title: Decreased vancomycin susceptibility in Staphylococcus aureus caused by IS256 tempering of WalKR expression.
abstract: 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.
authors: Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.
authors: Christopher R E
authors: CR
authors: McEvoy
authors: Brian
authors: B
authors: Tsuji
authors: Wei
authors: W
authors: Gao
authors: Torsten
authors: T
authors: Seemann
authors: Jessica L
authors: JL
authors: Porter
authors: Kenneth
authors: K
authors: Doig
authors: Dung
authors: D
authors: Ngo
authors: Benjamin P
authors: BP
authors: Howden
authors: Timothy P
authors: TP
authors: Stinear
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D017576:Daptomycin / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D016680:Genome, Bacterial; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D046928:Peptidoglycan Glycosyltransferase / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics; D017422:Sequence Analysis, DNA; D014640:Vancomycin / Q000494:pharmacology*; D020713:Vancomycin Resistance / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D014640:Vancomycin; D046928:Peptidoglycan Glycosyltransferase; C524260:SceD protein, Staphylococcus aureus; D011494:Protein Kinases; C531166:AgrC protein, Staphylococcus; D017576:Daptomycin
references: Int J Med Microbiol. 2007 Jul;297(4):205-15
references: 17418637
references: PLoS One. 2011 Feb 28;6(2):e17054
references: 21386961
references: PLoS Pathog. 2012 Feb;8(2):e1002505
references: 22319446
references: Antimicrob Agents Chemother. 2011 Aug;55(8):3870-81
references: 21628539
references: Antimicrob Agents Chemother. 2008 Jan;52(1):45-53
references: 17954695
references: Genome Res. 2004 Jun;14(6):1188-90
references: 15173120
references: Proteomics. 2006 Aug;6(15):4246-58
references: 16826566
references: Lancet. 1997 Dec 6;350(9092):1670-3
references: 9400512
references: J Bacteriol. 2010 Nov;192(21):5848-9
references: 20802046
references: Antimicrob Agents Chemother. 2012 Nov;56(11):5845-51
references: 22948864
references: J Infect Dis. 2010 May 1;201(9):1414-21
references: 20307206
references: Antimicrob Agents Chemother. 2012 Jul;56(7):3712-9
references: 22526313
references: PLoS Pathog. 2011 Nov;7(11):e1002359
references: 22102812
references: Antimicrob Agents Chemother. 2009 Mar;53(3):1231-4
references: 19124662
references: Nature. 1983 Oct 20-26;305(5936):709-12
references: 6226876
references: Clin Microbiol Rev. 2010 Jan;23(1):99-139
references: 20065327
references: PLoS One. 2012;7(1):e29573
references: 22253738
references: J Bacteriol. 2007 Nov;189(22):8257-69
references: 17827301
references: J Bacteriol. 2007 Oct;189(20):7316-25
references: 17675373
references: Gene. 1989 Apr 15;77(1):51-9
references: 2744487
references: Clin Infect Dis. 2012 Mar;54(6):755-71
references: 22302374
references: J Infect Dis. 2012 Jun;205(11):1677-87
references: 22492855
references: Science. 2003 Nov 28;302(5650):1569-71
references: 14645850
references: Int J Antimicrob Agents. 2005 Feb;25(2):177-9
references: 15664490
references: Clin Infect Dis. 2004 Feb 15;38(4):521-8
references: 14765345
references: Antimicrob Agents Chemother. 2008 Oct;52(10):3755-62
references: 18644967
references: J Med Microbiol. 2012 Mar;61(Pt 3):345-352
references: 22016559
references: Drug Resist Updat. 1998;1(2):135-50
references: 16904400
references: DNA Res. 2004 Feb 29;11(1):51-6
references: 15141945
references: PLoS Pathog. 2010 Jun 10;6(6):e1000944
references: 20548948
references: J Antimicrob Chemother. 2012 Sep;67(9):2101-6
references: 22643192
references: Infect Immun. 2012 Oct;80(10):3438-53
references: 22825451
references: J Antimicrob Chemother. 2010 Aug;65(8):1792-8
references: 20511366
references: J Bacteriol. 1999 Jun;181(12):3666-73
references: 10368139
references: J Microbiol Methods. 2005 Mar;60(3):291-8
references: 15649531
references: Antimicrob Agents Chemother. 2006 Sep;50(9):3039-47
references: 16940100
references: Int J Antimicrob Agents. 2012 Aug;40(2):108-13
references: 22633565
references: Annu Rev Biochem. 1998;67:509-44
references: 9759496
references: Proc Natl Acad Sci U S A. 2007 May 29;104(22):9451-6
references: 17517606
references: BMC Microbiol. 2008 Feb 27;8:39
references: 18304359
references: J Proteome Res. 2007 Dec;6(12):4690-702
references: 17997515
references: J Infect Dis. 2009 Feb 15;199(4):532-6
references: 19125671
references: J Bacteriol. 2004 Feb;186(4):1175-81
references: 14762013
references: J Antimicrob Chemother. 2013 Jan;68(1):12-6
references: 23045225
references: Mol Microbiol. 2008 Dec;70(6):1307-22
references: 19019149
references: Infect Immun. 2001 Dec;69(12):7851-7
references: 11705967