Howard S. Gold

Linezolid resistance in a clinical isolate of Staphylococcus aureus.

The new oxazolidinone antimicrobial, linezolid, has been approved for the treatment of infections caused by various gram-positive bacteria, including meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Although instances of linezolid resistance in VRE have been reported, resistance has not been encountered among clinical isolates of S aureus. We have characterised an MRSA isolate resistant to linezolid that was recovered from a patient treated with this agent for dialysis-associated peritonitis.

Accessory Gene Regulator (agr) Locus in Geographically Diverse Staphylococcus aureus Isolates with Reduced Susceptibility to Vancomycin

ABSTRACT The majority of infections with glycopeptide intermediate-level resistant Staphylococcus aureus (GISA) originate in biomedical devices, suggesting a possible increased ability of these strains to produce biofilm. Loss of function of the accessory gene regulator (agr) of S. aureus has been suggested to confer an enhanced ability to bind to polystyrene. We studied agr in GISA, hetero-GISA, and related glycopeptide-susceptible S. aureus isolates. All GISA strains from diverse geographic origins belong to agr group II. All GISA strains were defective in agr function, as demonstrated by their inability to produce delta-hemolysin. Hetero-GISA isolate A5940 demonstrated a nonsense mutation in agrA that was not present in a pulsed-field gel electrophoresis-indistinguishable vancomycin-susceptible isolate from the same patient. Various other agr point mutations were noted in several clinical GISA and hetero-GISA isolates. A laboratory-generated agr-null strain demonstrated a small but reproducible increase in vancomycin heteroresistance after growth in vitro in subinhibitory concentrations of vancomycin. This was not seen in the isogenic agr group II parent strain in which agr was intact. The in vitro bactericidal activity of vancomycin was attenuated in the agr-null strain compared to the parent strain. These findings imply that compromised agr function is advantageous to clinical isolates of S. aureus toward the development of vancomycin heteroresistance, perhaps through the development of vancomycin tolerance.

Fluoroquinolones and the risk for methicillin-resistant Staphylococcus aureus in hospitalized patients.

To determine whether fluoroquinolone exposure is a risk factor for the isolation of Staphylococcus aureus and whether the effect is different for methicillin-resistant S. aureus (MRSA) versus methicillin-susceptible S. aureus (MSSA), we studied two case groups. The first case group included 222 patients with nosocomially acquired MRSA. The second case group included 163 patients with nosocomially acquired MSSA. A total of 343 patients admitted concurrently served as controls. Outcome measures were the adjusted odds ratio (OR) for isolation of MRSA and MSSA after fluoroquinolone exposure. Exposure to both levofloxacin (OR 5.4; p < 0.0001) and ciprofloxacin (OR 2.2; p < 0.003) was associated with isolation of MRSA but not MSSA. After adjustment for multiple variables, both drugs remained risk factors for MRSA (levofloxacin OR 3.4; p < 0.0001; ciprofloxacin OR 2.5; p = 0.005) but not MSSA. Exposure to levofloxacin or ciprofloxacin is a significant risk factor for the isolation of MRSA, but not MSSA.

Antimicrobial Resistance to Linezolid

Acquired resistance to linezolid, the first approved oxazolidinone, has been selected in laboratory experiments and has been observed in clinical isolates of gram-positive cocci. This resistance has typically been associated with single-nucleotide changes in varying numbers of copies of the genes encoding 23S ribosomal RNA. In the current environment of increasingly prevalent resistance to standard antibiotics, linezolid is an important drug because of its activity against a number of clinically significant gram-positive cocci, including multidrug-resistant staphylococci and enterococci. Although resistance to linezolid remains uncommon, the development of resistance by clinical isolates should prompt increased attention to susceptibility testing for this agent and should be taken into account in consideration of the therapeutic use of this drug.

Linezolid Resistance in Sequential Staphylococcus aureus Isolates Associated with a T2500A Mutation in the 23S rRNA Gene and Loss of a Single Copy of rRNA

Linezolid is an important therapeutic option for infections caused by resistant gram-positive bacteria. We report the characterization of sequential methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates that developed resistance in a patient treated with a prolonged course of linezolid. Analysis of this series of clinical MRSA isolates detected, in the resistant isolates, the presence of a T2500A mutation in the domain V region of the 23S rRNA gene. In addition, the loss of a single copy of the 23S rRNA gene was found in 2 of the resistant isolates. As a result of these 2 factors, the proportion of mutant : wild-type 23S rRNA genes increased in association with an increase in the minimum inhibitory concentration of linezolid. The most recent isolate of this series was recovered 7 months after the patient discontinued linezolid and demonstrated reversion to a susceptible phenotype associated with a loss of the T2500A mutation.

Staphylococcus aureus Accessory Gene Regulator (agr) Group II: Is There a Relationship to the Development of Intermediate-Level Glycopeptide Resistance?

We previously determined that all 6 Staphylococcus aureus strains with confirmed intermediate-level resistance to glycopeptides (glycopeptide intermediate S. aureus [GISA]) from the United States that we tested belonged to accessory gene regulator (agr) group II. In the present study, we found that 56% of surveyed bloodstream methicillin-resistant S. aureus isolates (n = 148) at our hospital were agr group II, whereas only 24% of methicillin-susceptible S. aureus isolates (n = 33) were agr group II (P = .001). Population analysis of genetically engineered agr-null and parent wild-type strains of groups I, II, and IV revealed that, when agr function is lost, the agr group II knockout S. aureus was most likely to develop glycopeptide heteroresistance after growth in 1 microg/mL but not 16 microg/mL vancomycin. This strain was unique in showing decreased autolysis after growth in these conditions. This study suggests that some S. aureus strains have an intrinsic survival advantage under a glycopeptide selective pressure, which is possibly related to reduced autolysis after exposure to subinhibitory concentrations of glycopeptide.

Methicillin-Resistant Staphylococcus aureus: Comparison of Susceptibility Testing Methods and Analysis of mecA-Positive Susceptible Strains

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for themecA gene used as the “gold standard” assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecAwas weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed thatmecA- and PBP 2a-positive S. aureusisolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA.

Emergence of a Clinical Daptomycin-Resistant Staphylococcus aureus Isolate during Treatment of Methicillin-Resistant Staphylococcus aureus Bacteremia and Osteomyelitis

ABSTRACT The emergence of a clinically daptomycin-resistant Staphylococcus aureus isolate occurred during treatment of methicillin-resistant S. aureus bacteremia and probable vertebral osteomyelitis. The breakthrough isolate was indistinguishable from pretreatment daptomycin-susceptible isolates by pulsed-field gel electrophoresis. Daptomycin nonsusceptibility was confirmed by MIC and time-kill curve analyses.

Linezolid Resistance in Staphylococcus aureus: Characterization and Stability of Resistant Phenotype

Linezolid is an important therapeutic option for treatment of infections caused by glycopeptide- and beta-lactam-resistant gram-positive organisms. Linezolid resistance is caused by mutations within the domain V region of the 23S ribosomal RNA (rRNA) gene, which is present in multiple copies in most bacteria. Among clinical Staphylococcus aureus isolates, there has been only 1 reported case of linezolid resistance. In the present study, this isolate was further characterized by determination of the number of mutant 23S rRNA copies, assessment of the stability of the resistant phenotype, and comparison of its growth characteristics with those of linezolid-susceptible S. aureus. All 5 copies of the 23S rRNA gene contained a G2576U mutation in the domain V region. After serial passage on antibiotic-free medium, the isolate maintained resistance to high concentrations of linezolid. Compared with 2 linezolid-susceptible S. aureus isolates, the linezolid-resistant S. aureus isolate demonstrated no significant differences in in vitro growth characteristics.

Vancomycin-Resistant Enterococci: Mechanisms and Clinical Observations

Enterococci are not generally regarded as highly virulent bacterial pathogens. However, resistance to many antimicrobial drugs complicates treatment of enterococcal infections. Acquired resistance to high concentrations of glycopeptide antibiotics, specifically vancomycin, has exacerbated this problem. This article seeks to concisely review the mechanisms of that resistance and its effects on clinical management of enterococcal infections, as well as clinical microbiology and infection control.