{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T20:34:48Z","timestamp":1791491688615,"version":"4.3.4"},"reference-count":36,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,11,30]],"date-time":"2021-11-30T00:00:00Z","timestamp":1638230400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Vet. Sci."],"abstract":"<jats:p>The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the <jats:italic>mecA<\/jats:italic> gene, which encodes for the PBP2a protein and lends the resistance to \u03b2-lactams. Verifying the correspondence between gene harboring and protein expression and accelerating methicillin resistance diagnosis is critical to improve the management of antimicrobial administration and to reduce the spread of drug resistances. We tested the applicability of immunofluorescence targeting PBP2a protein to identify a new potential methicillin resistance screening test, ancillary to conventional culture methods. We collected 26 clinical <jats:italic>Staphylococcus pseudintermedius<\/jats:italic> (SP) isolates: 25 from canine pyoderma and 1 from dermatitis in a dog owner. SP is one of the most important etiological agents in canine pyoderma and can harbor the <jats:italic>mecA<\/jats:italic> gene. We performed PCR for <jats:italic>mecA<\/jats:italic> gene detection, broth microdilution (BMD) for phenotypic methicillin resistance, and immunofluorescence targeting PBP2a protein. Compared to the PCR as the gold standard, immunofluorescence showed an apparent prevalence of 34.6% vs. a true prevalence of 53.8%, with 100% specificity, 64.3% sensitivity, and 80.8% diagnostic accuracy. PBP2a expression showed isolate-dependent variability: in some isolates, most of the bacterial cells showed an intense and clearly membranous pattern, while in others only a few of them could be detected. Performing the assay in duplicate improved the diagnostic accuracy. Since the <jats:italic>mecA<\/jats:italic> gene is shared among the members of the <jats:italic>Staphylococcus<\/jats:italic> genus, the test can be applied to identify methicillin resistance independently from the staphylococcal species, both in human and animal samples. Being a rapid and easy method and providing the unique possibility to study the expression of PBP2a by directly visualizing the morphology, it could represent a new interesting tool for both research and diagnostics. To accelerate methicillin resistance diagnosis, it would be worth further testing of its performance on cytological samples.<\/jats:p>","DOI":"10.3389\/fvets.2021.740934","type":"journal-article","created":{"date-parts":[[2021,12,1]],"date-time":"2021-12-01T18:31:04Z","timestamp":1638383464000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test"],"prefix":"10.3389","volume":"8","author":[{"given":"Serenella","family":"Silvestri","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elisa","family":"Rampacci","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valentina","family":"Stefanetti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michele","family":"Trotta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caterina","family":"Fani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucia","family":"Levorato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiara","family":"Brachelente","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabrizio","family":"Passamonti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,11,30]]},"reference":[{"key":"B1","unstructured":"Ten Treats to Global Health in 20192019"},{"key":"B2","doi-asserted-by":"publisher","first-page":"e01788","DOI":"10.1128\/JCM.01788-17","article-title":"Epidemiology, clinical characteristics, antimicrobial susceptibility profiles of human clinical isolates of Staphylococcus intermedius group","volume":"56","author":"Yarbrough","year":"2018","journal-title":"J Clin Microbiol."},{"key":"B3","doi-asserted-by":"publisher","first-page":"103","DOI":"10.7883\/yoken.66.103","article-title":"Detection of methicillin-resistant Staphylococcus aureus using a specific anti-PBP2a chicken IgY antibody","volume":"66","author":"Yamada","year":"2013","journal-title":"Jpn J Infect Dis."},{"key":"B4","doi-asserted-by":"publisher","first-page":"3791","DOI":"10.1128\/AAC.02731-13","article-title":"The mecA homolog mecC confers resistance against \u03b2-lactams in Staphylococcus aureus irrespective of the genetic strain background","volume":"58","author":"Ballhausen","year":"2014","journal-title":"Antimicrob Agents Chemother."},{"key":"B5","doi-asserted-by":"publisher","first-page":"471","DOI":"10.1016\/j.diagmicrobio.2016.05.008","article-title":"Human infections due to Staphylococcus pseudintermedius, an emerging zoonosis of canine origin: report of 24 cases","volume":"85","author":"Somayaji","year":"2016","journal-title":"Diagn Microbiol Infect Dis."},{"key":"B6","first-page":"204","article-title":"Canine superficial pyoderma and therapeutic considerations","volume":"52","author":"Bajwa","year":"2016","journal-title":"Diagnostic Dermatology."},{"key":"B7","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1016\/j.tvjl.2018.04.002","article-title":"What has changed in canine pyoderma. A narrative review","volume":"235","author":"Loeffler","year":"2018","journal-title":"Vet J."},{"key":"B8","first-page":"65","article-title":"Antimicrobial susceptibility of Staphylococcus pseudintermedius colonizing healthy dogs in Saskatoon, Canada","volume":"57","author":"Priyantha","year":"2016","journal-title":"Can Vet J La Rev Vet Can."},{"key":"B9","doi-asserted-by":"publisher","first-page":"e00206","DOI":"10.1128\/AAC.01096-18","article-title":"Genetic determinants of high-level oxacillin resistance in methicillin-resistant Staphylococcus aureus","volume":"62","author":"Pardos de la Gandara","year":"2018","journal-title":"Antimicrob Agents Chemother"},{"key":"B10","doi-asserted-by":"publisher","first-page":"2680","DOI":"10.1128\/AAC.40.12.2680","article-title":"Suppression of methicillin resistance in a mecA-containing pre-methicillin-resistant Staphylococcus aureus strain is caused by the mecI-mediated repression of PBP 2 production","volume":"40","author":"Kuwahara-Arai","year":"1996","journal-title":"Antimicrob Agents Chemother."},{"key":"B11","doi-asserted-by":"publisher","first-page":"e23287","DOI":"10.1371\/journal.pone.0023287","article-title":"Methicillin-resistance in Staphylococcus aureus is not affected by the overexpression in trans of the mecA gene repressor: a surprising observation","volume":"6","author":"Oliveira","year":"2011","journal-title":"PLoS ONE."},{"key":"B12","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1186\/s12941-016-0167-8","article-title":"Rapid screening for antibiotic resistance elements on the RNA transcript, protein and enzymatic activity level","volume":"15","author":"Rohde","year":"2016","journal-title":"Ann Clin Microbiol Antimicrob."},{"key":"B13","doi-asserted-by":"publisher","first-page":"1145","DOI":"10.1093\/jac\/dkq078","article-title":"Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study","volume":"65","author":"Perreten","year":"2010","journal-title":"J Antimicrob Chemother."},{"key":"B14","doi-asserted-by":"publisher","first-page":"289","DOI":"10.12834\/VetIt.465.2275.6","article-title":"Investigation of the antibiotic resistance and biofilm formation of Staphylococcus pseudintermedius strains isolated from canine pyoderma","volume":"53","author":"Stefanetti","year":"2017","journal-title":"Vet Ital."},{"key":"B15","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1016\/j.vetmic.2017.09.015","article-title":"Frequency, antimicrobial susceptibility and clonal distribution of methicillin-resistant Staphylococcus pseudintermedius in canine clinical samples submitted to a veterinary diagnostic laboratory in Italy: A 3-year retrospective investigation","volume":"211","author":"Ventrella","year":"2017","journal-title":"Vet Microbiol."},{"key":"B16","doi-asserted-by":"publisher","first-page":"e0254382","DOI":"10.1371\/journal.pone.0254382","article-title":"Genomic insights into methicillin-resistant Staphylococcus pseudintermedius isolates from dogs and humans of the same sequence types reveals diversity in prophages and pathogenicity islands","volume":"16","author":"Phumthanakorn","year":"2021","journal-title":"PLoS ONE."},{"key":"B17","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1016\/j.meegid.2008.11.003","article-title":"Methicillin-resistant commensal staphylococci in healthy dogs as a potential zoonotic reservoir for community-acquired antibiotic resistance","volume":"9","author":"Epstein","year":"2009","journal-title":"Infect Genet Evol."},{"key":"B18","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1016\/j.rvsc.2011.06.017","article-title":"Screening for methicillin-resistant staphylococci in dogs admitted to a veterinary teaching hospital","volume":"93","author":"Vanderhaeghen","year":"2012","journal-title":"Res Vet Sci."},{"key":"B19","doi-asserted-by":"publisher","first-page":"857","DOI":"10.1002\/mbo3.258","article-title":"Methicillin-resistant Staphylococcus pseudintermedius (MRSP) from healthy dogs in Norway - occurrence, genotypes and comparison to clinical MRSP","volume":"4","author":"Kjellman","year":"2015","journal-title":"Microbiologyopen."},{"key":"B20","doi-asserted-by":"publisher","first-page":"e0034921","DOI":"10.1128\/JCM.00349-21","article-title":"Comparative performances of Vitek-2, disk diffusion, and broth microdilution for antimicrobial susceptibility testing of canine Staphylococcus pseudintermedius","volume":"59","author":"Rampacci","year":"2021","journal-title":"J Clin Microbiol"},{"key":"B21","doi-asserted-by":"publisher","first-page":"469","DOI":"10.1128\/JCM.01915-08","article-title":"Molecular diagnostic identification of Staphylococcus pseudintermedius","volume":"47","author":"Bannoehr","year":"2009","journal-title":"J Clin Microbiol"},{"key":"B22","doi-asserted-by":"publisher","first-page":"268","DOI":"10.1111\/j.1469-0691.2011.03570.x","article-title":"Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance","volume":"18","author":"Magiorakos","year":"2012","journal-title":"Clin Microbiol Infect."},{"key":"B23","doi-asserted-by":"publisher","first-page":"535","DOI":"10.1128\/JCM.02864-15","article-title":"Evaluation of oxacillin and cefoxitin disk and MIC breakpoints for prediction of methicillin resistance in human and veterinary isolates of Staphylococcus intermedius Group","volume":"54","author":"Wu","year":"2016","journal-title":"J Clin Microbiol."},{"key":"B24","doi-asserted-by":"publisher","first-page":"4089","DOI":"10.1128\/JCM.41.9.4089-4094.2003","article-title":"Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus","volume":"41","author":"Strommenger","year":"2003","journal-title":"J Clin Microbiol"},{"key":"B25","doi-asserted-by":"publisher","first-page":"745","DOI":"10.1128\/JCM.02869-15","article-title":"Evaluation of an immunochromatographic assay for rapid detection of penicillin-binding protein 2a in human and animal Staphylococcus intermedius Group, Staphylococcus lugdunensis, and Staphylococcus schleiferi clinical isolates","volume":"54","author":"Arnold","year":"2016","journal-title":"J Clin Microbiol."},{"key":"B26","doi-asserted-by":"publisher","first-page":"e01934","DOI":"10.1128\/JCM.01934-17","article-title":"CLSI methods development and standardization working group best practices for evaluation of antimicrobial susceptibility tests","volume":"56","author":"Humphries","year":"2018","journal-title":"J Clin Microbiol."},{"key":"B27","doi-asserted-by":"publisher","first-page":"159","DOI":"10.2307\/2529310","article-title":"The measurement of observer agreement for categorical data","volume":"33","author":"Landis","year":"1977","journal-title":"Biometrics."},{"key":"B28","first-page":"203","article-title":"Measures of diagnostic accuracy: basic definitions","volume":"19","author":"\u0160imundi\u0107","year":"2009","journal-title":"EJIFCC."},{"key":"B29","doi-asserted-by":"publisher","first-page":"e2019007","DOI":"10.4178\/epih.e2019007","article-title":"Diagnostic test accuracy: application and practice using R software","volume":"41","author":"Shim","year":"2019","journal-title":"Epidemiol Health."},{"key":"B30","volume-title":"R: A Language and Environment for Statistical Computing","author":"R Core","year":"2020"},{"key":"B31","volume-title":"Reflection Paper on Meticillin-Resistant Staphylococcus pseudintermedius","year":"2010"},{"key":"B32","volume-title":"Muller&Kirk's Small Animal Dermatology. 7th ed","author":"Miller","year":"2013"},{"key":"B33","doi-asserted-by":"publisher","first-page":"1961","DOI":"10.1128\/JCM.00068-17","article-title":"Detection of mecC-positive Staphylococcus aureus: what to expect from immunological tests targeting PBP2a?","volume":"55","author":"Dupieux","year":"2017","journal-title":"J Clin Microbiol."},{"key":"B34","doi-asserted-by":"publisher","first-page":"e00033","DOI":"10.1128\/mSphere.00033-19","article-title":"Characterization of agr Groups of Staphylococcus pseudintermedius Isolates from Dogs in Texas","volume":"4","author":"Little","year":"2019","journal-title":"mSphere."},{"key":"B35","doi-asserted-by":"publisher","first-page":"1591","DOI":"10.1128\/JCM.37.5.1591-1594.1999","article-title":"Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus","volume":"37","author":"Cavassini","year":"1999","journal-title":"J Clin Microbiol"},{"key":"B36","doi-asserted-by":"publisher","first-page":"3946","DOI":"10.1128\/JCM.39.11.3946-3951.2001","article-title":"Methicillin-resistant Staphylococcus aureus: Comparison of susceptibility testing methods and analysis of mecA-positive susceptible strains","volume":"39","author":"Sakoulas","year":"2001","journal-title":"J Clin Microbiol."}],"container-title":["Frontiers in Veterinary Science"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fvets.2021.740934\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,12,1]],"date-time":"2021-12-01T18:31:14Z","timestamp":1638383474000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fvets.2021.740934\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,11,30]]},"references-count":36,"alternative-id":["10.3389\/fvets.2021.740934"],"URL":"https:\/\/doi.org\/10.3389\/fvets.2021.740934","relation":{},"ISSN":["2297-1769"],"issn-type":[{"value":"2297-1769","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,11,30]]},"article-number":"740934"}}