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Identification of RNA signatures in Staphylococcus aureus to detect low-level glycopeptide resistance (hVISA and VISA): an integration of clinical and basic research

English title Identification of RNA signatures in Staphylococcus aureus to detect low-level glycopeptide resistance (hVISA and VISA): an integration of clinical and basic research
Applicant Renzoni Adriana
Number 169404
Funding scheme Project funding
Research institution Service des Maladies Infectieuses Département de Médecine Interne Hôpital Cantonal - HUG
Institution of higher education University of Geneva - GE
Main discipline Infectious Diseases
Start/End 01.03.2017 - 31.08.2020
Approved amount 444'083.00
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Keywords (4)

Molecular mechanisms; Markers for glycopeptide resistance; Staphylococcus aureus; Low-level glycopeptide resistance

Lay Summary (French)

Lead
Staphylocoque aureus est un agent infectieux. Ces infections sont traitées par différents antibiotiques, tels que les glycopeptides, les ß-lactamines et la daptomycine. Nous observons que leur utilisation mène, à la résistance à chaque antibiotique mais aussi à l'apparition d’une résistance croisée entre antibiotiques. Les glycopeptides restent encore la principale thérapie pour traiter S. aureus méthicilline-résistant. Leur utilisation mène à la sélection des bactéries résistantes de bas niveau appelées VISA et hVISA.
Lay summary

L’apparition de ces bactéries résistantes VISA/hVISA pendant le traitement antibiotique est risqué parce que : 1- Leur détection phénotypique est souvent difficile et aucun essai moléculaire fiable pour détecter une telle résistance n'est disponible; 2- La détection du phénotype hVISA est encore plus difficile dû au fait que, selon les méthodes cliniques de détection actuelles, ces bactéries sont classifiées comme susceptibles. De plus les bactéries hVISA montrent un phénotype instable compliquant leur détection; 3-Ces bactéries VISA/hVISA peuvent mener à une résistance croisée avec des nouveaux antibiotiques.

Grace aux précédents financements, nous avons identifié une signature moléculaire qui peut distinguer entre bactéries susceptibles et résistantes. Nous planifions d’utiliser cette signature pour développer une méthode de détection moléculaire directement dans des prélèvements sanguins de patients. L'instabilité de phénotype hVISA pose un problème majeur pour leur détection. Nous avons détecté deux gènes qui par leur fonction peuvent réguler l’instabilité d’hVISA. Nous planifions d’approfondir notre connaissance sur ces deux gènes a fin de comprendre l'instabilité du phénotype hVISA qui permettra de concevoir des meilleures méthodes de détection.

 Notre objectif à long terme est de développer des marqueurs moléculaires pour détecter l'apparition des bactéries VISA et hVISA pendant la thérapie du patient, avant que des bactéries complètement résistantes n’apparaissent. Notre étude aidera à optimiser le traitement antibiotiques des patients, estimer le risque de l’apparition de résistance croisée, et au final, découvrir des cibles  pour développer des nouveaux antibiotiques.

Direct link to Lay Summary Last update: 24.11.2016

Responsible applicant and co-applicants

Employees

Publications

Publication
Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin
Sierra Roberto, Prados Julien, Panasenko Olesya O, Andrey Diego O, Fleuchot Betty, Redder Peter, Kelley William L, Viollier Patrick H, Renzoni Adriana (2020), Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin, in Nucleic Acids Research, 48(15), 8545-8561.
YjbH Solubility Controls Spx in Staphylococcus aureus: Implication for MazEF Toxin-Antitoxin System Regulation
Panasenko Olesya O., Bezrukov Fedor, Komarynets Olga, Renzoni Adriana (2020), YjbH Solubility Controls Spx in Staphylococcus aureus: Implication for MazEF Toxin-Antitoxin System Regulation, in Frontiers in Microbiology, 11(113), 1-13.
Thermosensitive PBP2a requires extracellular folding factors PrsA and HtrA1 for Staphylococcus aureus MRSA β-lactam resistance
Roch Mélanie, Lelong Emmanuelle, Panasenko Olesya O., Sierra Roberto, Renzoni Adriana, Kelley William L. (2019), Thermosensitive PBP2a requires extracellular folding factors PrsA and HtrA1 for Staphylococcus aureus MRSA β-lactam resistance, in Communications Biology, 2(1), 417-417.
Linking toxin-antitoxin systems with phenotypes: A Staphylococcus aureus viewpoint
Sierra Roberto, Viollier Patrick, Renzoni Adriana (2019), Linking toxin-antitoxin systems with phenotypes: A Staphylococcus aureus viewpoint, in Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1862(7), 742-751.
Sub-Inhibitory Doses of Individual Constituents of Essential Oils Can Select for Staphylococcus aureus Resistant Mutants
Berdejo Daniel, Chueca Beatriz, Pagán Elisa, Renzoni Adriana, Kelley William, Pagán Rafael, Garcia-Gonzalo Diego (2019), Sub-Inhibitory Doses of Individual Constituents of Essential Oils Can Select for Staphylococcus aureus Resistant Mutants, in Molecules, 24(1), 170-170.
Whole-Genome Sequencing and Genetic Analysis Reveal Novel Stress Responses to Individual Constituents of Essential Oils in Escherichia coli
Chueca Beatriz, Renzoni Adriana, Berdejo Daniel, Pagán Rafael, Kelley William L., García-Gonzalo Diego (2018), Whole-Genome Sequencing and Genetic Analysis Reveal Novel Stress Responses to Individual Constituents of Essential Oils in Escherichia coli, in Applied and Environmental Microbiology, 84(7), 1-15.

Collaboration

Group / person Country
Types of collaboration
Université de Genéve Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Houston Methodist Research Institute United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
SSI Berne Talk given at a conference New molecular tools for viral diagnostics using CRISPR-Cas systems 03.09.2019 Berne, Switzerland Renzoni Adriana; Panasenko Olesya;
CUSO course Talk given at a conference MazEF toxin/antitoxin system in S. aureus: a magic postion for bacterial sleeping 19.11.2018 Geneva, Switzerland Renzoni Adriana; Panasenko Olesya;


Associated projects

Number Title Start Funding scheme
149762 Identification of molecular markers to detect emergence of low-level glycopeptide resistance in Staphylococcus aureus 01.02.2014 Project funding

Abstract

1. Summary of Research PlanBackground:Staphylococcus aureus infections are still a major problem worldwide, despite recent reduced incidence of methicillin-resistant (MRSA) or methicillin-susceptible (MSSA) S. aureus infections in some parts of the world. For decades, S. aureus has been responsible for several hospital-acquired or community-acquired infections treated with different antibiotics (glycopeptides, ß-lactams and daptomycin). Despite several antimicrobials available, antibiotic-resistance to a particular antibiotic has repeatedly brought complications for their successful treatment. Nowadays, cross-resistance, or resistance to a particular antibiotic that results in resistance to another antibiotic, is increasing the complexity of S. aureus antibiotic pharmacotherapy.Glycopeptides (vancomycin and teicoplanin) are still the first-line therapy for severe MRSA infections; however, under some special circumstances, they are also used to treat MSSA infections. Selection of low-level glycopeptide resistant strains, including heterogeneous vancomycin-intermediate (hVISA) or vancomycin-intermediate (VISA) in MSSA or MRSA strains, represents important risks: 1- Their phenotypic detection is often difficult and no reliable molecular assay for detecting such resistance is available; 2- Detection of the hVISA phenotype is even more difficult as they are classified as susceptible strains by current clinical detection methods and display an unstable phenotype complicating their detection; 3- Low-level glycopeptide resistant strains can potentially lead to cross-resistance to other antibiotics, such as daptomycin. Our long-term objective is to develop molecular markers to detect emergence of low-level glycopeptide resistant strains during glycopeptide therapy, before high-level resistant strains are selected or cross-resistance emerges.Specific Aims:Task 1. Develop an RNA signature detection method able to distinguish susceptible from hVISA and VISA strains directly from blood samples.Task 2. Study the function of yjbH and trfA genes to understand the instability of hVISA phenotype that will permit to design better detection methods or identify new molecular mechanisms regulating antibiotic resistance.Task 3. Discover the molecular link between PrsA and PBP2A, and identify the potential determinants linking PrsA with glycopeptides/daptomycin resistance. We recently showed the role of prsA in controlling membrane amounts of PBP2A, the master regulator of ß-lactam resistance in MRSA strains, and its role on glycopeptide and daptomycin resistance. This makes PrsA an ideal candidate to understand cell wall antibiotic cross-resistance. Experimental Design and/or Methods:Task 1. Previous clinical and basic research funding permitted us to isolate a collection of clinical S. aureus strains containing susceptible, hVISA and VISA strains, and to identify key molecular markers to detect low-level glycopeptide resistant strains. NanoString® technology will be applied to compare levels of expression of each molecular gene marker between susceptible, hVISA or VISA strains from our S. aureus collection and determine a threshold level of expression. These analyses will generate an RNA signature to distinguish between susceptible, hVISA and VISA strains directly from blood samples. Task 2. Selection of hVISA strains under ß-lactam antibiotic stress depends on trfA. Upon withdrawal of antibiotic stress, hVISA phenotype disappears and susceptibility is restored. Based on homology with Bacillus subtilis, YjbH protein aggregation is predicted to affect trfA transcription and hVISA phenotype. We will test if the antibiotic stress induces yjbH aggregation and consequently trfA transcription using biochemical and fluorescence microscopy techniques. As an adaptor protein, trfA is predicted to interact with other cellular proteins. We will study the potential direct interactions of TrfA with cell wall related candidate proteins, previously identified by TMT technology, using biochemical techniques such as enzyme-linked immunosorbent assay (ELISA). Task. 3. We will determine if PrsA interacts directly with PBP2A or with other proteins using ELISA, ligand overlay and pull down assays. We will further analyze the impact of PrsA on cellular localization of penicillin-binding proteins by fluorescence microscopy and its impact on peptidoglycan crosslinking by HPLC.Expected Value of the Proposed Project:(i) Develop and validate a reliable detection method of low-level glycopeptide-resistance in MRSA and MSSA isolates based on RNA signatures. Improved detection of low-level glycopeptide-resistance will optimize care of patients treated with glycopeptides, ?B-lactams and daptomycin; (ii) Estimate the risk of cross-resistance between glycopeptides, ß-lactams and daptomycin. (iii) Improve our understanding of the potential molecular mechanisms regulating antibiotic resistance; these results will be key for the discovery of novel targets for antibiotic development.
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