{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T20:54:53Z","timestamp":1780520093686,"version":"3.54.1"},"reference-count":40,"publisher":"Oxford University Press (OUP)","issue":"8","license":[{"start":{"date-parts":[[2021,4,14]],"date-time":"2021-04-14T00:00:00Z","timestamp":1618358400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2021,7,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence\/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA\/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin\/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G\u2192A transition occurred in the transcriptional repressor tetR\/acrR adjacent to REQ_RS13460.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1093\/jac\/dkab121","type":"journal-article","created":{"date-parts":[[2021,3,24]],"date-time":"2021-03-24T12:09:42Z","timestamp":1616587782000},"page":"2040-2048","source":"Crossref","is-referenced-by-count":7,"title":["Ethidium bromide exposure unmasks an antibiotic efflux system in <i>Rhodococcus equi<\/i>"],"prefix":"10.1093","volume":"76","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5023-2828","authenticated-orcid":false,"given":"Elisa","family":"Rampacci","sequence":"first","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, via San Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Luisa","family":"Marenzoni","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, via San Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rolando","family":"Cannalire","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, University of Naples \u201cFederico II\u201d, via D. Montesano 49, 80131 Napoli, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donatella","family":"Pietrella","sequence":"additional","affiliation":[{"name":"Department of Medicine and Surgery, University of Perugia, via Gambuli 1, 06156 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefano","family":"Sabatini","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5043-7233","authenticated-orcid":false,"given":"Stefano","family":"Giovagnoli","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tommaso","family":"Felicetti","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Pepe","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, via San Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabrizio","family":"Passamonti","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine, University of Perugia, via San Costanzo 4, 06126 Perugia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2021,4,14]]},"reference":[{"key":"2021071609142591600_dkab121-B1","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1016\/j.cmi.2018.04.033","article-title":"Diagnosis and management of pulmonary infection due to Rhodococcus equi","volume":"25","author":"Lin","year":"2018","journal-title":"Clin Microbiol Infect"},{"key":"2021071609142591600_dkab121-B2","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1016\/S1473-3099(10)70068-2","article-title":"Rhodococcus equi infection","volume":"10","author":"Yamshchikov","year":"2010","journal-title":"Lancet Infect Dis"},{"key":"2021071609142591600_dkab121-B3","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1016\/j.vetmic.2019.07.010","article-title":"Prevalence and risk factors associated with emergence of Rhodococcus equi resistance to macrolides and rifampicin in horse-breeding farms in Kentucky, USA","volume":"235","author":"Huber","year":"2019","journal-title":"Vet Microbiol"},{"key":"2021071609142591600_dkab121-B4","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1007\/BF00158580","article-title":"Distribution and antimicrobial susceptibility of Rhodococcus equi from clinical specimens","volume":"8","author":"McNeil","year":"1992","journal-title":"Eur J Epidemiol"},{"key":"2021071609142591600_dkab121-B5","doi-asserted-by":"crossref","first-page":"633","DOI":"10.18388\/abp.2014_1824","article-title":"Antimicrobial resistance in Rhodococcus equi","volume":"61","author":"Cisek","year":"2014","journal-title":"Acta Biochim Pol"},{"key":"2021071609142591600_dkab121-B6","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1093\/jac\/dki171","article-title":"Efflux-mediated antimicrobial resistance","volume":"56","author":"Poole","year":"2005","journal-title":"J Antimicrob Chemother"},{"key":"2021071609142591600_dkab121-B7","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1016\/j.ijantimicag.2009.07.010","article-title":"The role of efflux pumps in macrolide resistance in Mycobacterium avium complex","volume":"34","author":"Rodrigues","year":"2009","journal-title":"Int J Antimicrob Agents"},{"key":"2021071609142591600_dkab121-B8","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1054\/drup.2000.0175","article-title":"The role of efflux in macrolide resistance","volume":"3","author":"Zhong","year":"2000","journal-title":"Drug Resist Updat"},{"key":"2021071609142591600_dkab121-B9","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1038\/ja.2015.9","article-title":"Study of efflux pump gene expression in rifampicin-monoresistant Mycobacterium tuberculosis clinical isolates","volume":"68","author":"Li","year":"2015","journal-title":"J Antibiot (Tokyo)"},{"key":"2021071609142591600_dkab121-B10","doi-asserted-by":"crossref","first-page":"6942","DOI":"10.1128\/jb.172.12.6942-6949.1990","article-title":"Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones","volume":"172","author":"Yoshida","year":"1990","journal-title":"J Bacteriol"},{"key":"2021071609142591600_dkab121-B11","doi-asserted-by":"crossref","first-page":"1396","DOI":"10.1128\/AAC.41.6.1396","article-title":"Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae","volume":"41","author":"Baranova","year":"1997","journal-title":"Antimicrob Agents Chemother"},{"key":"2021071609142591600_dkab121-B12","doi-asserted-by":"crossref","first-page":"e1001145.","DOI":"10.1371\/journal.pgen.1001145","article-title":"The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions","volume":"6","author":"Letek","year":"2010","journal-title":"PLoS Genet"},{"key":"2021071609142591600_dkab121-B13","doi-asserted-by":"crossref","first-page":"133.","DOI":"10.3389\/fphys.2014.00133","article-title":"Membrane transport systems and the biodegradation potential and pathogenicity of genus Rhodococcus","volume":"5","author":"de Carvalho","year":"2014","journal-title":"Front Physiol"},{"key":"2021071609142591600_dkab121-B14","doi-asserted-by":"crossref","first-page":"2404","DOI":"10.1128\/AAC.43.10.2404","article-title":"Multiple novel inhibitors of the NorA multidrug transporter of Staphylococcus aureus","volume":"43","author":"Markham","year":"1999","journal-title":"Antimicrob Agents Chemother"},{"key":"2021071609142591600_dkab121-B15","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1093\/jac\/dkn217","article-title":"Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide","volume":"62","author":"Couto","year":"2008","journal-title":"J Antimicrob Chemother"},{"key":"2021071609142591600_dkab121-B16","year":"2018"},{"key":"2021071609142591600_dkab121-B17","doi-asserted-by":"crossref","first-page":"12149.","DOI":"10.1038\/s41598-018-30715-z","article-title":"In vitro performances of novel co-spray-dried azithromycin\/rifampicin microparticles for Rhodococcus equi disease treatment","volume":"8","author":"Rampacci","year":"2018","journal-title":"Sci Rep"},{"key":"2021071609142591600_dkab121-B18","year":"2017"},{"key":"2021071609142591600_dkab121-B19","year":"2018"},{"key":"2021071609142591600_dkab121-B20","year":"2018"},{"key":"2021071609142591600_dkab121-B21","first-page":"365","volume-title":"Antibiotics in Laboratory Medicine - Fifth Edition","author":"Pillai","year":"2005"},{"key":"2021071609142591600_dkab121-B22","first-page":"657","article-title":"An instrument-free method for the demonstration of efflux pump activity of bacteria","volume":"20","author":"Martins","year":"2006","journal-title":"In Vivo"},{"key":"2021071609142591600_dkab121-B23","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1590\/S1517-83822014000200040","article-title":"Genotypic and phenotypic detection of efflux pump in Rhodococcus equi","volume":"45","author":"Gressler","year":"2014","journal-title":"Braz J Microbiol"},{"key":"2021071609142591600_dkab121-B24","first-page":"3184","article-title":"Novel transferable erm(46) determinant responsible for emerging macrolide resistance in Rhodococcus equi","volume":"70","author":"Anastasi","year":"2015","journal-title":"J Antimicrob Chemother"},{"key":"2021071609142591600_dkab121-B25","doi-asserted-by":"crossref","first-page":"703.","DOI":"10.1128\/JCM.02925-13","article-title":"Appearance of multidrug-resistant virulent Rhodococcus equi clinical isolates obtained in China","volume":"52","author":"Liu","year":"2014","journal-title":"J Clin Microbiol"},{"key":"2021071609142591600_dkab121-B26","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.vetmic.2006.01.015","article-title":"A nucleotide mutation associated with fluoroquinolone resistance observed in gyrA of in vitro obtained Rhodococcus equi mutants","volume":"115","author":"Niwa","year":"2006","journal-title":"Vet Microbiol"},{"key":"2021071609142591600_dkab121-B27","doi-asserted-by":"crossref","first-page":"3520","DOI":"10.1128\/AAC.01670-09","article-title":"Mutant selection window and characterization of allelic diversity for ciprofloxacin-resistant mutants of Rhodococcus equi","volume":"54","author":"Niwa","year":"2010","journal-title":"Antimicrob Agents Chemother"},{"key":"2021071609142591600_dkab121-B28","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1093\/jac\/dkx400","article-title":"Active antimicrobial efflux in Staphylococcus epidermidis: building up of resistance to fluoroquinolones and biocides in a major opportunistic pathogen","volume":"73","author":"Costa","year":"2018","journal-title":"J Antimicrob Chemother"},{"key":"2021071609142591600_dkab121-B29","doi-asserted-by":"crossref","first-page":"3235","DOI":"10.1128\/AAC.00430-07","article-title":"Efflux-related resistance to norfloxacin, dyes, and biocides in bloodstream isolates of Staphylococcus aureus","volume":"51","author":"DeMarco","year":"2007","journal-title":"Antimicrob Agents Chemother"},{"key":"2021071609142591600_dkab121-B30","author":"Venc\u00e1","year":"2012"},{"key":"2021071609142591600_dkab121-B31","doi-asserted-by":"crossref","first-page":"835","DOI":"10.1590\/S0100-736X2015001000003","article-title":"Biofilm formation by Rhodococcus equi and putative association with macrolide resistance","volume":"35","author":"Gressler","year":"2015","journal-title":"Pesq Vet Bras"},{"key":"2021071609142591600_dkab121-B32","doi-asserted-by":"crossref","first-page":"284.","DOI":"10.3390\/pathogens8040284","article-title":"Phenotypic characterization of Rhodococcus equi biofilm grown in vitro and inhibiting and dissolving activity of azithromycin\/rifampicin treatment","volume":"8","author":"Rampacci","year":"2019","journal-title":"Pathogens"},{"key":"2021071609142591600_dkab121-B33","doi-asserted-by":"crossref","first-page":"37007","DOI":"10.1039\/C7RA03859C","article-title":"Investigation on the effect of known potent S. aureus NorA efflux pump inhibitors on the staphylococcal biofilm formation","volume":"7","author":"Sabatini","year":"2017","journal-title":"RSC Adv"},{"key":"2021071609142591600_dkab121-B34","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/S0065-2164(07)00006-8","article-title":"Bacterial efflux transport in biotechnology","volume":"63","author":"Van Dyk","year":"2008","journal-title":"Adv Appl Microbiol"},{"key":"2021071609142591600_dkab121-B35","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1038\/s41579-018-0048-6","article-title":"Multidrug efflux pumps: structure, function and regulation","volume":"16","author":"Du","year":"2018","journal-title":"Nat Rev Microbiol"},{"key":"2021071609142591600_dkab121-B36","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1006\/scdb.2000.0248","article-title":"Transcriptional regulation of multidrug efflux pumps in bacteria","volume":"12","author":"Grkovic","year":"2001","journal-title":"Semin Cell Dev Biol"},{"key":"2021071609142591600_dkab121-B37","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1128\/MMBR.69.2.326-356.2005","article-title":"The TetR family of transcriptional repressors","volume":"69","author":"Ramos","year":"2005","journal-title":"Microbiol Mol Biol Rev"},{"key":"2021071609142591600_dkab121-B38","doi-asserted-by":"crossref","first-page":"1608","DOI":"10.1016\/j.cellsig.2013.04.003","article-title":"The underling mechanism of bacterial TetR\/AcrR family transcriptional repressors","volume":"25","author":"Deng","year":"2013","journal-title":"Cell Signal"},{"key":"2021071609142591600_dkab121-B39","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1021\/acsinfecdis.9b00041","article-title":"Modifications on C6 and C7 positions of 3-phenylquinolone efflux pump inhibitors led to potent and safe antimycobacterial treatment adjuvants","volume":"5","author":"Felicetti","year":"2019","journal-title":"ACS Infect Dis"},{"key":"2021071609142591600_dkab121-B40","doi-asserted-by":"crossref","first-page":"7827","DOI":"10.1021\/acs.jmedchem.8b00791","article-title":"2-Phenylquinoline S. aureus NorA efflux pump inhibitors: evaluation of the importance of methoxy group introduction","volume":"61","author":"Felicetti","year":"2018","journal-title":"J Med Chem"}],"container-title":["Journal of Antimicrobial Chemotherapy"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/academic.oup.com\/jac\/article-pdf\/76\/8\/2040\/39065546\/dkab121.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/academic.oup.com\/jac\/article-pdf\/76\/8\/2040\/39065546\/dkab121.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T09:20:27Z","timestamp":1626427227000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jac\/article\/76\/8\/2040\/6225959"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,4,14]]},"references-count":40,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,4,14]]},"published-print":{"date-parts":[[2021,7,15]]}},"URL":"https:\/\/doi.org\/10.1093\/jac\/dkab121","relation":{},"ISSN":["0305-7453","1460-2091"],"issn-type":[{"value":"0305-7453","type":"print"},{"value":"1460-2091","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2021,8,1]]},"published":{"date-parts":[[2021,4,14]]}}}