Generation of a vancomycin-intermediate Staphylococcus aureus (VISA) strain by two amino acid exchanges in VraS

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. aureu

Synergetic Antimicrobial Activity and Mechanism of Clotrimazole-Linked CO-Releasing Molecules

This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) through fellowship PD/BD/ 148006/2019 (SSM), PTDC/SAU-INF/29313/2017 grant, and R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) cofounded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. The NMR data was acquired at CERMAX, Instituto de Tecnologia Quıḿ ica e Bioloǵ ica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/ SAICT/2016. This work was partially supported by the PPBIPortuguese Platform of BioImaging (PPBI-POCI-01- 0145-FEDER-022122) cofunded by national funds from OE “Orçamento de Estado” and by European funds from FEDER“Fundo Europeu de Desenvolvimento Regional”. LMS and SSM acknowledge funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 810856. H.B.-O., T.S., C.M., F.O., J.B., and M.A. gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project ID 398967434 (TRR 261, projects A01, A06, A10, and Z02). A.B. appreciates funding by the German Federal Ministry for Education and Research (project Gramneg. Design).Several metal-based carbon monoxide-releasing molecules (CORMs) are active CO donors with established antibacterial activity. Among them, CORM conjugates with azole antibiotics of type [Mn(CO)3(2,2′-bipyridyl)(azole)]+ display important synergies against several microbes. We carried out a structure-activity relationship study based upon the lead structure of [Mn(CO)3(Bpy)(Ctz)]+ by producing clotrimazole (Ctz) conjugates with varying metal and ligands. We concluded that the nature of the bidentate ligand strongly influences the bactericidal activity, with the substitution of bipyridyl by small bicyclic ligands leading to highly active clotrimazole conjugates. On the contrary, the metal did not influence the activity. We found that conjugate [Re(CO)3(Bpy)(Ctz)]+ is more than the sum of its parts: while precursor [Re(CO)3(Bpy)Br] has no antibacterial activity and clotrimazole shows only moderate minimal inhibitory concentrations, the potency of [Re(CO)3(Bpy)(Ctz)]+ is one order of magnitude higher than that of clotrimazole, and the spectrum of bacterial target species includes Gram-positive and Gram-negative bacteria. The addition of [Re(CO)3(Bpy)(Ctz)]+ to Staphylococcus aureus causes a general impact on the membrane topology, has inhibitory effects on peptidoglycan biosynthesis, and affects energy functions. The mechanism of action of this kind of CORM conjugates involves a sequence of events initiated by membrane insertion, followed by membrane disorganization, inhibition of peptidoglycan synthesis, CO release, and break down of the membrane potential. These results suggest that conjugation of CORMs to known antibiotics may produce useful structures with synergistic effects that increase the conjugate's activity relative to that of the antibiotic alone.publishersversionpublishe

Generation of a vancomycin-intermediate Staphylococcus aureus (VISA) strain by two amino acid exchanges in VraS.

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