Project

Staphylococcus aureus; low-level glycopeptide resistance; molecular mechanisms; detection of resistance phenotypes; glycopeptides; GISA; teicoplanin

Lead
Lay summary
Methicillin-resistant Staphylococcus aureus (MRSA) are major pathogens of hospital-acquired and community-acquired infections. Most MRSA-infected patients receive glycopeptide antibiotics, vancomycin or teicoplanin, which are "the drugs of last resort" for multi-resistant isolates. The therapeutic efficacy of vancomycin (or teicoplanin) is widely debated today, and there is a growing concern that their intensive use may promote a stepwise increase in glycopeptide resistance levels in hospital-acquired MRSA isolates. Emergence of low-level glycopeptide resistance in S. aureus (designated under the acronym of GISA) is currently viewed as the progressive selection of subpopulations of GISA precursors by long-term glycopeptide therapy, eventually leading to homogeneous GISA populations. The phenotypic detection of resistance in GISA, which frequently display cell wall thickening, is problematic. The molecular mechanisms of resistance in GISA are also not well understood, but seem to be linked with multiple genomic changes altering in an unknown manner transcription of several cell-wall regulation pathways. Using transcription profiling and genetic methods, we identified two novel genes playing a key role in emergence and expression of glycopeptide resistance in S. aureus. In addition, an innovative molecular strategy was developed to identify a very limited number of mutations potentially linked with expression of glycopeptide resistance. In this approach, comparison by whole genomic sequencing of GISA and non-GISA clonally-related isolates was combined with a systematic genetic approach, where all steps leading to glycopeptide resistance were reconstituted. We also demonstrated that a precise mutation in the bacterial cell-membrane sensor VraS could strongly inhibit the emergence of teicoplanin-resistant and vancomycin-resistant mutants, which suggests that pharmacological blockade of the VraS sensor might be used for preventing emergence of glycopeptide resistance in S. aureus. Finally, we recently developed and validated an improved phenotypic assay for detection of GISA isolates, which shows promising applications for predicting increased risk of glycopeptide therapy failure in patients with bacteremic and orthopedic MRSA infections. The development and validation of molecular and phenotypic assays for detection of glycopeptide-resistant MRSA isolates may be essential for optimizing antibiotic treatment protocols and infection control programs against multi-resistant MRSA. Improved understanding of the molecular mechanisms of glycopeptide resistance may help to unravel novel targets for development of new antibiotics.

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Last update: 21.02.2013

Active participation

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1.Summary of Research PlanBackground: Methicillin-resistant Staphylococcus aureus (MRSA) are major pathogens of hospital-acquired and community-acquired infections. Most MRSA-infected patients receive intravenously administered glycopeptide antibiotics, vancomycin or teicoplanin, which are “the drugs of last resort” for multi-resistant isolates. The therapeutic efficacy of vancomycin (or teicoplanin) is widely debated today, and there is a growing concern that their intensive use may eventually lead to the selection of MRSA isolates displaying increasing resistance to glycopeptides. Indeed, since 1997, low-level glycopeptide-resistant clinical isolates have been detected, designated as glycopeptide-intermediate S. aureus (GISA). However the molecular mechanisms underlying the GISA phenotype are still poorly understood. The potential underestimated emergence of GISA clinical isolates represents a special risk, because their phenotypic detection is difficult and no molecular assay for detecting such resistance is available. Working Hypothesis: Emergence of glycopeptide-resistant clones likely results from spontaneous mutations selected by antibiotic pressure. A number of candidate genes were identified as contributing to glycopeptide resistance, but their mode of action at the molecular level is not well understood. The recent discovery by our laboratory of two novel, teicoplanin resistance-contributing genes (trfA and trfB), whose inactivation could fully restore glycopeptide susceptibility, might help to elucidate the molecular basis of the GISA phenotype. Furthermore, the homology of S. aureus TrfA and TrfB with competence genes MecA1 and YjbF of B. subtilis should help to explore the relationships between glycopeptide resistance and putative competence pathway(s) of S. aureus.Specific Aims: We will explore the contribution of trfA and trfB (whose natural function in S. aureus is still unknown) to the expression and development of resistance to glycopeptides and other cell wall-active antibiotics in S. aureus laboratory and clinical isolates. Part A will analyze the potential links between glycopeptide resistance and putative competence regulatory circuits, decipher additional metabolic functional role(s) of trfA and trfB, study the transcriptional and translational regulation of both trfA and trfB genes and their interactions with other glycopeptide-resistance regulatory pathways. Molecular studies on the role of trfA and trfB in glycopeptide resistance will also be performed in clinically relevant MRSA isolates. Part B aims to implement reliable, phenotypic assays for detection of decreased glycopeptide susceptibility, on panels of S. aureus laboratory and MRSA clinical (from bloodstream-infected patients) isolates. Experimental Design and/or Methods:Part A: The potential links between glycopeptide resistance and putative competence regulatory circuits will be evaluated by inactivation of competence-related genes (ClpC, ComK), as well as identifying TrfA potential protein partners by ELISA and pull down assays. We will compare the transcriptional profiling of teicoplanin-resistant isolates with their trfA or/and trfB mutants and analyze their phenotypic properties by phenotypic microarrays (Biolog technology). TrfA and trfB transcription patterns will be monitored by real-time RT-PCR, northern blot and Smart-RACE technique, and expression of TrfA and TrfB proteins by Western blotting using specific rabbit anti-TrfA and TrfB antibodies. Real-time RT-PCR will be used to study the regulatory role of the global regulator vraR. Finally, we will generate trfAB mutations in different MRSA-GISA strain backgrounds and test their impact on glycopeptide and oxacillin resistance. Part B: A novel method, combining glycopeptide macrodilution MIC with a simplified population analysis assay on glycopeptide-containing agar, has been recently developed. This novel assay is currently validated on a large panel of MRSA isolates from bacteremic patients. Detailed molecular genetic studies will also be performed on eight clonally-related pairs of consecutive MRSA isolates, whose vancomycin and/or teicoplanin MICs increased during therapy of persistent or recurrent, bloodstream infectious episodes. Expected Value of the Proposed Project: Improved understanding of the molecular mechanisms of glycopeptide resistance may help to: (i) optimize care of patients treated with glycopeptides; (ii) estimate the risk of cross-resistance to currently developed glycopeptide derivatives; (iii) unravel novel targets for development of new antibiotics; (iii) develop molecular screening assays for detection of resistant isolates. More reliable methods for GISA detection may be essential for infection control programs targeting antibiotic-resistant pathogens.