Taiji Nakae

Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan

Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clinical isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concentration (MIC) of linezolid in these strains varied from 8 to 64xa0μg/ml. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S ribosomal RNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8xa0μg/ml had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, respectively. These results suggest that the level of linezolid resistance seems to be roughly correlated with the number of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.

Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa

MexXY, a drug efflux pump in Pseudomonas aeruginosa, confers resistance to aminoglycoside antibiotics. We recently reported that MexZ binds to the promoter region of the mexXY operon. Electrophoretic mobility shift assay (EMSA) using recombinant MexZ and oligonucleotide probes prepared from the intergenic region between mexZ and mexX revealed that MexZ binds to a 20 bp palindromic sequence. Culture of P. aeruginosa in the presence of tetracycline induced higher levels of MexX and MexZ, as measured by immunoblotting and EMSA, than in the absence of antibiotics. When MexZ was expressed by a mexZ expression plasmid, the plasmid-borne MexZ repressed drug-induced MexX production, further confirming that MexZ acts as a repressor of the mexXY operon. PA5471 protein has been reported to be essential for drug-induced MexXY production. Similarly to that report, we observed that plasmid-borne PA5471 induced both MexX and MexZ production in PAO1 cells. Interestingly, interaction between MexZ and PA5471 was observed in a yeast two-hybrid assay. Furthermore, EMSA and in vitro transcription assays revealed that interaction between PA5471 and MexZ reduced MexZ DNA-binding ability, leading to mexXY transcription. These findings contribute to the understanding of the molecular mechanisms underlying the transcriptional regulation of mexZ and mexXY by drug-induced PA5471 expression.

Antibiotic susceptibility survey of blood-borne MRSA isolates in Japan from 2008 through 2011

We conducted an antibiotic susceptibility survey of 830 blood-borne methicillin resistant Staphylococcus aureus collected from nationwide hospitals in Japan over a three-year period from January 2008 through May 2011. Antibiotic susceptibility was judged according to the criteria recommended by the Clinical Laboratory Standard Institute. Over 99% of the MRSA showed to be susceptible to teicoplanin, linezolid, sulfamethoxazole/trimethoprim and vancomycin, and over 97% of them were susceptible to daptomycin, arbekacin and rifampin. The majority of the MRSA strains showed resistant to minocycline, meropenem, imipenem, clindamycin, ciprofloxacin, cefoxitin, and oxacillin in the rates of 56.6, 72.9, 73.7, 78.7, 89.0, 99.5, and 99.9%, respectively. Among the MRSA strains, 72 showed reduced susceptibility to vancomycin, including 8 strains (0.96%) of vancomycin-intermediate S. aureus (VISA), 54 (6.51%) of heterogeneous vancomycin-intermediate S. aureus (hVISA), and 55 (5.63%) of β-lactam antibiotics-induced vancomycin resistant S. aureus (BIVR). Unexpectedly, among the 54 hVISA and 55 BIVR, 45 isolates (83.3% and 81.8%, respectively) showed both hVISA and BIVR phenotypes. A new trend of vancomycin resistance found in this study was that VISA strains were still prevalent among the bacteremic specimens. The high rates of the hVISA/BIVR two-phenotypic vancomycin resistance, and the prevalence of VISA in the bloodborne MRSA call attention in the MRSA epidemiology in Japan.

Emergence of Linezolid-Resistant Mutants in a Susceptible-Cell Population of Methicillin-Resistant Staphylococcus aureus

ABSTRACT Methicillin-resistant Staphylococcus aureus with a MIC of linezolid of 4 μg/ml, isolated from a patient who had undergone unsuccessful linezolid therapy, yielded linezolid-resistant mutants in blood agar at 48 h of incubation. The resistant clones showed a MIC of linezolid ranging from 8 to 64 μg/ml and accumulated the T2500A mutation(s) of the rRNA genes. Emergence of these resistant clones appears to be facilitated by a cryptic mutation or mutations associated with chloramphenicol resistance.

Development of an Immunochromatographic Strip for Simple Detection of Penicillin-Binding Protein 2′

ABSTRACT Infections with methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococcus (MR-CNS) are a serious problem in hospitals because these bacteria produce penicillin-binding protein 2′ (PBP2′ or PBP2a), which shows low affinity to β-lactam antibiotics. Furthermore, the bacteria show resistance to a variety of antibiotics. Identification of these pathogens has been carried out mainly by the oxacillin susceptibility test, which takes several days to produce a reliable result. We developed a simple immunochromatographic test that enabled the detection of PBP2′ within about 20 min. Anti-PBP2′ monoclonal antibodies were produced by a hybridoma of recombinant PBP2′ (rPBP2′)-immunized mouse spleen cells and myeloma cells. The monoclonal antibodies reacted only with PBP2′ of whole-cell extracts and showed no detectable cross-reactivity with extracts from other bacterial species tested so far. One of the monoclonal antibodies was conjugated with gold colloid particles, which react with PBP2′, and another antibody was immobilized on a nitrocellulose membrane, which captures the PBP2′-gold colloid particle complex on a nitrocellulose strip. This strip was able to detect 1.0 ng of rPBP2′ or 2.8 × 105 to 1.7 × 107 CFU of MRSA cells. The cross-reactivity test using 15 bacterial species and a Candida albicans strain showed no detectable false-positive results. The accuracy of this method in the detection of MRSA and MR-CNS appeared to be 100%, compared with the results obtained by PCR amplification of the PBP2′ gene, mecA. This newly developed immunochromatographic test can be used for simple and accurate detection of PBP2′-producing cells in clinical laboratories.

Two routes of MexS-MexT-mediated regulation of MexEF-OprN and MexAB-OprM efflux pump expression in Pseudomonas aeruginosa.

The NfxC‐type mutant of Pseudomonas aeruginosa produces the MexEF‐OprN efflux pump and down‐regulates expression of the quorum‐sensing‐dependent efflux pump MexAB‐OprM and production of virulence factors in the presence of an active transcriptional regulator, MexT. Consequently, these cells are resistant to chloramphenicol and hypersusceptible to β‐lactam antibiotics. An upper negative regulator, MexS, has been assumed to inactivate MexT in wild‐type strains, hence shutting down production of the MexEF‐OprN pump. This observation was, however, reported in only one clinical strain and not confirmed in well‐characterized laboratory strains. Moreover, it is not known whether MexS is involved in the quorum‐sensing‐dependent regulation of virulence factor production. To assess these issues, a plasmid carrying wild‐type mexS was introduced into three NfxC‐type mutants from laboratory strains, which carry an impaired mexS and unimpaired mexT. Unexpectedly, all the transformants produced an increased amount of MexEF‐OprN proteins. Three clinical NfxC strains were similarly transformed and although MexEF‐OprN was undetectable in two of these strains, one produced an increased amount of these proteins, similar to the laboratory strains. These results were interpreted to mean that P. aeruginosa takes two separate routes in MexT‐mediated regulation of mexEF‐oprN expression: the MexS‐bypassed pathway and MexS‐mediated pathway. On the other hand, the transformants of both the laboratory and clinically derived NfxC‐type cells produced increased amounts of MexAB‐OprM and virulence factors, suggesting that production of these proteins occurs via the MexS‐mediated pathway.

Mutation in the sdeS Gene Promotes Expression of the sdeAB Efflux Pump Genes and Multidrug Resistance in Serratia marcescens

ABSTRACT Serratia marcescens gained resistance to both biocides and antibiotics on expressing the SdeAB efflux pump, following exposure to increasingly higher concentrations of a biocide (H. Maseda et al., Antimicrob. Agents Chemother. 53:5230–5235, 2009). To reveal the regulatory mechanism of sdeAB expression, wild-type cells were subjected to transposon-mediated random mutagenesis, and a mutant with antibiotic resistance, which mimicked the phenotype of the previous biocide-resistant cells, was obtained. The transposon element was found in the chromosomal DNA downstream of the sdeAB operon. Sequencing revealed the presence of an open reading frame (ORF) encoding a protein with 159 amino acid residues that is highly similar to the BadM-type transcriptional repressor, designated sdeS. The level of sdeB::xylE reporter gene expression, undetectable in the wild-type cells, appeared to be fully comparable to that in the biocide-resistant cells. Nucleotide sequencing of the mutant revealed sdeS to have a single G-to-A base substitution at position 269 that converted Trp90 to a stop codon. Introduction of a plasmid-borne intact sdeS into the mutant cells and the biocide-resistant cells resulted in a reduction in sdeB::xylE reporter activity to an undetectable level. These results suggested that SdeS functions as a repressor of the sdeAB operon. It was concluded that the original biocide-resistant cells had an impaired sdeS and, therefore, a derepressed level of the SdeAB efflux pump.

Limited detectability of linezolid-resistant Staphylococcus aureus by the Etest method and its improvement using enriched media.

The aim of this study was to evaluate Etest for detectability of linezolid-resistant meticillin-resistant Staphylococcus aureus (MRSA). The MIC of linezolid obtained by the Etest method in 18 linezolid-resistant strains of MRSA was compared with that obtained using standard agar and broth dilution methods according to Clinical and Laboratory Standards Institute guidelines. The mean linezolid MIC obtained by Etest in 18 linezolid-resistant strains of MRSA using Mueller-Hinton (MH) agar was 12.6-fold lower than that obtained by the agar dilution method, with the result that 78u200a% of the linezolid-resistant strains were incorrectly classified as linezolid-susceptible. The MIC of linezolid by Etest on brain-heart infusion (BHI) agar had a mean value 2.5-fold lower than that obtained by the agar dilution method, suggesting that replacing MH agar with BHI agar considerably improved the detectability of linezolid-resistant MRSA. Use of blood agar (MH agar supplemented with 5u200a% sheep blood) and 48 h of incubation resulted in 100u200a% agreement with the agar and broth dilution methods. Thus, this study revealed that the Etest on MH agar and BHI agar yielded false-negative results in a significant fraction of the linezolid-resistant MRSA. Hence, the use of blood agar and prolonged incubation is highly recommended for the accurate detection of linezolid-resistant MRSA using Etest.

Low level β-lactamase production in methicillin-resistant staphylococcus aureus strains with β-lactam antibiotics-induced vancomycin resistance

BackgroundA class of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin only in the presence of β-lactam antibiotics (BIVR). This type of vancomycin resistance is mainly attributable to the rapid depletion of free vancomycin in the presence of β-lactam antibiotics. This means that β-lactam antibiotics remain active or intact in BIVR culture, although most MRSA cells are assumed to produce β-lactamase. We hypothesised that the BIVR cells either did not harbour the β-lactamase gene, blaZ, or the gene was quiescent. We tested this hypothesis by determining β-lactamase activity and conducting PCR amplification of blaZ.ResultsFive randomly selected laboratory stock BIVR strains showed an undetectable level of β-lactamase activity and were blaZ-negative. Five non-BIVR stock strains showed an average β-lactamase activity of 2.59u2009±u20090.35 U. To test freshly isolated MRSA, 353 clinical isolates were collected from 11 regionally distant hospitals. Among 25 BIVR strains, only 16% and 8% were blaZ positive and β-lactamase-positive, respectively. In contrast, 95% and 61% of 328 non-BIVR strains had the blaZ gene and produced active β-lactamase, respectively. To know the mechanism of low β-lactamase activity in the BIVR cells, they were transformed with the plasmid carrying the blaZ gene. The transformants still showed a low level of β-lactamase activity that was several orders of magnitude lower than that of blaZ-positive non-BIVR cells. Presence of the β-lactamase gene in the transformants was tested by PCR amplification of blaZ using 11 pairs of primers covering the entire blaZ sequence. Yield of the PCR products was consistently low compared with that using blaZ-positive non-BIVR cells. Nucleotide sequencing of blaZ in one of the BIVR transformants revealed 10 amino acid substitutions. Thus, it is likely that the β-lactamase gene was modified in the BIVR cells to downregulate active β-lactamase production.ConclusionsWe concluded that BIVR cells gain vancomycin resistance by the elimination or inactivation of β-lactamase production, thereby preserving β-lactam antibiotics in milieu, stimulating peptidoglycan metabolism, and depleting free vancomycin to a level below the minimum inhibitory concentration of vancomycin.

Trends in empirical chemotherapy of bacterial meningitis in children aged more than 4 months in Japan : a survey from 1997 through 2008

Bacterial meningitis is a serious problem in pediatric clinics and, therefore, needs urgent and empirical chemotherapy. We investigated 1,116 cases of empirical chemotherapy of patients aged older than 4xa0months from 1997 through 2008 by sending questionnaires. A single antibiotic treatment was carried out in less than 30% of the cases throughout the years, whereas the combination of two antibiotics had been practiced in more than 70% of the cases. The main antibiotics used were cephalosporins, carbapenems, and ampicillin. Combinatory use of ampicillin and cephalosporin was carried out in 74.7–82.7% of cases in 1997–2000, but sharply declined thereafter to 0–13.8% in 2004–2008. However, the combination of carbapenem and cephalosporin compensated for the decline, increasing from 3.8–6.6% in 1998–1999 to 79.5–89.9% in 2005–2008. The breakdown in the use of cephalosporins, carbapenems, and ampicillin in two-drug combinatory therapy was as follows. (i) Use of cefotaxime was 61.8–75.3% in 1997–2001, but decreased to nearly 50%, equivalent to the level of ceftriaxone use in 2003–2008. (ii) Use of ampicillin dropped from 74.7–92.3% in 1997–2000 to 4.6% in 2008, and this decreased level was compensated for by the use of carbapenems. Overall, combinatory chemotherapy of the third-generation cephalosporins and carbapenems seems to be practical. The discussion in this report includes the difference between Japan and the United States in the prevalence of the causative agents and the use of antibiotics. These studies provide information on trends in the treatment of children’s meningitis in Japan and will be useful for the design of future empirical chemotherapy.