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The mechanism of vancomycin resistance of VRSA without <jats:italic>vanA<\/jats:italic> gene acquisition was not fully elucidated. Therefore, we aimed to determine the mechanism of vancomycin resistance of VRSA besides that by <jats:italic>vanA<\/jats:italic> gene acquisition. In this study, we obtained vancomycin-resistant strains (V036-V64; MIC\u2009=\u200964\u00a0\u00b5g \/ml) from susceptible strain (V036; MIC\u2009=\u20090.5\u00a0\u00b5g \/ml) by exposure of vancomycin in vitro and examined the phenotypic characteristics and antibiotic susceptibility profiles of the resistant strain (V036-V64). To identify the genetic variations caused vancomycin resistance, we determined the complete genome sequences of V036 and V036-V64 and analyzed for single-nucleotide polymorphisms (SNPs) between two strains. Morphologically, V036-V64 had a twofold thicker cell wall compared with V036. Linezolid, rifampicin, and ceftaroline had similar MIC ranges against V036-V64 and V036, but V036-V64 showed lower susceptibilities to daptomycin and telavancin. We detected eight single-nucleotide polymorphisms differing between V036-V64 and V036: <jats:italic>rimM<\/jats:italic> (G16D), <jats:italic>ssaA2<\/jats:italic> (G128A), <jats:italic>rpsK<\/jats:italic> (P60R), <jats:italic>rpoB<\/jats:italic> (R917C), <jats:italic>walK<\/jats:italic> (T492R), <jats:sc>d<\/jats:sc>-alanyl-<jats:sc>d<\/jats:sc>-alanine carboxypeptidase (L307I), <jats:italic>vraT<\/jats:italic> (A152V), and chromosome segregation ATPase (T440I). This study demonstrates that, under selective pressure, by the accumulation of mutations in genes related to cell wall synthesis, vancomycin-susceptible <jats:italic>S. aureus<\/jats:italic> can develop thicker cell walls and, hence, develop high vancomycin resistance. Thus, we highlight a novel <jats:italic>vanA<\/jats:italic>-negative mechanism for VRSA emergence.<\/jats:p>","DOI":"10.1007\/s00203-020-01906-y","type":"journal-article","created":{"date-parts":[[2020,6,13]],"date-time":"2020-06-13T07:02:35Z","timestamp":1592031755000},"page":"2255-2261","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Single-nucleotide polymorphisms in a vancomycin-resistant Staphylococcus aureus strain based on whole-genome sequencing"],"prefix":"10.1007","volume":"202","author":[{"given":"Jung Wook","family":"Kim","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7831-5905","authenticated-orcid":false,"given":"Kwang Jun","family":"Lee","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2020,6,13]]},"reference":[{"key":"1906_CR1","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1007\/s12223-013-0275-1","volume":"59","author":"AA Adegoke","year":"2014","unstructured":"Adegoke AA, Okoh AI (2014) Species diversity and antibiotic resistance properties of Staphylococcus aureus of farm animal origin in Nkonkobe Municipality, South Africa. 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