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.

The action mechanism of daptomycin

“…112 Several mutations in different locations of the yycFG operon in daptomycinresistant S. aureus were reported by Howden et al, although their exact contribution to resistance is unclear. 113 Nevertheless, a single amino acid change in YycG was sufficient to increase the MIC from 0.5 to 2 µg/L, a value that is sufficient for clinical resistance. In Enterococcus faecium, mutations in the yycFG operon have also been found in daptomycin-resistant strains.…”

Relationship between Vancomycin-Resistant Staphylococcus aureus, Vancomycin-Intermediate S. aureus, High Vancomycin MIC, and Outcome in Serious S. aureus Infections

“…These changes are particularly noted after prior exposure to vancomycin (4,13). Multiple genetic mutations have been implicated in the pathogenesis of these cell wall modifications, usually occurring in genes important for cell wall metabolism such as vraRS (15) and graRS; however, recent attention has focused on the essential S. aureus regulator walKR (14).…”

Mechanisms of vancomycin resistance in Staphylococcus aureus

“…Comparison of such susceptible and resistant isolates can then be used to identify the altered genetic determinant in the VISA strain. This approach has identified a variable number and kinds of mutations in different VISA isolates (18,28,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40).…”

“…In another study, the predominant genetic change was in the yvqF/vraSR system (41), while Watanabe et al showed that most of the 38 VISA strains recovered from 10 different countries carried a mutated rpoB (42). In most, but not all, cases, the relevant mutated genes appear to be directly or indirectly involved with the biosynthesis/metabolism of the staphylococcal cell wall, specifically, two-component sensory regulatory systems controlling the transcription of genes in cell wall synthesis (32,33,43). However, mutations in the RNA polymerase gene rpoB were also detected in VISA isolates (42).…”