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We attempted to find a relationship between the microbiological properties of bloodstream isolates of methicillin-resistant Staphylococcus aureus (MRSA) and the efficacy of vancomycin in the treatment of bacteremia. Vancomycin susceptibility testing was performed, and bactericidal activity was determined for 30 isolates from 30 different patients with MRSA bacteremia for whom clinical and microbiological outcome data were available. The majority of these patients had been previously enrolled in multicenter prospective studies of MRSA bacteremia refractory to conventional vancomycin therapy. Logistic regression found a statistically significant relationship between treatment success with vancomycin and decreases in both vancomycin MICs (<0.5 g/ml versus 1.0 to 2.0 g/ml; P ‫؍‬ 0.02) and degree of killing (reduction in 1og 10 CFU/milliliter) by vancomycin over 72 h of incubation in vitro (P ‫؍‬ 0.03). For MRSA isolates with vancomycin MICs < 0.5 g/ml, vancomycin was 55.6% successful in the treatment of bacteremia whereas vancomycin was only 9.5% effective in cases in which vancomycin MICs for MRSA were 1 to 2 g/ml.

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 electrophoresisindistinguishable 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.Since 1997, several case reports have appeared describing Staphylococcus aureus clinical isolates with reduced susceptibility to glycopeptide antibiotics (5, 11, 13-15, 32, 35, 36). The mechanisms responsible for this low-level resistance are poorly understood but do not involve the vanA, vanB, vanC, vanD, vanE, or vanG genes that confer vancomycin resistance in enterococci (12-15). The preponderance of data suggests that the mechanism for intermediate-level vancomycin resistance in S. aureus may relate to sequestration of the antimicrobial agent by nonamidated muropeptides within a thickened cell wall (8,12,13).Our review of clinical case histories of several patients with glycopeptide intermediate-level resistant S. aureus (GISA) strains revealed that, in addition to prolonged exposure to vancomycin, most of these patients had infections originating in or involving biomedical devices such as artificial heart valves, central venous catheters, biliary stents, and dialysis catheters (5, 11, 13-15, 32, 35, 36).The accessory gene regulator (agr) locus of S. aureus is a quorum-sensing gene cluster of five genes (hld, agrB, agrD, agrC, and agrA) that upregulates production of secreted virulence factors, including the alpha-, beta-, and delta-hemolysins, and downregulates production of cell-associated virulence factors (16,23,25,26,28,31). Polymorphisms in agrD and agrC define four S. aureus agr groups (25). Published reports have noted that agr group I strains comprised a significant majority of clinical isolates (...

Isolates from patients with PB differed from those in patients with RB, in several in vitro characteristics. Further studies will be necessary to define how these factors might affect clinical outcome.

BackgroundDaptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.MethodsWhole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates.ResultsOn average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol.ConclusionPoint mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus.

Nonmammalian model systems of infection such as Galleria mellonella (caterpillars of the greater wax moth) have significant logistical and ethical advantages over mammalian models. In this study, we utilize G. mellonella caterpillars to study host-pathogen interactions with the gram-negative organism Acinetobacter baumannii and determine the utility of this infection model to study antibacterial efficacy. After infecting G. mellonella caterpillars with a reference A. baumannii strain, we observed that the rate of G. mellonella killing was dependent on the infection inoculum and the incubation temperature postinfection, with greater killing at 37°C than at 30°C (P ‫؍‬ 0.01). A. baumannii strains caused greater killing than the less-pathogenic species Acinetobacter baylyi and Acinetobacter lwoffii (P < 0.001). Community-acquired A. baumannii caused greater killing than a reference hospital-acquired strain (P < 0.01). Reduced levels of production of the quorum-sensing molecule 3-hydroxy-C 12 -homoserine lactone caused no change in A. baumannii virulence against G. mellonella. Treatment of a lethal A. baumannii infection with antibiotics that had in vitro activity against the infecting A. baumannii strain significantly prolonged the survival of G. mellonella caterpillars compared with treatment with antibiotics to which the bacteria were resistant. G. mellonella is a relatively simple, nonmammalian model system that can be used to facilitate the in vivo study of host-pathogen interactions in A. baumannii and the efficacy of antibacterial agents.

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Pseudomonas aeruginosa is a leading cause of nosocomial infections. The risk of emergence of antibiotic resistance may vary with different antibiotic treatments. To compare the risks of emergence of resistance associated with four antipseudomonal agents, ciprofloxacin, ceftazidime, imipenem, and piperacillin, we conducted a cohort study, assessing relative risks for emergence of resistantP. aeruginosa in patients treated with any of these drugs. A total of 271 patients (followed for 3,810 days) with infections due to P. aeruginosa were treated with the study agents. Resistance emerged in 28 patients (10.2%). Adjusted hazard ratios for the emergence of resistance were as follows: ceftazidime, 0.7 (P = 0.4); ciprofloxacin, 0.8 (P = 0.6); imipenem, 2.8 (P = 0.02); and piperacillin, 1.7 (P = 0.3). Hazard ratios for emergence of resistance to each individual agent associated with treatment with the same agent were as follows: ceftazidime, 0.8 (P = 0.7); ciprofloxacin, 9.2 (P = 0.04); imipenem, 44 (P = 0.001); and piperacillin, 5.2 (P= 0.01). We concluded that there were evident differences among antibiotics in the likelihood that their use would allow emergence of resistance in P. aeruginosa. Ceftazidime was associated with the lowest risk, and imipenem had the highest risk.