{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:07:42Z","timestamp":1785424062506,"version":"3.56.0"},"posted":{"date-parts":[[2025,5,28]]},"group-title":"Microbiology","reference-count":38,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,5,28]],"date-time":"2025-05-28T00:00:00Z","timestamp":1748390400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"award":["NIAMS R01 AR082167"],"award-info":[{"award-number":["NIAMS R01 AR082167"]}],"id":[{"id":"https:\/\/ror.org\/01an3r305","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R03 AR077602"],"award-info":[{"award-number":["R03 AR077602"]}],"id":[{"id":"https:\/\/ror.org\/01an3r305","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,5,28]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Methicillin resistant\n                  <jats:italic>Staphylococcus aureus<\/jats:italic>\n                  (MRSA) sepsis has a high rate of morbidity and mortality. Multiple clinical studies have demonstrated improved outcomes when MRSA sepsis is treated with dual antibiotic therapy that includes a \u03b2-lactam antibiotic such as cefazolin. This is a paradox as MRSA should be inherently resistant to this class of antibiotics. We report a serendipitous observation revealed a phenotype where MRSA became sensitive to cefazolin when cultured in a physiologic relevant media of fetal bovine serum as well as in synovial fluid. This could be observed across multiple clinical isolates. Expected resistance was maintained when cultured in Muller Hinton Broth (MHB). MRSA \u03b2-lactam antibiotic resistance is mediated by PBP2a, a penicillin-binding protein encoded by\n                  <jats:italic>mecA<\/jats:italic>\n                  . We hypothesized that this phenotype of antibiotic sensitivity in physiologic medium was based, in part, on levels of PBP2a expression and post-translational modifications of peptidoglycan wall teichoic acid (WTA). We therefore conducted quantitative RT-PCR analysis and Western blotting which demonstrated limited\n                  <jats:italic>mecA<\/jats:italic>\n                  expression in the mRNA level and limited PBP2a protein level when cultured in FBS or synovial fluid as compared to the clinical microbiology standard MHB, respectively. Whole genome sequencing of loss of function mutants generated through serial passaging in FBS revealed that the\n                  <jats:italic>clp<\/jats:italic>\n                  family of proteins and\n                  <jats:italic>rpo<\/jats:italic>\n                  genes were involved in \u03b2-lactam resistance. Cell wall peptidoglycan analysis suggested that WTA glycosylation was altered between \u03b2-lactam resistant and sensitive MRSA phenotypes. Together, this suggests pathways for\n                  <jats:italic>clpP<\/jats:italic>\n                  ,\n                  <jats:italic>rpoB,<\/jats:italic>\n                  and WTA glycosylation can be new potential targets for MRSA treatment.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    This study demonstrates that MRSA has phenotypic variation in\n                    <jats:italic>mecA<\/jats:italic>\n                    expression, PBP2a protein levels, and patterns of wall teichoic acid glycosylation based on environmental conditions which can result in a phenotype of \u03b2-lactam antibiotic sensitivity. The presence of an antibiotic resistance gene does not necessarily result in antibiotic resistance. This provides a possible explanation for the clinical observations of potential therapeutic benefit of \u03b2-lactam antibiotics in dual therapy treatment of MRSA sepsis and suggests new therapeutic strategies to combat multidrug resistant bacteria. More importantly, identifying genes potentially responsible for this phenotype offers new potential therapeutic targets for the treatment of multidrug resistant infection.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.05.28.656574","type":"posted-content","created":{"date-parts":[[2025,5,29]],"date-time":"2025-05-29T01:35:27Z","timestamp":1748482527000},"source":"Crossref","is-referenced-by-count":1,"title":["Methicillin-Resistant\n                  <i>Staphylococcus aureus<\/i>\n                  has Phenotypic Variation in\n                  <i>mecA<\/i>\n                  Expression that Alters Antibiotic Sensitivity"],"prefix":"10.64898","author":[{"given":"Dongzhu","family":"Ma","sequence":"first","affiliation":[{"name":"Arthritis and Arthroplasty Design Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rekha","family":"Arya","sequence":"additional","affiliation":[{"name":"Arthritis and Arthroplasty Design Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Beth Ann","family":"Knapick","sequence":"additional","affiliation":[{"name":"Arthritis and Arthroplasty Design Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadine","family":"Sadaka","sequence":"additional","affiliation":[{"name":"Arthritis and Arthroplasty Design Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jewelia","family":"Rempuszewski","sequence":"additional","affiliation":[{"name":"Arthritis and Arthroplasty Design Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudette","family":"Moul","sequence":"additional","affiliation":[{"name":"Arthritis and Arthroplasty Design Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan B.","family":"Mandell","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Neel B","family":"Shah","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Division of Infectious Disease, University of Pittsburgh Medical Center, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James B.","family":"Doub","sequence":"additional","affiliation":[{"name":"Division of Clinical Care and Research, Institute of Human Virology, University of Maryland, Baltimore, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charlie","family":"Gish","sequence":"additional","affiliation":[{"name":"Arthritis and Arthroplasty Design Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dana","family":"Parker","sequence":"additional","affiliation":[{"name":"Arthritis and Arthroplasty Design Laboratory, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefanie A.","family":"Sydlik","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hunter","family":"Wood","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7726-0765","authenticated-orcid":false,"given":"Vaughn S.","family":"Cooper","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony R.","family":"Richardson","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert M.Q.","family":"Shanks","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenneth L.","family":"Urish","sequence":"additional","affiliation":[{"name":"Arthritis and Arthroplasty Design Group, The Bone and Joint Center, Magee-Womens Hospital of the University of Pittsburgh Medical Center; 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