Mechanism and suppression of lysostaphin resistance in oxacillin-resistant Staphylococcus aureus.
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The potential for the development of resistance in oxacillin-resistant Staphylococcus aureus (ORSA) to lysostaphin, a glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus, was examined in vitro and in an in vivo model of infection. Following in vitro exposure of ORSA to subinhibitory concentrations of lysostaphin, lysostaphin-resistant mutants were idenitifed among all isolates examined. Resistance to lysostaphin was associated with a loss of resistance to beta-lactams and a change in the muropeptide interpeptide cross bridge from pentaglycine to a single glycine. Mutations in femA, the gene required for incorporation of the second and third glycines into the cross bridge, were found following PCR amplification and nucleotide sequence analysis. Complementation of lysostaphin-resistant mutants with pBBB31, which encodes femA, restored the phenotype of oxacillin resistance and lysostaphin susceptibility. Addition of beta-lactam antibiotics to lysostaphin in vitro prevented the development of lysostaphin-resistant mutants. In the rabbit model of experimental endocarditis, administration of a low dose of lysostaphin for 3 days led predictably to the appearance of lysostaphin-resistant ORSA mutants in vegetations. Coadministration of nafcillin with lysostaphin prevented the emergence of lysostaphin-resistant mutants and led to a mean reduction in aortic valve vegetation counts of 7.5 log(10) CFU/g compared to those for untreated controls and eliminated the isolation of lysostaphin-resistant mutants from aortic valve vegetations. Treatment with nafcillin and lysostaphin given alone led to mean reductions of 1.35 and 1.65 log(10) CFU/g respectively. In ORSA, resistance to lysostaphin was associated with mutations in femA, but resistance could be suppressed by the coadministration of beta-lactam antibiotics. (+info)
Evaluation of a new selective medium for methicillin-resistant Staphyloccocus aureus.
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The aims of this study were to evaluate the performance of a new medium, desferrioxamine oxacillin tellurite egg-yolk mannitol salt agar (DOTEMSA) in detecting methicillin-resistant Staphylococcus aureus (MRSA) and then to compare this medium against the Public Health Laboratory Service (PHLS) recommendation of mannitol salt agar (Oxoid) with oxacillin (OMSA) and Baird-Parker medium with ciprofloxacin (BPC) for the isolation of MRSA. The individual selective agents contained in DOTEMSA were tested against isolates of coagulase-negative staphylococci (CNS) and the medium with all constituents was challenged with various bacteria. Routine screening specimens were plated out on OMSA, BPC and DOTEMSA and the plates were incubated and examined at 24 and 48 h. Tellurite, desferrioxamine and oxacillin each inhibited the majority of CNS isolates; only three (of 103) grew in the presence of all three agents. Sixty-two of 63 isolates of MRSA grew on DOTEMSA and 59 produced lipase. Most other bacteria were inhibited. In all, 184 MRSA isolates were isolated from 540 screening specimens. The sensitivity of OMSA, BPC and DOTEMSA was 42%, 81% and 51% at 24 h, and 60%, 89% and 89% at 48 h. At 48 h, the combination of BPC and DOTEMSA detected 99% of MRSA isolates. Seventy, 49 and one non-MRSA isolates needed investigation for each of the three media respectively. A proposed strategy for MRSA screening would use BPC and DOTEMSA, examining BPC at 24 h and both media at 48 h. Provisional reports could then be issued at 24 h on the basis of rapid agglutination tests to confirm isolates as S. aureus from BPC and at 48 h on the basis of typical colonies from DOTEMSA. (+info)
Detection of methicillin/oxacillin resistance in staphylococci.
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Methicillin/oxacillin-resistant staphylococci are heterogeneous in their expression of resistance to beta-lactam agents and the test conditions have a major effect on the expression and therefore the detection of resistance. Conflicting recommendations regarding the most reliable method for routine use are partly related to differences between strains and there may be a variable interaction between the factors affecting the expression of resistance, including the agent tested, the medium, the NaCl concentration, the inoculum, temperature and period of incubation and the reading of endpoints. 'Borderline' resistant strains may have altered PBPs or be penicillinase hyperproducers, and these can be difficult to distinguish from resistant strains that carry the mecA gene. Recommended methods for MIC and disc diffusion testing are described, although it is unlikely that any single method will detect all resistant strains. Some rapid and/or automated methods are also available, including latex agglutination techniques for the detection of PBP2a. The gold standard method for the detection of resistance mediated by mecA is PCR, which is most commonly used as a reference method at present. (+info)
Correlation between genotype and phenotypic categorization of staphylococci based on methicillin susceptibility and resistance.
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Positive correlation between methicillin and oxacillin susceptibility test results and the detection of the mecA gene was observed for Staphylococcus aureus, S. epidermidis, and S. haemolyticus as well as among mecA(+) strains of other species of coagulase-negative staphylococci (CNS). However, at least 50% of the mecA-negative strains of these other species of CNS were falsely classified as methicillin and oxacillin resistant. (+info)
In vitro antimicrobial activity of the aminoglycoside arbekacin tested against oxacillin-resistant Staphylococcus aureus isolated in Brazilian hospitals.
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Arbekacin is an aminoglycoside used in Japan for treating infections caused by gentamicin and oxacillin-resistant S. aureus (ORSA). The objective of this study was to determine the in vitro antimicrobial activity of arbekacin against 454 clinical isolates of ORSA. The isolates were consecutively collected between January and July, 2000, from patients hospitalized in 8 Brazilian medical centers. The antimicrobial susceptibility testing was performed by disk diffusion method according to NCCLS recommendations. The vast majority of the isolates, 453 strains (99.8%), were considered susceptible to arbekacin based on the criteria proposed by the Requirements for Antibiotic Products of Japan. Only 1 isolate (0.2%) was classified as resistant. On the other hand, high rates of resistance were demonstrated for other aminoglycosides, such as gentamicin (97.6% resistance) and amikacin (97.0% resistance). Resistance rate was also high for ciprofloxacin (98.0%). All isolates were considered susceptible to vancomycin. The excellent in vitro antimicrobial activity of arbekacin demonstrated in this study indicates that this antimicrobial agent may play an important role in the treatment of severe ORSA infections, especially those that show poor clinical response with vancomycin monotherapy. Since the aminoglycosides should not be used as monotherapy to treat Gram positive infections, further studies evaluating in vitro and in vivo synergistic activity of arbekacin combinations are necessary to clarify the clinical role of this aminoglycoside. (+info)
Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolone exposure.
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To study the effect of fluoroquinolone exposure on the expression of mec(A)-encoded oxacillin resistance, population analysis profiling was performed on four strains of fluoroquinolone-susceptible, mec(A)-positive, heteroresistant Staphylococcus aureus. Growth in the presence of 0.5 x MIC of a fluoroquinolone resulted in >10-fold increase in the proportion of the population that grew on agar containing oxacillin 128 mg/L. Ciprofloxacin exhibited a greater effect than moxifloxacin, levofloxacin and gatifloxacin (average 3400-, 220-, 170- and 49-fold increase in oxacillin-resistant colonies versus the control, respectively). The increase was directly proportional to the fluoroquinolone concentration and could be detected as early as 8 h after exposure to the fluoroquinolone. At 8 h, the absolute number of colonies that grew on oxacillin 128 mg/L was similar whether or not the isolate was exposed to the fluoroquinolone, but the total cfu on non-selective media decreased. The resultant oxacillin-resistant colonies also showed a 1.5- to 3-fold increase in fluoroquinolone MIC. No oxacillin resistance was observed on two similarly treated fluoroquinolone-susceptible, mec(A)-negative strains. It appears that fluoroquinolones influence oxacillin resistance by selective inhibition or killing of the more susceptible subpopulations in heteroresistant S. aureus. The surviving populations are more resistant to both oxacillin and fluoroquinolone. The mechanisms of resistance to the two agents may be unrelated but tend to be associated. This could explain in part the observed increases in fluoroquinolone-resistant MRSA. (+info)
Comparative synergistic activity of nafcillin, oxacillin, and methicillin in combination with gentamicin against.
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The effectiveness of three semisynthetic, penicillinase-resistant penicillins alone and in combination with gentamicin was tested against 29 clinical isolates of enterococci. The minimal inhibitory concentrations of nafcillin were considerably lower than those of oxacillin and methicillin but were slightly higher than those of penicillin. At clinically achievable concentrations, the combination of nafcillin plus gentamicin produced enhanced killing against 13 of 14 strains of enterococci and was synergistic (by very rigid criteria) against 10 of 14 strains. In contrast, combinations of oxacillin plus gentamicin were synergistic against only 3 of 14 strains, and methicillin plus gentamicin produced synergistic killing against only 1 of 14 strains. (+info)
Comparison of the Vitek gram-positive susceptibility 106 card, the MRSA-Screen latex agglutination test, and mecA analysis for detecting oxacillin resistance in a geographically diverse collection of clinical isolates of coagulase-negative staphylococci.
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The Vitek automated susceptibility testing system with a modified gram-positive susceptibility (GPS) 106 card (bioMerieux Vitek, Inc., Hazelwood. Mo.) and a rapid slide latex agglutination test (MRSA-Screen test; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their abilities to detect oxacillin resistance in coagulase-negative staphylococci (CoNS). The reference broth microdilution method and the detection of the mecA gene by PCR ("gold standard" reference result) were used to compare the results obtained with the commercial products. A total of 123 clinical isolates consisting of eight species were selected from U.S. surveillance collections. Among the mecA-positive isolates (95 strains), 30 isolates were initially negative on the MRSA-Screen test read at 3 min. When the agglutination reaction was extended for 10 min, 26 of the 30 isolates became positive. For a different four isolates, the oxacillin MIC was < or =0.25 microg/ml on the Vitek GPS 106 card. Among the mecA-negative isolates (28 strains), for two Staphylococcus warneri, two S. lugdunensis, and two S. saprophyticus strains MICs were > or =0.5 microg/ml by the reference broth microdilution method. Four of these isolates were also categorized as resistant with the Vitek GPS 106 card and two isolates were positive by the MRSA-Screen test. Overall, the MRSA-Screen test, GPS 106 card, and reference broth microdilution method had sensitivities of 95.7 (result at 10 min), 95.7, and 100%, respectively, and specificities of 92.8, 85.7, and 78.5%, respectively. Although the MRSA-Screen test required a slight procedural modification, both commercial methods achieved a sensitivity and specificity at detecting oxacillin resistance in CoNS at a level that was acceptable for clinical laboratory use. (+info)