{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T17:20:20Z","timestamp":1788801620170,"version":"build-2803163510"},"reference-count":76,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,6]],"date-time":"2026-06-06T00:00:00Z","timestamp":1780704000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100016247","name":"Wroc\u0142aw University of Environmental and Life Sciences","doi-asserted-by":"publisher","award":["No. N070\/0011\/24"],"award-info":[{"award-number":["No. N070\/0011\/24"]}],"id":[{"id":"10.13039\/100016247","id-type":"DOI","asserted-by":"publisher"}]},{"award":["No. N070\/0011\/24"],"award-info":[{"award-number":["No. N070\/0011\/24"]}],"id":[{"id":"https:\/\/ror.org\/05cs8k179","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Background:\u00a0Enterococcus faecalis is one of the most frequent causes of catheter-associated urinary tract infections, largely due to its ability to form biofilms on indwelling urinary catheter surfaces, which enhance bacterial persistence and antimicrobial tolerance. Sub-minimum inhibitory concentrations (sub-MICs) of antimicrobials frequently occur in clinical settings, and growing evidence suggests that such suboptimal exposures can induce bacterial biofilm formation. We hypothesized that exposure to sub-MICs of amoxicillin, ciprofloxacin, and nitrofurantoin, antimicrobials commonly employed in the treatment of urinary tract infections, would enhance the biofilm-forming capacity of E. faecalis strains. Objective: To investigate the effects of sub-MICs of amoxicillin, ciprofloxacin, and nitrofurantoin on biofilm formation and biofilm-associated gene expression. The study focused on key biofilm-related genes, including those encoding aggregation substance protein (asa1), collagen adhesin (ace), E. faecalis surface protein (esp), gelatinase (gelE), cytolysin activator A (cylA), endocarditis antigen A (efaA), and the endocarditis- and biofilm-associated pili subunit A (ebpA) in E. faecalis. Methods: Two strains, E. faecalis ATCC 29212 and strain 54, were exposed to 1\/8\u00d7 and 1\/4\u00d7 MIC of amoxicillin, ciprofloxacin, and nitrofurantoin in either artificial urine medium (AUM) or tryptone soya broth (TSB). Bacterial growth kinetics were monitored by optical density measurements, while biofilm formation was quantified using a microtiter plate biofilm assay. The expression of biofilm-associated genes was analyzed using quantitative reverse transcription PCR (RT-qPCR) at 24 and 48 h following exposure to sub-MICs of amoxicillin under flow conditions mimicking the urinary tract milieu. Results: Exposure to sub-MICs of the three antimicrobials did not significantly affect bacterial growth in either strain or culture medium. Sub-MICs of amoxicillin significantly enhanced biofilm formation, with the most pronounced effect observed at 1\/4\u00d7 MIC in both AUM and TSB. In contrast, ciprofloxacin and nitrofurantoin exerted inhibitory effects on biofilm formation across both media. Gene expression analysis demonstrated time- and strain-dependent responses to amoxicillin exposure. E. faecalis ATCC 29212 exhibited a moderate, coordinated upregulation of adhesion- and biofilm-associated genes, particularly at 48 h. By comparison, E. faecalis strain 54 showed a stronger and more dynamic transcriptional response, characterized by early and sustained induction of key biofilm-related genes, including esp and gelE, as well as a pronounced late upregulation of ebpA. Conclusions: These findings emphasize the importance of maintaining therapeutically effective antimicrobial concentrations, as sub-inhibitory amoxicillin exposure may promote biofilm-associated persistence and potentially compromise treatment efficacy.<\/jats:p>","DOI":"10.3390\/molecules31121986","type":"journal-article","created":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T02:32:46Z","timestamp":1780885966000},"page":"1986","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Sub-Minimum Inhibitory Concentrations of Amoxicillin Modulate Biofilm Formation and the Expression of Biofilm-Associated Genes in Enterococcus faecalis"],"prefix":"10.3390","volume":"31","author":[{"given":"Desiye T.","family":"Tegegne","sequence":"first","affiliation":[{"name":"Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroc\u0142aw University of Environmental and Life Sciences, ul. Norwida 31, 50-375 Wroclaw, Poland"},{"name":"Animal Biotechnology Research Program, National Agricultural Biotechnology Research Center, Ethiopian Institute of Agricultural Research, Holeta P.O. Box 249, Ethiopia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6494-5757","authenticated-orcid":false,"given":"Sylwia","family":"Banaszkiewicz","sequence":"additional","affiliation":[{"name":"Department of Food Hygiene and Consumer Health Protection, Faculty of Veterinary Medicine, Wroc\u0142aw University of Environmental and Life Sciences, ul. Norwida 31, 50-375 Wroclaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacek","family":"Bania","sequence":"additional","affiliation":[{"name":"Department of Food Hygiene and Consumer Health Protection, Faculty of Veterinary Medicine, Wroc\u0142aw University of Environmental and Life Sciences, ul. Norwida 31, 50-375 Wroclaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5813-1404","authenticated-orcid":false,"given":"B\u0142a\u017cej","family":"Po\u017aniak","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroc\u0142aw University of Environmental and Life Sciences, ul. Norwida 31, 50-375 Wroclaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1186\/2047-2994-3-23","article-title":"Catheter associated urinary tract infections","volume":"3","author":"Nicolle","year":"2014","journal-title":"Antimicrob. Resist. Infect. 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