Anne Berscheid1, Patrice François2, Axel Strittmatter3, Gerhard Gottschalk3, Jacques Schrenzel2, Peter Sass4, Gabriele Bierbaum5. 1. Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University of Bonn, Bonn, Germany. 2. Genomic Research Laboratory, Infectious Diseases Service, Geneva University Hospitals and the University of Geneva, Geneva, Switzerland. 3. Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany. 4. Institute for Pharmaceutical Biology, University of Düsseldorf, Düsseldorf, Germany. 5. Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University of Bonn, Bonn, Germany g.bierbaum@uni-bonn.de.
Abstract
OBJECTIVES: Staphylococcus aureus is a notorious bacterial pathogen and antibiotic-resistant isolates complicate current treatment strategies. We characterized S. aureus VC40, a laboratory mutant that shows full resistance to glycopeptides (vancomycin and teicoplanin MICs ≥32 mg/L) and daptomycin (MIC = 4 mg/L), to gain deeper insights into the underlying resistance mechanisms. METHODS: Genomics and transcriptomics were performed to characterize changes that might contribute to development of resistance. The mutations in vraS were reconstituted into a closely related parental background. In addition, antimicrobial susceptibility testing, growth analyses, transmission electron microscopy, lysostaphin-induced lysis and autolysis assays were performed to characterize the phenotype of resistant strains. RESULTS: Genome sequencing of strain VC40 revealed 79 mutations in 75 gene loci including genes encoding the histidine kinases VraS and WalK that control cell envelope-related processes. Transcriptomics indicated the increased expression of their respective regulons. Although not reaching the measured MIC for VC40, reconstitution of the L114S and D242G exchanges in VraS(VC40) into the susceptible parental background (S. aureus NCTC 8325) resulted in increased resistance to glycopeptides and daptomycin. The expression of VraS(VC40) led to increased transcription of the cell wall stress stimulon, a thickened cell wall, a decreased growth rate, reduced autolytic activity and increased resistance to lysostaphin-induced lysis in the generated mutant. CONCLUSIONS: We show that a double mutation of a single gene locus, namely vraS, is sufficient to convert the vancomycin-susceptible strain S. aureus NCTC 8325 into a vancomycin-intermediate S. aureus.
OBJECTIVES:Staphylococcus aureus is a notorious bacterial pathogen and antibiotic-resistant isolates complicate current treatment strategies. We characterized S. aureus VC40, a laboratory mutant that shows full resistance to glycopeptides (vancomycin and teicoplanin MICs ≥32 mg/L) and daptomycin (MIC = 4 mg/L), to gain deeper insights into the underlying resistance mechanisms. METHODS: Genomics and transcriptomics were performed to characterize changes that might contribute to development of resistance. The mutations in vraS were reconstituted into a closely related parental background. In addition, antimicrobial susceptibility testing, growth analyses, transmission electron microscopy, lysostaphin-induced lysis and autolysis assays were performed to characterize the phenotype of resistant strains. RESULTS: Genome sequencing of strain VC40 revealed 79 mutations in 75 gene loci including genes encoding the histidine kinases VraS and WalK that control cell envelope-related processes. Transcriptomics indicated the increased expression of their respective regulons. Although not reaching the measured MIC for VC40, reconstitution of the L114S and D242G exchanges in VraS(VC40) into the susceptible parental background (S. aureus NCTC 8325) resulted in increased resistance to glycopeptides and daptomycin. The expression of VraS(VC40) led to increased transcription of the cell wall stress stimulon, a thickened cell wall, a decreased growth rate, reduced autolytic activity and increased resistance to lysostaphin-induced lysis in the generated mutant. CONCLUSIONS: We show that a double mutation of a single gene locus, namely vraS, is sufficient to convert the vancomycin-susceptible strain S. aureus NCTC 8325 into a vancomycin-intermediate S. aureus.
Authors: Stefano G Giulieri; Romain Guérillot; Jason C Kwong; Ian R Monk; Ashleigh S Hayes; Diane Daniel; Sarah Baines; Norelle L Sherry; Natasha E Holmes; Peter Ward; Wei Gao; Torsten Seemann; Timothy P Stinear; Benjamin P Howden Journal: mBio Date: 2020-12-08 Impact factor: 7.867
Authors: Michael Hort; Ute Bertsche; Senada Nozinovic; Alina Dietrich; Anne Sophie Schrötter; Laura Mildenberger; Katharina Axtmann; Anne Berscheid; Gabriele Bierbaum Journal: Microbiol Spectr Date: 2021-10-20