1887

Abstract

We used genomics to study the evolution of meticillin-resistant (MRSA) during a complex, protracted clinical infection. Preparing closed MRSA genomes from days 0 and 115 allowed us to precisely reconstruct all genetic changes that occurred. Twenty-three MRSA blood cultures were also obtained during treatment, yielding 44 colony morphotypes that varied in size, haemolysis and antibiotic susceptibility. A subset of 15 isolates was sequenced and shown to harbour a total of 37 sequence polymorphisms. Eighty per cent of all mutations occurred from day 45 onwards, which coincided with the appearance of discrete chromosome expansions, and concluded in the day 115 isolate with a 98 kb tandem DNA duplication. In all heterogeneous vancomycin-intermediate isolates, the chromosomal amplification spanned at least a 20 kb region that notably included , a gene involved in resistance to antimicrobial peptides, and , an essential DNA replication gene with an unusual V463 codon insertion. Restoration of the chromosome after serial passage under non-selective growth was accompanied by increased susceptibility to antimicrobial peptide killing and reduced vancomycin resistance, two signature phenotypes that help explain the clinical persistence of this strain. Elevated expression of the V463 was deleterious to the cell and reduced colony size, but did not alter ciprofloxacin susceptibility. In this study, we identified large DNA expansions as a clinically relevant mechanism of resistance and persistence, demonstrating the extent to which bacterial chromosomes remodel in the face of antibiotic and host immune pressures.

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2015-08-03
2020-04-03
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References

  1. Adler M., Anjum M., Berg O.G., Andersson D.I., Sandegren L.. 2014; High fitness costs and instability of gene duplications reduce rates of evolution of new genes by duplication-divergence mechanisms. Mol Biol Evol31:1526–1535 [CrossRef][PubMed]
    [Google Scholar]
  2. Andersson D.I., Hughes D.. 2009; Gene amplification and adaptive evolution in bacteria. Annu Rev Genet43:167–195 [CrossRef][PubMed]
    [Google Scholar]
  3. Bae T., Schneewind O.. 2006; Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid55:58–63 [CrossRef][PubMed]
    [Google Scholar]
  4. Bayer A.S., Mishra N.N., Sakoulas G., Nonejuie P., Nast C.C., Pogliano J., Chen K.T., Ellison S.N., Yeaman M.R., Yang S.J.. 2014; Heterogeneity of mprF sequences in methicillin-resistant Staphylococcus aureus clinical isolates: role in cross-resistance between daptomycin and host defense antimicrobial peptides. Antimicrob Agents Chemother58:7462–7467 [CrossRef][PubMed]
    [Google Scholar]
  5. Berglund C., Söderquist B.. 2008; The origin of a methicillin-resistant Staphylococcus aureus isolate at a neonatal ward in Sweden-possible horizontal transfer of a staphylococcal cassette chromosome mec between methicillin-resistant Staphylococcus haemolyticus Staphylococcus aureus. Clin Microbiol Infect14:1048–1056 [CrossRef][PubMed]
    [Google Scholar]
  6. Bloemendaal A.L., Brouwer E.C., Fluit A.C.. 2010; Methicillin resistance transfer from Staphylocccus epidermidis to methicillin-susceptible Staphylococcus aureus in a patient during antibiotic therapy. PLoS One5:e11841 [CrossRef][PubMed]
    [Google Scholar]
  7. Bolger A.M., Lohse M., Usadel B.. 2014; Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics30:2114–2120 [CrossRef][PubMed]
    [Google Scholar]
  8. Cameron D.R., Ward D.V., Kostoulias X., Howden B.P., Moellering R.C. Jr, Eliopoulos G.M., Peleg A.Y.. 2012; Serine/threonine phosphatase Stp1 contributes to reduced susceptibility to vancomycin and virulence in Staphylococcus aureus. J Infect Dis205:1677–1687 [CrossRef][PubMed]
    [Google Scholar]
  9. Chain P.S., Grafham D.V., Fulton R.S., Fitzgerald M.G., Hostetler J., Muzny D., Ali J., Birren B., Bruce D.C., other authors. 2009; Genomics. Genome project standards in a new era of sequencing. Science326:236–237 [CrossRef][PubMed]
    [Google Scholar]
  10. Chambers H.F., Deleo F.R.. 2009; Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol7:629–641 [CrossRef][PubMed]
    [Google Scholar]
  11. Cui L., Isii T., Fukuda M., Ochiai T., Neoh H.M., Camargo I.L., Watanabe Y., Shoji M., Hishinuma T., Hiramatsu K.. 2010; An RpoB mutation confers dual heteroresistance to daptomycin and vancomycin in Staphylococcus aureus. Antimicrob Agents Chemother54:5222–5233 [CrossRef][PubMed]
    [Google Scholar]
  12. Cui L., Neoh H.M., Iwamoto A., Hiramatsu K.. 2012; Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria. Proc Natl Acad Sci U S A109:E1647–E1656 [CrossRef][PubMed]
    [Google Scholar]
  13. Didier J.P., Villet R., Huggler E., Lew D.P., Hooper D.C., Kelley W.L., Vaudaux P.. 2011; Impact of ciprofloxacin exposure on Staphylococcus aureus genomic alterations linked with emergence of rifampin resistance. Antimicrob Agents Chemother55:1946–1952 [CrossRef][PubMed]
    [Google Scholar]
  14. Ernst C.M., Peschel A.. 2011; Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids. Mol Microbiol80:290–299 [CrossRef][PubMed]
    [Google Scholar]
  15. Friedman L., Alder J.D., Silverman J.A.. 2006; Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob Agents Chemother50:2137–2145 [CrossRef][PubMed]
    [Google Scholar]
  16. Gao W., Chua K., Davies J.K., Newton H.J., Seemann T., Harrison P.F., Holmes N.E., Rhee H.W., Hong J.I., other authors. 2010; Two novel point mutations in clinical Staphylococcus aureus reduce linezolid susceptibility and switch on the stringent response to promote persistent infection. PLoS Pathog6:e1000944 [CrossRef][PubMed]
    [Google Scholar]
  17. Gao W., Cameron D.R., Davies J.K., Kostoulias X., Stepnell J., Tuck K.L., Yeaman M.R., Peleg A.Y., Stinear T.P., Howden B.P.. 2013; The RpoB H481Y rifampicin resistance mutation and an active stringent response reduce virulence and increase resistance to innate immune responses in Staphylococcus aureus. J Infect Dis207:929–939 [CrossRef][PubMed]
    [Google Scholar]
  18. Green J.M., Hollandsworth R., Pitstick L., Carter E.L.. 2010; Purification and characterization of the folate catabolic enzyme p-aminobenzoyl-glutamate hydrolase from Escherichia coli. J Bacteriol192:2407–2413 [CrossRef][PubMed]
    [Google Scholar]
  19. Gullberg E., Cao S., Berg O.G., Ilbäck C., Sandegren L., Hughes D., Andersson D.I.. 2011; Selection of resistant bacteria at very low antibiotic concentrations. PLoS Pathog7:e1002158 [CrossRef][PubMed]
    [Google Scholar]
  20. Helle L., Kull M., Mayer S., Marincola G., Zelder M.E., Goerke C., Wolz C., Bertram R.. 2011; Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus. Microbiology157:3314–3323 [CrossRef][PubMed]
    [Google Scholar]
  21. Howden B.P., Stinear T.P., Allen D.L., Johnson P.D., Ward P.B., Davies J.K.. 2008; Genomic analysis reveals a point mutation in the two-component sensor gene graS that leads to intermediate vancomycin resistance in clinical Staphylococcus aureus. Antimicrob Agents Chemother52:3755–3762 [CrossRef][PubMed]
    [Google Scholar]
  22. Howden B.P., McEvoy C.R., Allen D.L., Chua K., Gao W., Harrison P.F., Bell J., Coombs G., Bennett-Wood V., other authors. 2011; Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR. PLoS Pathog7:e1002359 [CrossRef][PubMed]
    [Google Scholar]
  23. Huson D.H., Bryant D.. 2006; Application of phylogenetic networks in evolutionary studies. Mol Biol Evol23:254–267 [CrossRef][PubMed]
    [Google Scholar]
  24. LaMarre J.M., Howden B.P., Mankin A.S.. 2011; Inactivation of the indigenous methyltransferase RlmN in Staphylococcus aureus increases linezolid resistance. Antimicrob Agents Chemother55:2989–2991 [CrossRef][PubMed]
    [Google Scholar]
  25. Langmead B., Salzberg S.L.. 2012; Fast gapped-read alignment with Bowtie 2. Nat Methods9:357–359 [CrossRef][PubMed]
    [Google Scholar]
  26. Locke J.B., Hilgers M., Shaw K.J.. 2009; Novel ribosomal mutations in Staphylococcus aureus strains identified through selection with the oxazolidinones linezolid and torezolid (TR-700). Antimicrob Agents Chemother53:5265–5274 [CrossRef][PubMed]
    [Google Scholar]
  27. Lowy F.D.. 1998; Staphylococcus aureus infections. N Engl J Med339:520–532 [CrossRef][PubMed]
    [Google Scholar]
  28. McAdam P.R., Holmes A., Templeton K.E., Fitzgerald J.R.. 2011; Adaptive evolution of Staphylococcus aureus during chronic endobronchial infection of a cystic fibrosis patient. PLoS One6:e24301 [CrossRef][PubMed]
    [Google Scholar]
  29. McEvoy C.R., Tsuji B., Gao W., Seemann T., Porter J.L., Doig K., Ngo D., Howden B.P., Stinear T.P.. 2013; Decreased vancomycin susceptibility in Staphylococcus aureus caused by IS256 tempering of WalKR expression. Antimicrob Agents Chemother57:3240–3249 [CrossRef][PubMed]
    [Google Scholar]
  30. Mishra N.N., Yang S.J., Sawa A., Rubio A., Nast C.C., Yeaman M.R., Bayer A.S.. 2009; Analysis of cell membrane characteristics of in vitro-selected daptomycin-resistant strains of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother53:2312–2318 [CrossRef][PubMed]
    [Google Scholar]
  31. Monk I.R., Tree J.J., Howden B.P., Stinear T.P., Foster T.J.. 2015; Complete bypass of restriction systems for major Staphylococcus aureus lineages. MBio6:e00308–e00315 [CrossRef][PubMed]
    [Google Scholar]
  32. Mwangi M.M., Wu S.W., Zhou Y., Sieradzki K., de Lencastre H., Richardson P., Bruce D., Rubin E., Myers E., other authors. 2007; Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing. Proc Natl Acad Sci U S A104:9451–9456 [CrossRef][PubMed]
    [Google Scholar]
  33. Paulander W., Nissen Varming A., Baek K.T., Haaber J., Frees D., Ingmer H.. 2013; Antibiotic-mediated selection of quorum-sensing-negative Staphylococcus aureus. MBio3:e00459–12[PubMed]
    [Google Scholar]
  34. Ruzin A., Severin A., Moghazeh S.L., Etienne J., Bradford P.A., Projan S.J., Shlaes D.M.. 2003; Inactivation of mprF affects vancomycin susceptibility in Staphylococcus aureus. Biochim Biophys Acta1621:117–121 [CrossRef][PubMed]
    [Google Scholar]
  35. Sandegren L., Andersson D.I.. 2009; Bacterial gene amplification: implications for the evolution of antibiotic resistance. Nat Rev Microbiol7:578–588 [CrossRef][PubMed]
    [Google Scholar]
  36. Schmitz F.J., Jones M.E., Hofmann B., Hansen B., Scheuring S., Lückefahr M., Fluit A., Verhoef J., Hadding U., other authors. 1998; Characterization of grlA grlB gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC. Antimicrob Agents Chemother42:1249–1252[PubMed]
    [Google Scholar]
  37. Seemann T.. 2014; Prokka: rapid prokaryotic genome annotation. Bioinformatics30:2068–2069 [CrossRef][PubMed]
    [Google Scholar]
  38. Seggewiss J., Becker K., Kotte O., Eisenacher M., Yazdi M.R., Fischer A., McNamara P., Al Laham N., Proctor R., other authors. 2006; Reporter metabolite analysis of transcriptional profiles of a Staphylococcus aureus strain with normal phenotype and its isogenic hemB mutant displaying the small-colony-variant phenotype. J Bacteriol188:7765–7777 [CrossRef][PubMed]
    [Google Scholar]
  39. Smith E.E., Buckley D.G., Wu Z., Saenphimmachak C., Hoffman L.R., D'Argenio D.A., Miller S.I., Ramsey B.W., Speert D.P., other authors. 2006; Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A103:8487–8492 [CrossRef][PubMed]
    [Google Scholar]
  40. Sun S., Ke R., Hughes D., Nilsson M., Andersson D.I.. 2012; Genome-wide detection of spontaneous chromosomal rearrangements in bacteria. PLoS One7:e42639 [CrossRef][PubMed]
    [Google Scholar]
  41. Traber K.E., Lee E., Benson S., Corrigan R., Cantera M., Shopsin B., Novick R.P.. 2008; agr function in clinical Staphylococcus aureus isolates. Microbiology154:2265–2274 [CrossRef][PubMed]
    [Google Scholar]
  42. Weigel L.M., Clewell D.B., Gill S.R., Clark N.C., McDougal L.K., Flannagan S.E., Kolonay J.F., Shetty J., Killgore G.E., Tenover F.C.. 2003; Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science302:1569–1571 [CrossRef][PubMed]
    [Google Scholar]
  43. Young B.C., Golubchik T., Batty E.M., Fung R., Larner-Svensson H., Votintseva A.A., Miller R.R., Godwin H., Knox K., other authors. 2012; Evolutionary dynamics of Staphylococcus aureus during progression from carriage to disease. Proc Natl Acad Sci U S A109:4550–4555 [CrossRef][PubMed]
    [Google Scholar]
  44. Zhou Y., Liang Y., Lynch K.H., Dennis J.J., Wishart D.S.. 2011; phast: a fast phage search tool. Nucleic Acids Res39:(Suppl)W347–W352 [CrossRef][PubMed]
    [Google Scholar]
  45. Stinear, T. FigShare http://dx.doi.org/10.6084/m9.figshare.1394626 (2015) Table S4
  46. Stinear, T. FigShare http://dx.doi.org/10.6084/m9.figshare.1391325 (2015) JKD6229.gb
  47. Stinear, T. FigShare http://dx.doi.org/10.6084/m9.figshare.1391322 (2015) JKD6210.gb
  48. Stinear, T. FigShare http://dx.doi.org/10.6084/m9.figshare.1391323 (2015) JKD6210p.gb
  49. Seeman, T. GitHub https://github.com/tseemann/prokka (2015)
  50. Stinear, T. European Nucleotide Archive http://www.ebi.ac.uk/ena/data/view/ERP010276 (2015)
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