Robert C. Moellering

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.

Relationship of MIC and Bactericidal Activity to Efficacy of Vancomycin for Treatment of Methicillin-Resistant Staphylococcus aureus Bacteremia

ABSTRACT 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 μg/ml versus 1.0 to 2.0 μg/ml; P = 0.02) and degree of killing (reduction in 1og10 CFU/milliliter) by vancomycin over 72 h of incubation in vitro (P = 0.03). For MRSA isolates with vancomycin MICs ≤ 0.5 μg/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 μg/ml. Patients with MRSA that was more effectively killed at 72 h by vancomycin in vitro had a higher clinical success rate with vancomycin therapy in the treatment of bacteremia (log10 < 4.71 [n = 9], 0%; log10 4.71 to 6.26 [n = 13], 23.1%; log10 > 6.27 [n = 8], 50%). We conclude that a significant risk for vancomycin treatment failure in MRSA bacteremia begins to emerge with increasing vancomycin MICs well within the susceptible range. Elucidating the mechanisms involved in intermediate-level glycopeptide resistance in S. aureus should begin by examining bacteria that begin to show changes in vancomycin susceptibility before the development of obvious resistance. Prognostic information for vancomycin treatment outcome in MRSA bacteremia may also be obtained by testing the in vitro bactericidal potency of vancomycin.

Vancomycin Therapeutic Guidelines: A Summary of Consensus Recommendations from the Infectious Diseases Society of America, the American Society of Health-System Pharmacists, and the Society of Infectious Diseases Pharmacists

Practice guidelines for therapeutic monitoring of vancomycin treatment for Staphylococcus aureus infection in adult patients were reviewed by an expert panel of the Infectious Diseases Society of America, the American Society of Health-System Pharmacists, and the Society of Infectious Diseases Pharmacists. A literature review of existing evidence regarding vancomycin dosing and monitoring of serum concentrations, in addition to patient outcomes combined with expert opinion regarding the drugs pharmacokinetic, pharmacodynamic, and safety record, resulted in new recommendations for targeting and adjustment of vancomycin therapy.

Outbreak of ceftazidime resistance caused by extended-spectrum beta-lactamases at a Massachusetts chronic-care facility.

During a 4-month period in late 1988, we isolated ceftazidime-resistant strains of Klebsiella pneumoniae and other members of the family Enterobacteriaceae from 29 patients at a chronic-care facility in Massachusetts. Ceftazidime resistance resulted from two distinct extended-spectrum beta-lactamases of the TEM type which efficiently hydrolyzed the cephalosporin: YOU-1 with a pI of 5.57 and YOU-2 with a pI of 5.2. Genes encoding these enzymes were present on different but closely related high-molecular-weight, multiple antibiotic resistance plasmids of the H12 incompatibility group and were transferable by conjugation in vitro. Agarose gel electrophoresis of extracts from clinical isolates indicated that this outbreak arose from plasmid transmission among different strains of the family Enterobacteriaceae rather than from dissemination of a single resistant isolate. Isolation rates of ceftazidime-resistant organisms transiently decreased after use of this drug was restricted, but resistant isolates continued to be recovered 7 months after empiric use of ceftazidime ceased. Images

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.

Increasing resistance to beta-lactam antibiotics among clinical isolates of Enterococcus faecium: a 22-year review at one institution.

To identify any change in the antibiotic resistance of Enterococcus faecium, we examined the antibiotic susceptibilities of clinical strains (n = 84) isolated at one institution during the 22 years since 1968. A significant increase in resistance to penicillin was observed during the study period: the MICs of penicillin for 50 and 90% of isolates tested were 16 and 64 micrograms/ml, respectively, from 1969 to 1988 (n = 48; geometric mean MIC, 14 micrograms/ml) , whereas they were 256 and 512 micrograms/ml, respectively, from 1989 to 1990 (n = 36; geometric mean MIC, 123 micrograms/ml) (P less than 0.001). A comparable increase in resistance to ampicillin was also noted (P less than 0.001). No strains produced detectable beta-lactamase. In contrast, susceptibilities to vancomycin, teicoplanin, and ciprofloxacin remained stable. High-level resistance to gentamicin was observed in none of 48 isolates from 1969 to 1988, but was present in 22 of 36 strains (61%) from 1989 to 1990 (P less than 0.001) and was significantly associated with resistance (MIC, greater than or equal to 128 micrograms/ml) to penicillin (P less than 0.001). To assess the potential evolution of antibiotic resistance in this species, clinical isolates (n = 24) were compared with strains isolated in 1968 from a human population in the Solomon Islands that was never exposed to antibiotics. Solomon Island isolates were significantly more susceptible than all clinical strains to penicillin, ampicillin, and vancomycin (P less than 0.001 for each), but they exhibited no differences in susceptibility to teicoplanin or ciprofloxacin. The penicillin-binding affinity of penicillin-binding protein 5 (PBP 5) in penicillin-resistant clinical strains (MIC, 512 micrograms/ml) was notably lower than that in strains with more typical susceptibilities, suggesting an alteration in this PBP as a possible mechanism for increased penicillin resistance. Solomon Island strains most susceptible to penicillin demonstrated a prominent PBP 5* and the absence of PBP 5. These changes in the antibiotic resistance of E. faecium emphasize the importance of identifying this species in patients with serious enterococcal infections and the necessity of assessing its susceptibility to both beta-lactams and aminoglycosides if effective therapy is to be identified. Images

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.

Whole Genome Characterization of the Mechanisms of Daptomycin Resistance in Clinical and Laboratory Derived Isolates of Staphylococcus aureus

Background Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus. Methods Whole 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. Results On 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. Conclusion Point 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.

Prokaryote–eukaryote interactions identified by using Caenorhabditis elegans

Prokaryote–eukaryote interactions are ubiquitous and have important medical and environmental significance. Despite this, a paucity of data exists on the mechanisms and pathogenic consequences of bacterial–fungal encounters within a living host. We used the nematode Caenorhabditis elegans as a substitute host to study the interactions between two ecologically related and clinically troublesome pathogens, the prokaryote, Acinetobacter baumannii, and the eukaryote, Candida albicans. After co-infecting C. elegans with these organisms, we observed that A. baumannii inhibits filamentation, a key virulence determinant of C. albicans. This antagonistic, cross-kingdom interaction led to attenuated virulence of C. albicans, as determined by improved nematode survival when infected with both pathogens. In vitro coinfection assays in planktonic and biofilm environments supported the inhibitory effects of A. baumannii toward C. albicans, further showing a predilection of A. baumannii for C. albicans filaments. Interestingly, we demonstrate a likely evolutionary defense by C. albicans against A. baumannii, whereby C. albicans inhibits A. baumannii growth once a quorum develops. This counteroffensive is at least partly mediated by the C. albicans quorum-sensing molecule farnesol. We used the C. elegans–A. baumannii–C. albicans coinfection model to screen an A. baumannii mutant library, leading to the identification of several mutants attenuated in their inhibitory activity toward C. albicans. These findings present an extension to the current paradigm of studying monomicrobial pathogenesis in C. elegans and by use of genetic manipulation, provides a whole-animal model system to investigate the complex dynamics of a polymicrobial infection.

In vitro activity of CP-99,219, a new fluoroquinolone, against clinical isolates of gram-positive bacteria.

The in vitro activity of the fluoroquinolone CP-99,219 against gram-positive bacteria was compared with those of five other antimicrobial agents. Against ciprofloxacin-susceptible staphylococci and against streptococci, MICs were < or = 0.12 and < or = 0.5 microgram/ml, respectively. CP-99,219 was also more active than ciprofloxacin against ciprofloxacin-resistant staphylococci, most enterococci, Leuconostoc spp., and lactobacilli.