be designed with plans for system maintenance taken into account. It also suggests that SOP and on-the job training are essential in order to avoid operator errors.