Low-level glycopeptide resistance; Molecular mechanisms; Markers for glycopeptide resistance; Staphylococcus aureus; Cross-resistance
Andrey D O, François P, Manzano C, Bonetti E J, Harbarth S, Schrenzel J, Kelley W L, Renzoni A (2017), Antimicrobial activity of ceftaroline against methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 2013-2014 at the Geneva University Hospitals., in European journal of clinical microbiology & infectious diseases : official publication of the Europe
, 36(2), 343-350.
Renzoni A., Kelley W. L., Martinez M.P., Fatouraei M, Paz L., Fernandez R, Rosato R.R., Margollin W., Foster S. J., Rosato A.E. (2017), Molecular Bases Determining Daptomycin Resistance-Mediated Resensitization to β-Lactams (Seesaw Effect) in Methicillin-Resistant Staphylococcus aureus., in Antimicrobial Agents and Chemotherapy
Villanueva Maite, Jousselin Ambre, Baek Kristoffer T, Prados Julien, Andrey Diego O, Renzoni Adriana, Ingmer Hanne, Frees Dorte, Kelley William L (2016), Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus., in Journal of bacteriology
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Kelley William L, Jousselin Ambre, Barras Christine, Lelong Emmanuelle, Renzoni Adriana (2015), Missense mutations in PBP2A Affecting ceftaroline susceptibility detected in epidemic hospital-acquired methicillin-resistant Staphylococcus aureus clonotypes ST228 and ST247 in Western Switzerland archived since 1998., in Antimicrobial agents and chemotherapy
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Jousselin Ambre, Manzano Caroline, Biette Alexandra, Reed Patricia, Pinho Mariana G, Rosato Adriana E, Kelley William L, Renzoni Adriana (2015), The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A., in Antimicrobial agents and chemotherapy
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Roch Mélanie, Clair Perrine, Renzoni Adriana, Reverdy Marie-Elisabeth, Dauwalder Olivier, Bes Michèle, Martra Annie, Freydière Anne-Marie, Laurent Frédéric, Reix Philippe, Dumitrescu Oana, Vandenesch François (2014), Exposure of Staphylococcus aureus to subinhibitory concentrations of β-lactam antibiotics induces heterogeneous vancomycin-intermediate Staphylococcus aureus., in Antimicrobial agents and chemotherapy
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1.Summary of Research PlanBackground: Methicillin-resistant Staphylococcus aureus (MRSA) are major pathogens of hospital infections and are associated with high risks of mortality and complications. Despite the recent introduction of antimicrobial agents, glycopeptide antibiotics (vancomycin or teicoplanin) remain the first-line therapy for severely MRSA-infected patients. Their intensive use leads to the selection of low-level glycopeptide-resistant isolates, designated as glycopeptide-intermediate S. aureus (GISA). The therapeutic efficacy of glycopeptides is widely debated, and there is a growing concern that their use will select resistance, not only to glycopeptides but also to daptomycin via an unknown cross-resistance mechanism. The emergence of GISA clinical isolates during antimicrobial therapy represents a special risk, because their phenotypic detection is frequently difficult and no reliable molecular assay for detecting such resistance is available. Working Hypothesis: Development of the GISA phenotype is currently viewed as a stepwise, endogenous process, characterized by the sequential occurrence and glycopeptide-mediated selection of resistance-conferring mutations. Genetic changes linked with early steps of GISA development lead to low-level glycopeptide resistance as detected by standard phenotypic assays. Since reduced susceptibility to teicoplanin seems to occur before emergence of vancomycin low-level resistance, detection of teicoplanin resistance may be a useful early marker of GISA emergence. Specific Aims: Part A. By a whole-genome sequencing approach, we plan to identify the prevalence of genes essential for the stepwise development of low-level glycopeptide resistance in panels of bacteremic and orthopaedic clinical isolates that developed the GISA phenotype during glycopeptide therapy. We also plan to build a preliminary molecular-detection method of GISA phenotype by a multi-gene expression analysis. Part B. Exploring the function of the newly identified glycopeptide resistance genes trfA and prsA should help to design an integrated molecular model describing the stepwise development of low-level glycopeptide resistance. Experimental Design and/or Methods: Part A. Whole genome sequencing will be done in collaboration with Fasteris SA, to identify the most frequently GISA altered genes in clinical strains. For each identified change we will further validate by genetic means its association with the glycopeptide resistant phenotype. Following validation, the prevalence of each mutation in GISA strains will be evaluated. This analysis should hopefully lead to identification of genetic changes contributing preferentially to a teicoplanin resistance phenotype compared to those that may concomitantly lead to a vancomycin resistance phenotype. Furthermore, we will test the value of previously identified glycopeptide resistance genes for the development of a molecular-based detection method for glycopeptide resistance emergence. We will analyse expression of selected candidate genes by RT-PCR, first in a cohort of non-GISA strains to establish baseline levels of expression. Following the baseline determination, we will analyse expression of candidate genes using a cohort of NARSA GISA strains. Part B. Based on homologies with B. subtilis, molecular functions of both trfA and prsA genes are predicted to interact with other cellular proteins. We plan to identify their corresponding molecular partners using biochemical techniques such as ELISA, ligand overlay and pull down assays. Furthermore, the TrfA or PrsA-mediated effects on cell wall ultrastructure will be measured by transmission electron microscopy, zymograms, Triton-X susceptibility profiles and high performance liquid chromatography. The effect of TrfA and PrsA on extracellular cell-wall related protein secretion will be evaluated by proteomic analysis of extracellular protein profiles using nano-liquid chromatography and mass spectroscopy.Expected Value of the Proposed Project: (i) Develop and validate genetic and phenotypic assays for detection of glycopeptide-resistant MRSA isolates. Improved GISA detection may be essential for infection control programs targeting antibiotic-resistant pathogens. (ii) Optimize care of patients treated with glycopeptides; (iii) Estimate the risk of cross-resistance to daptomycin. (iv) Improve understanding of the molecular mechanisms of glycopeptide resistance may help to unravel novel targets for development of new antibiotics.