title: Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.
abstract: Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne, Victoria, Australia. Benjamin.Howden@austin.org.au
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Howden
authors.forename: Christopher R E
authors.initials: CR
authors.lastname: McEvoy
authors.forename: David L
authors.initials: DL
authors.lastname: Allen
authors.forename: Kyra
authors.initials: K
authors.lastname: Chua
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.forename: Paul F
authors.initials: PF
authors.lastname: Harrison
authors.forename: Jan
authors.initials: J
authors.lastname: Bell
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Coombs
authors.forename: Vicki
authors.initials: V
authors.lastname: Bennett-Wood
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Porter
authors.forename: Roy
authors.initials: R
authors.lastname: Robins-Browne
authors.forename: John K
authors.initials: JK
authors.lastname: Davies
authors.forename: Torsten
authors.initials: T
authors.lastname: Seemann
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Stinear
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018441:Biofilms; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D017576:Daptomycin / Q000494:pharmacology / Q000627:therapeutic use; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008826:Microbial Sensitivity Tests; D058889:Molecular Typing; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D013203:Staphylococcal Infections / Q000188:drug therapy / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D014640:Vancomycin / Q000494:pharmacology / Q000627:therapeutic use; D020713:Vancomycin Resistance / Q000235:genetics; D037521:Virulence Factors
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D037521:Virulence Factors; C119939:YycF protein, Bacteria; D014640:Vancomycin; D017576:Daptomycin
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