Tracking thein vivoevolution of multidrugresistance inStaphylococcus aureusbywhole-genome sequencingMichael M. Mwangi*†, Shang Wei Wu‡§, Yanjiao Zhou‡§, Krzysztof Sieradzki‡, Herminia de Lencastre‡¶,Paul Richardson!, David Bruce!, Edward Rubin!, Eugene Myers**, Eric D. Siggia*†, and Alexander Tomasz‡††*Physics Department, Cornell University, Ithaca, NY 14850;†Center for Studies in Physics and Biology and‡Laboratory of Microbiology, The RockefellerUniversity, New York, NY 10021;§Department of Microbiology, Tianjin Medical University, Tianjin 300070, People’s Republic of China;¶Laboratoryof Molecular Genetics, Instituto de Tecnologia Quı´mica e Biolo´ gica, Universidade Nova de Lisboa, Oeiras, Portugal;!United States Departmentof Energy Joint Genomic Institute, Walnut Creek, CA 94598; and **Howard Hughes Medical Institute, Janelia Farm Research Campus,Ashburn, VA 20146Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved April 13, 2007 (received for review November 6, 2006)The spread of multidrug-resistant Staphylococcus aureus (MRSA)strains in the clinical environment has begun to pose serious limitsto treatment options. Yet virtually nothing is known about howresistance traits are acquired in vivo. Here, we apply the power ofwhole-genome sequencing to identify steps in the evolution ofmultidrug resistance in isogenic S. aureus isolates recovered peri-odically from the bloodstream of a patient undergoing chemother-apy with vancomycin and other antibiotics. After extensive ther-apy, the bacterium developed resistance, and treatment failed.Sequencing the first vancomycin susceptible isolate and the lastvancomycin nonsusceptible isolate identified genome wide only 35point mutations in 31 loci. These mutations appeared in a sequen-tial order in isolates that were recovered at intermittent timesduring chemotherapy in parallel with increasing levels of resis-tance. The vancomycin nonsusceptible isolates also showed a100-fold decrease in susceptibility to daptomycin, although thisantibiotic was not used in the therapy. One of the mutated lociassociated with decreasing vancomycin susceptibility (the vraRoperon) was found to also carry mutations in six additionalvancomycin nonsusceptible S. aureus isolates belonging to differ-ent genetic backgrounds and recovered from different geographicsites. As costs drop, whole-genome sequencing will become auseful tool in elucidating complex pathways of in vivo evolution inbacterial pathogens.Staphylococcus aureus has remained one of the most frequentcauses of a wide range of bot h hospital- and community-acquired infections, from superficial skin and other sof t tissueinfections to life threatening toxic shock, pneumonia, endocar-ditis, and septicemia. Th e spectacular adaptive capacity of thispathogen resulted in the emergence and worldwide spread oflineages that acquired resistance to the majority of availableantimicrobial agents. The choice of therapy against such multi-drug-resistant S. aureus (MRSA) strains has been narrowed to afew antibacterial agents, among them the glycopeptide antibioticvancomycin, which has become the mainstay of therapy world-wide. MRSA strains with reduced susceptibility to vancomycinhave been reported in clinical specimen sinc e the lat e 1990s (1).In most of these so-called vancomycin i ntermediate-resistant S.aureus (VISA) is olates, decr ease in drug susceptibility, as ex-pressed by the increase in the minimal inhibitory concentration(MIC) of vancomycin, is sufficient to cause complications intherapy and treatment failure (2–7) . V ISA-type resistance ha snow been identified in each of the globally spread pandemicclones of MRSA (8).The genetic basis of VISA-type resistance to va ncomycin isunknown. Unlike the most recently described and currently stillrare VRSA isolates which carry the Tn1546-linked resistancemechanism (9, 10), the VISA-type isola tes do not seem to carryacquired genetic elements related to drug resistance: theirreduced susceptibility to vancomycin appears to be based on agradual adaptive process.Examination of VISA-type isolat es recovered from many partsof t he world showed a number of different pheno typic alter-ations, inclu ding changes in cell morphology and changes in thecomposition, thickness, a nd/or turnover of cell walls (11, 12).Nevertheless, associating these altered properties with the mech-anism of resistance has rem ained problema tic because of the lackof availability of an isogenic vancomycin susceptible ‘‘parental’’isolate that could be used as a valid comparison. For instance,comparing the seq uences of the first c linical VISA isolate MU50to t he genetically related vancomycin susceptible strain N315identified over 174 ORFs that carried nonsynonymous changes(13, 14). However, MRSA st rain N315 was isolated 15 yearsearlier than strain Mu50 and from a different patient. Thus, it isnot cle ar how many of the 174 mutations are related to themechanism of drug resistance versus the different evolutionaryhistory of the strains.Recently we obtained a series of MRSA isolates from theblood stream of a patient with congenital heart disease who wastreated extensively with vanco mycin without success (15). Avai l-able clinical data suggests that the primary si te of infection wasendocarditis.‡‡In addition to vancomycin, the patient alsoreceived a single dose of rifampin and a course of therapy withthe!-lactam antibiotic imipenem. After !12 weeks of therapyand replacement of a heart valve, the patient died because ofcomplications of the underlying disease.The first isolate JH1 recovered before the beginning of chemo-therapy was fully susceptible to vancomycin (MIC " 1"g/ml).Vancomycin therapy was begun between the culture isolation ofJH1 and JH2. The last isolate JH9 recovered at the end ofchemotherapy showed decreased susceptibility to vancomycin(MIC " 8"g/ml). Comparison of the series of JH isolates by severalgenetic typing techniques indicated that they were isogenic (15, 16).The JH lineage was also related, although more remotely, to thefully sequenced MRSA strains N315 and MU50 (17).Author contributions: A.T. designed research; M.M.M., S.W.W., and Y.Z. performed re-search; K.S., P.R., D.B., and E.R. contributed new reagents/analytic tools; M.M.M. H.d.L.,E.M., and E.D.S. analyzed data; and M.M.M., E.D.S., and A.T. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct