21.15109/ARP/QM0ZUOBuni, DominikaDominikaBuni0009-0004-1692-7828Veterinary Medical Research InstituteKovács, Áron BotondÁron BotondKovács0000-0001-6013-819XVeterinary Medical Research InstituteWehmann, EnikőEnikőWehmann0000-0002-1229-1146Veterinary Medical Research InstituteGrózner, DénesDénesGrózner0000-0002-2154-7537Veterinary Medical Research InstituteBányai, KrisztiánKrisztiánBányai0000-0002-6270-1772Veterinary Medical Research InstituteNagy, Eszter ZsófiaEszter ZsófiaNagy57950528900Veterinary Medical Research InstituteBradbury, JanetJanetBradbury0000-0003-2416-220XInstitute of Infection, Veterinary and Ecological Sciences, University of LiverpoolBottinelli, MarcoMarcoBottinelli0000-0003-4654-206XIstituto Zooprofilattico Sperimentale delle VenezieStefani, ElisabettaElisabettaStefani0009-0004-4939-7480Istituto Zooprofilattico Sperimentale delle VenezieCatania, SalvatoreSalvatoreCatania0000-0003-3258-172XIstituto Zooprofilattico Sperimentale delle VenezieLysnyansky, InnaInnaLysnyansky0000-0002-6923-3676Department of Avian Diseases, Kimron Veterinary InstituteKovács, LászlóLászlóKovács0009-0009-9281-2543University of Veterinary Medicine BudapestGyuranecz, MiklósMiklósGyuranecz0000-0001-9085-5923Veterinary Medical Research InstituteKreizinger, ZsuzsaZsuzsaKreizinger0000-0002-6365-0719Veterinary Medical Research InstituteSupplementary data – M. iowae antimicrobial resistance genetic markers – Buni, 2026ARP2026Agricultural SciencesMycoplasma iowaeAntimicrobial resistanceEfflux pumpsMismatch amplification mutation assay (MAMA)OrthovanadateTurkey pathogenKreizinger, ZsuzsaZsuzsaKreizingerVeterinary Medical Research Institute2026-06-112026-06-1210.1371/journal.pone.03473451319632297777675671573730373123862645818651381316131173351934910840text/markdownapplication/vnd.openxmlformats-officedocument.spreadsheetml.sheettext/csvtext/csvtext/csvapplication/vnd.openxmlformats-officedocument.spreadsheetml.sheettext/csvapplication/vnd.openxmlformats-officedocument.spreadsheetml.sheettext/csvapplication/vnd.openxmlformats-officedocument.spreadsheetml.sheetapplication/vnd.openxmlformats-officedocument.spreadsheetml.sheettext/csvtext/csvtext/csvapplication/vnd.openxmlformats-officedocument.spreadsheetml.sheet1.0CC BY-NC 4.0<i>Mycoplasma iowae</i> is an economically significant pathogen that causes reduced hatchability, late embryo mortality and leg deformities, chondrodystrophy and skeletal lesions in poults. While prevention is essential in the control of infection, the appropriate administration of antibiotics may reduce economic losses during outbreaks. As a first step in the exploration of antimicrobial resistance mechanisms in <i>M. iowae</i>, target modification and efflux pump activity were examined in the present study. Point mutations were analyzed in previously described antibiotic binding sites in the whole genome sequences of 99 <i>M. iowae</i> strains. Mismatch amplification mutation assays (MAMAs) were designed and validated for the differentiation of mutations corresponding to elevated minimum inhibitory concentration (MIC) values for fluoroquinolones. Broth microdilution assays were performed to evaluate the effect of efflux pump inhibitors. In the presence of orthovanadate (OV), MIC values were significantly lower than in the absence of OV for spiramycin, tilmicosin, tylosin and oxytetracycline, which may indicate the presence of an active efflux system in <i>M. iowae</i>. Putative promoter regions of efflux-related genes were predicted and characterized. Genetic mutations, previously described in other bacteria, were described to be associated with elevated fluoroquinolone, macrolide and lincomycin MICs in <i>M. iowae</i>, although certain resistant phenotypes remained unexplained, promoting future examinations for deeper insights. The developed MAMAs may support rapid identification of <i>M. iowae</i> strains with elevated MIC values for fluoroquinolones. The better understanding of the efflux pump mechanisms enables the development of alternative methods for the support of therapy against this pathogen.National Research, Development and Innovation OfficeFK21 (137809)National Research, Development and Innovation OfficeTKP2021-EGA-01Eötvös Loránd Research NetworkSA-27/2021Recovery and Resilience FacilityRRF-2.3.1-21-2022-00001Recovery and Resilience FacilityRRF-2.3.1-21-2022-00006