title: Comprehensive identification of mutations responsible for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA)-to-VISA conversion in laboratory-generated VISA strains derived from hVISA clinical strain Mu3.
abstract: Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10(-6) or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.
authors.affiliation: Department of Bacteriology, Faculty of Medicine, Juntendo University, Tokyo, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Matsuo
authors.forename: Longzhu
authors.initials: L
authors.lastname: Cui
authors.forename: Jeeyoung
authors.initials: J
authors.lastname: Kim
authors.forename: Keiichi
authors.initials: K
authors.lastname: Hiramatsu
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002473:Cell Wall / Q000648:ultrastructure; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D014640:Vancomycin / Q000494:pharmacology*; D020713:Vancomycin Resistance / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D014640:Vancomycin; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit
references.citation: Antimicrob Agents Chemother. 2006 Feb;50(2):428-38
references.pmid: 16436693
references.citation: Microbiol Mol Biol Rev. 1999 Mar;63(1):174-229
references.pmid: 10066836
references.citation: Clin Infect Dis. 2004 Aug 15;39(4):539-45
references.pmid: 15356818
references.citation: J Antimicrob Chemother. 2001 Nov;48(5):617-25
references.pmid: 11679550
references.citation: Antimicrob Agents Chemother. 2011 Sep;55(9):4188-95
references.pmid: 21746940
references.citation: Nucleic Acids Res. 1994 Apr 11;22(7):1287-95
references.pmid: 8165145
references.citation: Lancet. 1998 Apr 18;351(9110):1212
references.pmid: 9643727
references.citation: J Clin Microbiol. 2000 Oct;38(10):3879-81
references.pmid: 11015427
references.citation: Antimicrob Agents Chemother. 2008 Jan;52(1):45-53
references.pmid: 17954695
references.citation: Antimicrob Agents Chemother. 2012 Oct;56(10):5212-23
references.pmid: 22850507
references.citation: Antimicrob Agents Chemother. 2000 Oct;44(10):2845-7
references.pmid: 10991869
references.citation: J Antimicrob Chemother. 1998 Aug;42(2):199-209
references.pmid: 9738837
references.citation: J Bacteriol. 1974 Dec;120(3):1370-9
references.pmid: 4373439
references.citation: PLoS Pathog. 2011 Nov;7(11):e1002359
references.pmid: 22102812
references.citation: J Bacteriol. 1995 Feb;177(3):517-23
references.pmid: 7836281
references.citation: Antimicrob Agents Chemother. 2009 Mar;53(3):1231-4
references.pmid: 19124662
references.citation: Clin Microbiol Rev. 2010 Jan;23(1):99-139
references.pmid: 20065327
references.citation: Microb Drug Resist. 2008 Mar;14(1):1-6
references.pmid: 18346009
references.citation: J Clin Microbiol. 2003 Apr;41(4):1687-93
references.pmid: 12682161
references.citation: PLoS Pathog. 2012 Feb;8(2):e1002505
references.pmid: 22319446
references.citation: Antimicrob Agents Chemother. 2000 Sep;44(9):2276-85
references.pmid: 10952568
references.citation: Gene. 2005 Nov 21;361:13-37
references.pmid: 16213112
references.citation: Annu Rev Microbiol. 1981;35:365-403
references.pmid: 6170248
references.citation: J Biol Chem. 2000 Jun 16;275(24):18145-52
references.pmid: 10751408
references.citation: J Antimicrob Chemother. 1998 Sep;42(3):315-20
references.pmid: 9786471
references.citation: J Bacteriol. 2003 Dec;185(24):7103-10
references.pmid: 14645269
references.citation: Open Biol. 2012 Dec 05;2(12):120142
references.pmid: 23271832
references.citation: Antimicrob Agents Chemother. 2011 Aug;55(8):3870-81
references.pmid: 21628539
references.citation: J Bacteriol. 2012 Mar;194(5):1256-7
references.pmid: 22328755
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Aug 1;62(Pt 8):710-5
references.pmid: 16880539
references.citation: Int J Antimicrob Agents. 2012 Jun;39(6):478-85
references.pmid: 22534508
references.citation: S Afr Med J. 2000 Nov;90(11):1113
references.pmid: 11196030
references.citation: Antimicrob Agents Chemother. 2008 Oct;52(10):3755-62
references.pmid: 18644967
references.citation: Proc Natl Acad Sci U S A. 2010 Nov 2;107(44):18991-6
references.pmid: 20944066
references.citation: PLoS Pathog. 2007 Jul 27;3(7):e102
references.pmid: 17676995
references.citation: J Antimicrob Chemother. 2000 Jun;45(6):887-90
references.pmid: 10837445
references.citation: Proc Natl Acad Sci U S A. 2002 May 28;99(11):7687-92
references.pmid: 12032344
references.citation: Proc Natl Acad Sci U S A. 2007 May 29;104(22):9451-6
references.pmid: 17517606
references.citation: Infect Control Hosp Epidemiol. 2001 Jul;22(7):443-8
references.pmid: 11583214
references.citation: J Clin Microbiol. 2003 Jan;41(1):5-14
references.pmid: 12517819
references.citation: N Engl J Med. 1999 Feb 18;340(7):517-23
references.pmid: 10021472
references.citation: J Clin Microbiol. 2011 Jul;49(7):2680-4
references.pmid: 21525224
references.citation: Lancet. 1997 Dec 6;350(9092):1670-3
references.pmid: 9400512
references.citation: Biol Pharm Bull. 2010;33(2):198-203
references.pmid: 20118540
references.citation: Antimicrob Agents Chemother. 2010 Dec;54(12):5222-33
references.pmid: 20837752
references.citation: J Bacteriol. 1994 Sep;176(18):5788-95
references.pmid: 8083170
references.citation: J Clin Microbiol. 2009 Jul;47(7):2311-6
references.pmid: 19403764
references.citation: J Bacteriol. 2010 Mar;192(5):1459-62
references.pmid: 20061474
references.citation: J Clin Microbiol. 1998 Apr;36(4):1020-7
references.pmid: 9542929
references.citation: Antimicrob Agents Chemother. 2001 Nov;45(11):3070-5
references.pmid: 11600358
references.citation: Plasmid. 2006 Jan;55(1):58-63
references.pmid: 16051359