-
[show abstract]
[hide abstract]
ABSTRACT: The genetic mechanisms that contribute to reduced susceptibility to vancomycin in Staphylococcus aureus are complex and heterogeneous. In addition, debate is emerging as to the true effect of reduced susceptibility to vancomycin on staphylococcal virulence. To investigate this, comparative genomics was performed on a collection of vancomycin-exposed isogenic S. aureus pairs (14 strains in total). Previously described mutations were observed in genes such as vraG, agrA, yvqF, and rpoB; however, a new mechanism was identified involving a serine/threonine phosphatase, Stp1. After constructing an stp1 deletion mutant, we showed that stp1 is important in vancomycin susceptibility and cell wall biosynthesis. Gene expression studies showed that stp1 also regulates virulence genes, including a hemolysin, superantigen-like protein, and phenol-soluble modulin, and that the deletion mutant is attenuated in virulence in vivo. Stp1 provides a new link between vancomycin susceptibility and virulence in S. aureus.
The Journal of Infectious Diseases 04/2012; 205(11):1677-87. · 6.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.
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.
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.
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.
PLoS ONE 01/2012; 7(1):e28316. · 4.09 Impact Factor
-
The Journal of Infectious Diseases 01/2010; 201(2):312-313. · 6.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: With the current high prevalence of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) strains but in light of the general belief that beta-lactam antibiotics are more effective than vancomycin against infections caused by methicillin-susceptible S. aureus (MSSA) isolates, clinicians may utilize antistaphylococcal penicillins in combination with vancomycin for the empirical treatment of S. aureus infections. Vancomycin is considered to kill MSSA more slowly than oxacillin. Thus, we sought to evaluate the interaction of the combination of oxacillin and vancomycin on bacterial killing in vitro. Ten clinical isolates of MSSA isolated in the year 2000 were investigated. The killing observed at 24 h by vancomycin at 20 microg/ml, oxacillin at 16 microg/ml, or the combination did not differ (approximately 2.5 to 3.5 log10 CFU/ml). In a separate experiment, we assessed bacterial killing in a dynamic model simulating the free plasma concentration profiles expected following the administration of a combination of vancomycin at 1 g every 12 h and oxacillin at 1 g every 6 h. The time-kill profiles of these regimens against S. aureus ATCC 29213 were comparable to those observed in the fixed-concentration experiments. Using these methods, we found no evidence that vancomycin antagonized the bactericidal effect of oxacillin or that there was any benefit from use of the combination.
Antimicrobial Agents and Chemotherapy 11/2009; 54(2):773-7. · 4.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Most cases of reduced vancomycin susceptibility in Staphylococcus aureus reported in the literature have been in methicillin-resistant strains. We report the development of reduced vancomycin susceptibility in a series of clonally related, methicillin-susceptible S. aureus (MSSA) clinical isolates. This isogenic series permitted us to determine whether the evolution of reduced vancomycin susceptibility in MSSA is similar to that seen in MRSA.
Differences in vancomycin population analysis profiles; chemical autolysis; vancomycin, oxacillin, and daptomycin minimum inhibitory concentrations; and bactericidal activities were examined.
Progressive vancomycin resistance correlated with increasing daptomycin nonsusceptibility. Chemical autolysis and the bactericidal activity of vancomycin, oxacillin, and daptomycin were reduced in the final, vancomycin-intermediate S. aureus isolate, compared with the vancomycin-susceptible MSSA progenitor.
Clinicians should recognize that reduced vancomycin susceptibility can occur in S. aureus irrespective of background methicillin susceptibility and that development of intermediate vancomycin susceptibility in MSSA may result in increased tolerance to several classes of anti-staphylococcal antibiotics.
Clinical Infectious Diseases 10/2009; 49(8):1169-74. · 9.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments.
Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994, we determined the multilocus sequence type; the presence of 16 putative virulence genes (hyl(Efm), esp(Efm), and fms genes); resistance to ampicillin (AMP) and vancomycin (VAN); and high-level resistance to gentamicin and streptomycin.
Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the United States. The earliest CC17 isolates were part of an outbreak that occurred in 1982 in Richmond, Virginia. The characteristics of CC17 isolates included increases in resistance to AMP, the presence of hyl(Efm) and esp(Efm), emergence of resistance to VAN, and the presence of at least 13 of 14 fms genes. Eight of 41 of the early isolates with resistance to AMP, however, were not in CC17.
Although not all early US AMP isolates were clonally related, E. faecium CC17 isolates have been circulating in the United States since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment.
The Journal of Infectious Diseases 10/2009; 200(10):1566-73. · 6.41 Impact Factor
-
George M Eliopoulos
[show abstract]
[hide abstract]
ABSTRACT: Several new antimicrobials demonstrate in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and other Gram-positive bacteria. Data from large surveys indicate that linezolid, daptomycin, and tigecycline are almost universally active against MRSA. Linezolid and tigecycline inhibit both Enterococcus faecium and Enterococcus faecalis at low concentrations; daptomycin is somewhat more potent against the latter. The investigational agents dalbavancin and telavancin are more potent than vancomycin against vancomycin-susceptible organisms. Dalbavancin inhibits vanB type VRE at low concentrations, but is not active against vanA type VRE. Telavancin is less active against VRE than against vancomycin-susceptible enterococci, but minimum inhibitory concentrations are lower than those of vancomycin against VRE. With continued careful use of available antimicrobials, the vast majority of these organisms should remain susceptible to 1 or more of the agents discussed for the foreseeable future.
The Journal of infection 09/2009; 59 Suppl 1:S17-24. · 4.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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 degrees C than at 30 degrees 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.
Antimicrobial Agents and Chemotherapy 04/2009; 53(6):2605-9. · 4.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In the present study, we demonstrated the utility of the nonmammalian model system Galleria mellonella for studying the pathogenesis of Staphylococcus aureus infection. By use of clinical and laboratory strains that had been exposed to vancomycin, we showed that both agr functional status and vancomycin minimum inhibitory concentration are determinants associated with the virulence of S. aureus in G. mellonella. These results show that G. mellonella can be effectively used to facilitate the in vivo study of S. aureus virulence and, more specifically, the relationship between antibiotic drug resistance and the pathogenesis of infection.
The Journal of Infectious Diseases 02/2009; 199(4):532-6. · 6.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Proceedings of the National Academy of Sciences 10/2008; 105(38):14585-90. · 9.68 Impact Factor
-
Zukile Gqada,
Ben Marais,
Elizabeth Wasserman,
Colleen Bamford,
Heidi Orth,
George Sakoulas,
Warren Rose,
Michael J Rybak,
Satish Pillai,
Jeff Alder,
Robert C Moellering, George M Eliopoulos
Journal of clinical microbiology 08/2008; 46(7):2471; author reply 2471-2. · 4.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We examined sequential methicillin-susceptible Staphylococcus aureus isolates from a patient with mitral valve endocarditis recovered during persistent bacteremia on standard therapy and relapse after treatment with daptomycin. An isolate obtained after 5 days of antimicrobial therapy, but before exposure to daptomycin, showed subtle physiological changes in response to daptomycin, with significant regrowth in the daptomycin killing assay compared to the treatment-naive strain. Once daptomycin was started, the population became more heterogeneous and tested as nonsusceptible. These organisms were examined in a simulated-vegetation in vitro pharmacodynamic model, which confirmed progressive decreases in killing with daptomycin concentrations that simulate those attained in humans with 6-mg/kg of body weight daily dosing. Early surgical intervention or combination therapy or both might have prevented the loss of daptomycin susceptibility.
Journal of clinical microbiology 02/2008; 46(1):220-4. · 4.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The G2576T mutation in domain V of 23S rRNA has been most often associated with the rare cases of linezolid resistance in Staphylococcus aureus. In a linezolid-susceptible S. aureus (A8761B) possessing a single mutated (G2576T) copy, originally derived from a resistant clinical isolate, we assessed the persistence of the mutation on further passage on antibiotic-free medium and the selection of resistance upon re-exposure of the susceptible strain to linezolid.
The stability of the mutant rRNA copy was tested through 40 serial passages on antibiotic-free medium. The re-emergence of linezolid-resistant mutants was examined after serial passage on successively increasing linezolid concentrations. The efficacy of novobiocin, at subinhibitory concentrations, to prevent or delay the emergence of resistant mutants was examined. Strain relatedness was confirmed by PFGE and domain V of individual rRNA copies was sequenced.
After 40 passages in antibiotic-free medium, the linezolid MIC of derived strain A9584 remained stable at 2 mg/L and the G2576T mutation persisted in one 23S rRNA gene copy (copy number 2). Upon re-exposure of the strain to increasing concentrations of linezolid, linezolid resistance (MIC of 64 mg/L) emerged rapidly. In a representative derivative (A9753), the G2576T mutation was found in four of the five rRNA copies. All laboratory derivates were closely related by PFGE. When A9584 was applied to plates containing linezolid at 4 x MIC, resistant colonies emerged at a frequency of 8 x 10(-6). Novobiocin at 1/4 x MIC prevented the emergence of resistant colonies.
The persistence of the G2576T mutation in one rRNA operon copy in the absence of selective pressure suggests that the mutation has a minimal impact on the organism's fitness in vitro. Resistance to linezolid, associated with acquisition of multiple mutant copies, emerges rapidly upon re-exposure to linezolid. Novobiocin, predicted to interfere with gene conversion, may reduce the likelihood of rapid development of linezolid resistance.
Journal of Antimicrobial Chemotherapy 10/2007; 60(3):649-51. · 5.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A previous study documented the presence of mutations in mprF that accompanied the loss of daptomycin susceptibility among Staphylococcus aureus isolates following exposure to the drug. An association between the development of glycopeptide-intermediate S. aureus and daptomycin nonsusceptibility has also been recently described. We report that among three clinical S. aureus isolates which developed vancomycin heteroresistance, as well as daptomycin nonsusceptibility despite a lack of exposure to this drug, there were no mutations resulting in amino acid substitutions in MprF.
Antimicrobial Agents and Chemotherapy 06/2007; 51(6):2223-5. · 4.84 Impact Factor
-
George M Eliopoulos
Current Infectious Disease Reports 10/2006; 8(5):333-6.
-
[show abstract]
[hide abstract]
ABSTRACT: We studied vancomycin and daptomycin susceptibility in methicillin-resistant Staphylococcus aureus from patients exposed to vancomycin, glycopeptide-intermediate S. aureus, and S. aureus passaged in vancomycin-containing medium. A correlation between vancomycin and daptomycin heteroresistance was noted in some strains, suggesting that exposure of S. aureus to vancomycin may affect susceptibility to daptomycin.
Antimicrobial Agents and Chemotherapy 05/2006; 50(4):1581-5. · 4.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To evaluate microbiological properties of methicillin-resistant Staphylococcus aureus (MRSA) during prolonged vancomycin therapy.
We evaluated vancomycin susceptibility and heteroresistance, accessory gene regulator (agr) function, autolysis, biofilm production and in vitro vancomycin killing in serial MRSA bloodstream isolates obtained over a 30 month period from a patient with a chronic endovascular infection.
Despite the fact that the MRSA in this patient had the same genetic background as other clinical glycopeptide intermediate-resistant S. aureus (GISA) isolates, vancomycin administered for 9 months, maintaining serum concentrations >10 mg/L, did not select for GISA. Minimal changes in vancomycin susceptibility were detected using agar dilution and population analysis methods. We noted increases in delta haemolysin production, autolysis and the bactericidal effects of vancomycin in vitro against the MRSA obtained after prolonged vancomycin suppressive therapy was discontinued.
Despite the lack of development of detectable resistance, MRSA exposed to vancomycin for prolonged periods may begin to develop vancomycin tolerance and decreased autolysis. In addition, suppression of agr function appears to end after vancomycin is stopped. Whether these changes are prerequisites for attenuated vancomycin efficacy and the development of glycopeptide resistance warrants further study. The development of vancomycin resistance may be more difficult under conditions where vancomycin serum concentrations are maintained >10 mg/L.
Journal of Antimicrobial Chemotherapy 04/2006; 57(4):699-704. · 5.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: For the past 2 decades, vancomycin has served as the cornerstone of therapy against serious methicillin-resistant Staphylococcus aureus infections. This role is increasingly challenged by questions of efficacy, including reduced efficacy against infections caused by glycopeptide-intermediate S. aureus strains. In an evaluation of clinical glycopeptide-intermediate S. aureus isolates and serial, clinical methicillin-resistant S. aureus isolates obtained from patients receiving vancomycin for the treatment of bacteremia, we found that loss of function of the accessory gene regulator operon may confer a survival advantage to S. aureus under vancomycin selection pressure, particularly in strains with the accessory gene regulator group II genotype. Other advantages in a nosocomial setting may include enhancement of biofilm formation and promotion of physiologic changes supporting colonization. We conclude that loss of accessory gene regulator function in methicillin-resistant S. aureus might, in part, explain the decreased efficacy of vancomycin in the therapy of methicillin-resistant S. aureus bacteremia, thus highlighting the need to reevaluate the criteria of susceptibility to vancomycin.
Clinical Infectious Diseases 02/2006; 42 Suppl 1:S40-50. · 9.15 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Loss of agr function, vancomycin exposure, and abnormal autolysis have been linked with both development of the GISA phenotype and low-level resistance in vitro to thrombin-induced platelet microbicidal proteins (tPMPs). We examined the potential in vitro interrelationships among these parameters in well-characterized, isogenic laboratory-derived and clinical Staphylococcus aureus isolates. The laboratory-derived S. aureus strains included RN6607 (agrII-positive parent) and RN6607V (vancomycin-passaged variant; hetero-GISA), RN9120 (RN6607 agr::tetM; agr II knockout parent), RN9120V (vancomycin-passaged variant), and RN9120-GISA (vancomycin passaged, GISA). Two serial isolates from a vancomycin-treated patient with recalcitrant, methicillin-resistant S. aureus (MRSA) endocarditis were also studied: A5937 (agrII-positive initial isolate) and A5940 (agrII-defective/hetero-GISA isolate obtained after prolonged vancomycin administration). In vitro tPMP susceptibility phenotypes were assessed after exposure of strains to either 1 or 2 mug/ml. Triton X-100- and vancomycin-induced lysis profiles were determined spectrophotometrically. For agrII-intact strain RN6607, vancomycin exposure in vitro was associated with modest increases in vancomycin MICs and reduced killing by tPMP, but no change in lysis profiles. In contrast, vancomycin exposure of agrII-negative RN9120 yielded a hetero-GISA phenotype and was associated with defects in lysis and reduced in vitro killing by tPMP. In the clinical isolates, loss of agrII function during prolonged vancomycin therapy was accompanied by emergence of the hetero-GISA phenotype and reduced tPMP killing, with no significant change in lysis profiles. An association was identified between loss of agrII function and the emergence of hetero-GISA phenotype during either in vitro or in vivo vancomycin exposure. In vitro, these events were associated with defective lysis and reduced susceptibility to tPMP. The precise mechanism(s) underlying these findings is the subject of current investigations.
Antimicrobial Agents and Chemotherapy 08/2005; 49(7):2687-92. · 4.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Linezolid resistance in rare isolates of Staphylococcus aureus has been associated with G2576T mutations in domain V of the 23S rRNA gene. We report the analysis of a clinical S. aureus isolate that developed linezolid resistance (MIC of linezolid of 12 mg/L) after a 25 day course of the drug. Sequencing identified G2576T mutations in four of the five copies of the 23S rRNA gene.
To examine the stability of this resistance, we serially passaged this original isolate 60 times over a 75 day period on antimicrobial-free medium.
After 30 passages, the MIC of linezolid had decreased to 8 mg/L and only two of the five copies of the 23S rRNA gene contained the G2576T mutation. After 60 passages, the MIC of linezolid fell to 2 mg/L and only one of the five 23S rRNA gene copies contained the mutation. The original and two passaged staphylococci were indistinguishable by pulsed-field gel electrophoresis.
In the absence of antibiotic pressure, linezolid resistance was unstable in a clinical isolate that did not have all copies of the 23S rRNA gene mutated, although a single copy of mutant rDNA was maintained. Gene conversion was probably the mechanism for this reversion, using the wild-type 23S rRNA gene sequences to replace the G2576T mutation by homologous recombination.
Journal of Antimicrobial Chemotherapy 11/2004; 54(4):818-20. · 5.07 Impact Factor
