Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS 256 Tempering of WalKR Expression

Abstract

f Vancomycin-intermediate Staphylococcus aureus (VISA) strains often arise by mutations in the essential two-component regulator walKR; however their impact on walKR function has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptible S. aureus (VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256 insertions in the walKR 5= untranslated region (5= UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction in walKR gene expression in the IS256 mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256 insertions had replaced two SigA-like walKR promoters with weaker, hybrid promoters. Removal of IS256 reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation of ssaA but no change in expression of sak and sceD in both IS256 mutants. Whole-genome sequencing of the two mutants revealed an additional IS256 insertion within agrC for one mutant, and we confirmed that this mutation abolished agr function. These data provide the first substantial analysis of walKR promoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction in walKR expression; however, the mechanisms by which this occurs remain to be determined.

Biofilm formation of Brazilian meticillin-resistant Staphylococcus aureus strains: prevalence of biofilm determinants and clonal profiles

“…Other studies have associated mutations caused by the presence of the IS256 and the occurrence of a nonfunctional agr quorum-sensing system (McEvoy et al, 2013). As a result, agr-dysfunctional strains have a tendency to form biofilms in vitro, which has been attributed to, among other factors, the decreased production of proteases responsible for digestion of biofilm extracellular matrix, overexpression of protein A and the increased accumulation of fibronectin-binding proteins (Toledo-Arana et al, 2005;Merino et al, 2009;Vergara-Irigaray et al, 2009;Ferreira et al, 2013).…”

Diversity and Evolution of the Tn 5801-tet (M)-Like Integrative and Conjugative Elements among Enterococcus, Streptococcus, and Staphylococcus

“…Among type B variants, only the mutator family IS256 was consistently detected upstream of orf13 (types B20, B18, and B19), and this stability persisted even when some particular variants (e.g., B20) were disseminated across genera. Some ISs from the IS256 family have been implicated as modulators of gene expression, suggesting that future studies might reveal the role of this IS in the function of type B Tn5801-like elements (50)(51)(52)(53).…”

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).…”