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.
Highlights
In hospitals worldwide infections with methicillin-resistant S. aureus (MRSA) remain a significant cause of morbidity and mortality, with a small number of clones accounting for a large number of hospital acquired infections
While the emergence of vancomycinintermediate sequence type (MLST) 239 (ST239) S. aureus (VISA) in Australia has been in strains of the ST239 clone [4], the first VISA strain Mu50 was reported from Japan in 1997 [5], and a number of other reported VISA strains belong to the same clonal complex as Mu50 (CC5) [6,7,8]
The treatment of serious infections caused by Staphylococcus aureus is complicated by the development of antibiotic resistance, and recently resistance to one of the last available antibiotics to treat resistant S. aureus infections, vancomycin, has emerged
Summary
In hospitals worldwide infections with methicillin-resistant S. aureus (MRSA) remain a significant cause of morbidity and mortality, with a small number of clones accounting for a large number of hospital acquired infections. In Australasia, multi-locus sequence type (MLST) 239 (ST239) is the major hospital acquired clone of MRSA, and has been present in the region for over 30 years. This clone is resistant to almost all antibiotic classes; the mainstay of therapy for serious MRSA infections has been the glycopeptide antibiotic vancomycin. Resistant strains have recently emerged [1], and the level of resistance is low there is an impact on treatment outcome [2] These vancomycin-intermediate S. aureus (VISA, vancomycin MIC 4–8 mg/ml) and heterogenous-VISA (hVISA, vancomycin MIC #2 mg/ml with a ‘‘resistant subpopulation’’) strains are increasingly common, the genetics of resistance are incompletely defined [3]. While the emergence of VISA in Australia has been in strains of the ST239 clone [4], the first VISA strain Mu50 was reported from Japan in 1997 [5], and a number of other reported VISA strains belong to the same clonal complex as Mu50 (CC5) [6,7,8]
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