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 64 µg/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 8 µg/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.

Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.

The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound-ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate-was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method.

Complete Genome Sequence of Pseudomonas aeruginosa Strain 8380, Isolated from the Human Gut.

Pseudomonas aeruginosa shows multidrug resistance, which is mainly attributable to its expression of xenobiotic efflux pumps. However, it is unclear how silent pumps are expressed in clinical isolates. Here, we sequenced the complete genome of P. aeruginosa strain 8380, which was isolated from a human gut.

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 ß-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.

Immunochromatographic detection of the group B streptococcus antigen from enrichment cultures.

Group B Streptococcus (GBS; Streptococcus agalactiae) is a leading cause of serious neonatal infections. The Centers for Disease Control and Prevention recommends GBS screening for all pregnant women during the 35th to 37th weeks of gestation. Although GBS screening has been performed mainly by the culture-based method, it takes several days to obtain a reliable result. In this study, we developed a rapid immunochromatographic test (ICT) for the detection of GBS-specific surface immunogenic protein in 15 min using an overnight enrichment culture. The ICT was prepared using two anti-Sip monoclonal antibodies. This ICT was able to detect recombinant Sip levels of 0.5 ng/ml, or about 10(6) CFU/ml of GBS cells, in tests with 9 GBS strains of different serotypes. The cross-reactivity test using 26 species of microorganism showed no detectable false-positive result. Reactivity of the ICT with 229 GBS strains showed one false-negative result that was attributable to the production of truncated Sip. Among 260 enrichment cultures of vaginal swabs, 17 produced red to orange pigments in Granada medium, and they were all GBS and Sip positive. Among 219 pigment-negative cultures, 12 were GBS positive and 10 were Sip positive. Two Sip-negative cultures contained GBS cells below the limit of detection by the ICT. Among 207 GBS-negative cultures, only one was Sip positive, which was attributable to GBS cell debris. Thus, the sensitivity and specificity of the ICT appeared to be 93.1% and 99.6%, respectively. The newly developed ICT is readily applicable to clinical use in the detection of GBS.

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 ß-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.

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

BACKGROUND: A class of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin only in the presence of ß-lactam antibiotics (BIVR). This type of vancomycin resistance is mainly attributable to the rapid depletion of free vancomycin in the presence of ß-lactam antibiotics. This means that ß-lactam antibiotics remain active or intact in BIVR culture, although most MRSA cells are assumed to produce ß-lactamase. We hypothesised that the BIVR cells either did not harbour the ß-lactamase gene, blaZ, or the gene was quiescent. We tested this hypothesis by determining ß-lactamase activity and conducting PCR amplification of blaZ. RESULTS: Five randomly selected laboratory stock BIVR strains showed an undetectable level of ß-lactamase activity and were blaZ-negative. Five non-BIVR stock strains showed an average ß-lactamase activity of 2.59 ± 0.35 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 ß-lactamase-positive, respectively. In contrast, 95% and 61% of 328 non-BIVR strains had the blaZ gene and produced active ß-lactamase, respectively. To know the mechanism of low ß-lactamase activity in the BIVR cells, they were transformed with the plasmid carrying the blaZ gene. The transformants still showed a low level of ß-lactamase activity that was several orders of magnitude lower than that of blaZ-positive non-BIVR cells. Presence of the ß-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 ß-lactamase gene was modified in the BIVR cells to downregulate active ß-lactamase production. CONCLUSIONS: We concluded that BIVR cells gain vancomycin resistance by the elimination or inactivation of ß-lactamase production, thereby preserving ß-lactam antibiotics in milieu, stimulating peptidoglycan metabolism, and depleting free vancomycin to a level below the minimum inhibitory concentration of vancomycin.

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 78 % 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 5 % sheep blood) and 48 h of incubation resulted in 100 % 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.

Emergence of linezolid-resistant mutants in a susceptible-cell population of methicillin-resistant Staphylococcus aureus.

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.

Mutation in the sdeS gene promotes expression of the sdeAB efflux pump genes and multidrug resistance in Serratia marcescens.

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.

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 4 months 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.

Development of an immunochromatographic strip for simple detection of penicillin-binding protein 2'.

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 ß-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 × 10(5) to 1.7 × 10(7) 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.

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 64 µg/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 8 µg/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.

A 25-year trace of methicillin-resistant Staphylococcus aureus dissemination in a geriatric hospital in Japan.

We analyzed 218 strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from the septicemia patients in a geriatric hospital for 25 years. These strains were classified into 11 major DNA types, A through K, and 27 minor types. The strains belonging to group A and B isolated before 1990 were susceptible to imipenem (IPM), fluoroquinolone, and most other antibiotics tested, except that they were markedly resistant to gentamicin. Strains mostly isolated in 1985 and thereafter were classified into group C through K, and they were mainly resistant to IPM, fluoroquinolones, and clindamycin. Analysis of the MRSA marker gene, staphylococcal cassette chromosome mec (SCCmec), of these strains revealed that the strains in groups A and B had mainly type IV and type I, respectively, and that strains in groups C through J had mainly type II. These results suggested that the strains holding type II SCCmec were resistant to IPM, fluoroquinolone, and clindamycin and they were dominant-resistant type after late 1980s. The antibiotic resistance profiles of MRSA dramatically changed during late 1980s, and these were correlated with the SCCmec types. The lesson from this study would be that consistent execution of surveillance study is needed to update the resistant profiles.

Transcriptional regulation of the mexEF-oprN multidrug efflux pump operon by MexT and an unidentified repressor in nfxC-type mutant of Pseudomonas aeruginosa.

Expression of the MexEF-OprN efflux pump in Pseudomonas aeruginosa seems to be upregulated by MexT. The region upstream of the mexEF-oprN operon contains mexT arranged in tandem with mexEF-oprN and separated by 230 bp of mexT-mexE intergenic DNA. Therefore, it is likely that this intergenic DNA contains the promoter-operator element of mexEF-oprN. To characterize how the expression of the mexEF-oprN operon was controlled, we analyzed the mexT-mexE intergenic DNA by constructing a series of intergenic DNA deletions connected with the mexE∷lacZ reporter and made two important discoveries. The first was that the central region of the DNA contained two nod boxes. The mexT-proximal nod box was identified as the MexT-binding site by gel-shift assays using purified MexT. The mexT-distal nod box was required for the transcription of the mexEF-oprN operon, but not for the binding of MexT, suggesting that this region accommodates the binding of the RNA polymerase. The second observation is that there is a 13 bp inverted repeat sequence separated by 10 bp immediately upstream of the mexE gene. Deletion of this region caused a sudden rise in MexEF-OprN production, suggesting that this region accommodates the binding of a putative repressor protein.

Identification of the active component that induces vancomycin resistance in MRSA.

A fraction of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin (VCM) in the presence of ß-lactam antibiotics (BIVR) at low concentrations. We hypothesized that the BIVR phenomenon might be exerted by a peptidoglycan derivative(s) generated as a consequence of ß-lactam antibiotic action. To verify this hypothesis, we isolated the fraction that mimicked the effect of ß-lactam antibiotics by the enzymatic treatment of the crude cell wall. The active components were purified by a combination of reverse phase chromatographies, mass spectrum and amino-acid analyses, and were identified to be a muropeptide with the following formula: N-acetyglucosamyl-N-acetylmuramyl--Ala-D-isoGln-L-Lys-(ɛ-NH-4Gly)-D-Ala-2Gly. This is the very first identification of the active component, which induces VCM resistance in MRSA. We found that the BIVR cells are highly sensitive to this compound rendering the cells resistant to VCM compared with non-BIVR MRSA.

Tat pathway-mediated translocation of the sec pathway substrate protein MexA, an inner membrane component of the MexAB-OprM xenobiotic extrusion pump in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is equipped with the Sec and Tat protein secretion systems, which translocate the xenobiotic transporter MexAB-OprM and the pathogenic factor phospholipase C (PlcH), respectively. When the signal sequence of MexA was replaced with that of PlcH, the hybrid protein was successfully expressed and recovered from the periplasmic fraction, suggesting that the hybrid protein had been translocated across the inner membrane. MexA-deficient cells harboring the plasmid carrying the plcH-mexA fusion gene showed antibiotic resistance comparable to that of the wild-type cells. This result suggested that MexA secreted via the Tat machinery was properly assembled and functioned as a subunit of the MexAB-OprM efflux pump. A mutation was introduced into the chromosomal tatC gene encoding an inner membrane component of the Tat protein secretion machinery in mexA-deficient cells, and they were transformed with the plasmid carrying the plcH-mexA fusion gene. The transformants showed antibiotic susceptibility comparable to that of mexA-deficient cells, indicating that the hybrid protein was not transported to the periplasm. Whole-cell lysate of the mexA-tatC double mutant harboring the plcH-mexA plasmid produced mainly unprocessed PlcH-MexA. The periplasmic fraction showed no detectable anti-MexA antibody-reactive material. On the basis of these results, we concluded that MexA could be translocated across the inner membrane through the Tat pathway and assembled with its cognate partners, MexB and OprM, and that this complex machinery was fully functional. This hybrid protein translocation system has the potential to be a powerful screening tool for antimicrobial agents targeting the Tat system, which is not present in mammalian cells.

Rapid detection of vaginal Candida species by newly developed immunochromatography.

For the diagnosis of vulvovaginal candidiasis, we developed a simple immunochromatographic method that enables the detection of vaginal Candida spp. within about 30 min. Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of this method appeared to be 80.3, 99.3, 98.0, and 92.0%, respectively.

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.

Rapid depletion of free vancomycin in medium in the presence of beta-lactam antibiotics and growth restoration in Staphylococcus aureus strains with beta-lactam-induced vancomycin resistance.

A class of methicillin-resistant Staphylococcus aureus strains shows vancomycin resistance in the presence of beta-lactam antibiotics (beta-lactam-induced VAN-resistant methicillin-resistant S. aureus [BIVR]). Two possible explanations may be offered: (i) vancomycin in culture medium is depleted, and (ii) the D-Ala-D-Ala terminal of the peptidoglycan network is replaced with D-Ala-D-lactate. We tested these hypotheses by quantifying free vancomycin in the medium through the course of cell growth and by PCR amplification of the van genes. Growth of the BIVR cells to an absorption level of approximately 0.3 at 578 nm required about 24 h in the presence of vancomycin alone at the MIC (4.0 microg/ml). However, growth was achieved in only about 10 h when 1/1,000 to 1/2,000 the MIC of beta-lactam antibiotic was added 2 h prior to the addition of vancomycin, suggesting that the beta-lactams shortened the time to recovery from vancomycin-mediated growth inhibition. Free vancomycin in the culture medium decreased to 2.3 microg/ml in the first 8 h in the culture containing vancomycin alone, yet cell growth was undetectable. When the vancomycin concentration dropped below approximately 1.5 microg/ml at 24 h, the cells began to grow. In the culture supplemented with the beta-lactam 2 h prior to the addition of vancomycin, the drug concentration continuously dropped from 4 to 0.5 microg/ml in the first 8 h, and the cells began to grow at a vancomycin concentration of approximately 1.7 microg/ml or at 4 h of incubation. The gene encoding the enzyme involved in D-Ala-D-lactate synthesis was undetectable. Based on these results, we concluded that BIVR is attributable mainly to a rapid depletion of vancomycin in the medium triggered or promoted by beta-lactam antibiotics.