{"title":"Role and regulation of membrane efflux pumps in Staphylococcus pseudintermedius clinical isolates","authors":"Elisa Rampacci , Tommaso Felicetti , Francesco Birettoni , Riccardo Zelli , Rolando Arcelli , Stefano Sabatini , Fabrizio Passamonti","doi":"10.1016/j.vetmic.2025.110574","DOIUrl":null,"url":null,"abstract":"<div><div>The overexpression of efflux pumps (EPs) is one of the strategies through which <em>Staphylococcus</em> spp. survive antibiotic treatment. This process is regulated by intricate and still not fully understood mechanisms. This study aimed to evaluate the efflux capability of <em>Staphylococcus pseudintermedius</em> clinical isolates, investigate its implications in multidrug resistance, and elucidate the molecular mechanisms underlying EP expression. Additionally, potential therapeutic approaches for inhibiting staphylococcal EPs were assessed. Phenotypic characterization of efflux activity included ethidium bromide (EtBr) MIC screening, fluorometry-based accumulation and efflux assays, and synergy tests with EP inhibitors (EPIs). The genetic basis of high efflux phenotypes was explored using WGS and qPCR. Based on EtBr MIC, nine out of 109 isolates (8.3 %) progressed to fluorometry tests, confirming high phenotypic efflux in six isolates (Spearman r<sub>s</sub>= 0.886). These were classified into distinct multilocus sequence types (ST71, ST496, ST551 and ST1062), with two isolates representing novel STs, designated ST2837 and ST2838. Synergism was observed when combining an EPI with EtBr, ciprofloxacin, clindamycin, and chloramphenicol; however, no EPI restored full susceptibility to these antibiotics. All isolates contained at least one plasmid-encoded EP gene, with <em>qacG</em> being the most common (6/6), followed by <em>qacH</em> (3/6), <em>tetK</em> (3/6), <em>smr</em> (2/6), <em>qacJ</em> (1/6), and <em>msrA/B</em> (1/6). Variable expression of EP genes was noted, with MFS-JC286_RS07290 overexpressed in all isolates, while MFS-JC286_RS11100 and MFS-JC286_RS11015 showed the highest expression levels. Comparative alignments suggested that the observed high efflux is unlikely due to mutational events upstream. These findings imply a potential role for transcriptional regulators, highlighting new research directions in this area.</div></div>","PeriodicalId":23551,"journal":{"name":"Veterinary microbiology","volume":"306 ","pages":"Article 110574"},"PeriodicalIF":2.7000,"publicationDate":"2025-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Veterinary microbiology","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0378113525002093","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
The overexpression of efflux pumps (EPs) is one of the strategies through which Staphylococcus spp. survive antibiotic treatment. This process is regulated by intricate and still not fully understood mechanisms. This study aimed to evaluate the efflux capability of Staphylococcus pseudintermedius clinical isolates, investigate its implications in multidrug resistance, and elucidate the molecular mechanisms underlying EP expression. Additionally, potential therapeutic approaches for inhibiting staphylococcal EPs were assessed. Phenotypic characterization of efflux activity included ethidium bromide (EtBr) MIC screening, fluorometry-based accumulation and efflux assays, and synergy tests with EP inhibitors (EPIs). The genetic basis of high efflux phenotypes was explored using WGS and qPCR. Based on EtBr MIC, nine out of 109 isolates (8.3 %) progressed to fluorometry tests, confirming high phenotypic efflux in six isolates (Spearman rs= 0.886). These were classified into distinct multilocus sequence types (ST71, ST496, ST551 and ST1062), with two isolates representing novel STs, designated ST2837 and ST2838. Synergism was observed when combining an EPI with EtBr, ciprofloxacin, clindamycin, and chloramphenicol; however, no EPI restored full susceptibility to these antibiotics. All isolates contained at least one plasmid-encoded EP gene, with qacG being the most common (6/6), followed by qacH (3/6), tetK (3/6), smr (2/6), qacJ (1/6), and msrA/B (1/6). Variable expression of EP genes was noted, with MFS-JC286_RS07290 overexpressed in all isolates, while MFS-JC286_RS11100 and MFS-JC286_RS11015 showed the highest expression levels. Comparative alignments suggested that the observed high efflux is unlikely due to mutational events upstream. These findings imply a potential role for transcriptional regulators, highlighting new research directions in this area.
期刊介绍:
Veterinary Microbiology is concerned with microbial (bacterial, fungal, viral) diseases of domesticated vertebrate animals (livestock, companion animals, fur-bearing animals, game, poultry, fish) that supply food, other useful products or companionship. In addition, Microbial diseases of wild animals living in captivity, or as members of the feral fauna will also be considered if the infections are of interest because of their interrelation with humans (zoonoses) and/or domestic animals. Studies of antimicrobial resistance are also included, provided that the results represent a substantial advance in knowledge. Authors are strongly encouraged to read - prior to submission - the Editorials (''Scope or cope'' and ''Scope or cope II'') published previously in the journal. The Editors reserve the right to suggest submission to another journal for those papers which they feel would be more appropriate for consideration by that journal.
Original research papers of high quality and novelty on aspects of control, host response, molecular biology, pathogenesis, prevention, and treatment of microbial diseases of animals are published. Papers dealing primarily with immunology, epidemiology, molecular biology and antiviral or microbial agents will only be considered if they demonstrate a clear impact on a disease. Papers focusing solely on diagnostic techniques (such as another PCR protocol or ELISA) will not be published - focus should be on a microorganism and not on a particular technique. Papers only reporting microbial sequences, transcriptomics data, or proteomics data will not be considered unless the results represent a substantial advance in knowledge.
Drug trial papers will be considered if they have general application or significance. Papers on the identification of microorganisms will also be considered, but detailed taxonomic studies do not fall within the scope of the journal. Case reports will not be published, unless they have general application or contain novel aspects. Papers of geographically limited interest, which repeat what had been established elsewhere will not be considered. The readership of the journal is global.