article · BMC Infectious Diseases
Exposure to low concentrations of antibiotics, below the level required to stop bacterial growth, can stimulate Staphylococcus aureus to form protective biofilms. Researchers examined five classes of antibiotics, namely azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, across five clinical isolates of the bacterium. Testing concentrations at twelve and a half, twenty-five, and fifty per cent of minimal inhibitory concentrations, they assessed biofilm responses using crystal violet staining, quantitative polymerase chain reaction, and spread plate counting. The findings revealed that sub-inhibitory doses induced biofilm development in sixty-four per cent of conditions evaluated by crystal violet staining. Imipenem and ciprofloxacin provoked the highest induction rates, followed by doxycycline, azithromycin, and gentamicin. This response increased total biofilm biomass rather than viable cell counts, showing that sub-inhibitory drug levels enhance the protective structure without diminishing viable bacteria.
When antibiotics fail to achieve concentrations sufficient to clear bacteria, they may inadvertently trigger Staphylococcus aureus to construct sturdier protective biofilms. Recognising that low drug exposures stimulate biomass without reducing viable bacterial numbers is critical for clinicians and researchers. It underscores the danger of inadequate antibiotic dosing, which can strengthen bacterial defences, worsen infection persistence, and hinder successful eradication.
This work represents early-stage laboratory research that could inform the design of antimicrobial dosing protocols and screening assays for anti-biofilm agents. The primary users would be pharmaceutical developers and clinical researchers seeking to evaluate drug efficacy and prevent treatment-induced resistance mechanisms. Because the evidence is derived exclusively from in vitro testing on five clinical isolates, practical translation into therapeutic guidelines or commercial products remains at a preliminary stage.
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BACKGROUND: Biofilm formation is an essential virulence factor that creates a highly protected growth mode for Staphylococcus aureus (S. aureus) to survive in any hostile environment. Antibiotic sub-minimal inhibitory concentration (sub-MIC) may modulate the biofilm formation ability of bacterial pathogens, thereby affecting bacterial pathogenesis and infection outcomes. Intense antimicrobial therapy to treat biofilm-associated infections can control the pathogenic infection aggravation but cannot guarantee its complete eradication. OBJECTIVE: This study aimed to assess the sub-MICs effect of 5 different antimicrobial classes on biofilm-forming capacity among Staphylococcus aureus clinical isolates using three different biofilm quantitation techniques. METHODS: In this study, the effects of 5 different antimicrobial agents, namely, azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, at sub-MICs of 12.5%, 25%, and 50% were tested on 5 different clinical isolates of S. aureus. The biofilms formed in the absence and presence of different antimicrobial sub-MICs were then assessed using the following three different techniques: the crystal violet (CV) staining method, the quantitative PCR (qPCR) method, and the spread plate method (SPM). RESULTS: Biofilm formation was significantly induced in 64% of the tested conditions using the CV technique. On the other hand, the qPCR quantifying the total bacterial count and the SPM quantifying the viable bacterial count showed significant induction only in 24% and 17.3%, respectively (Fig. 1). The difference between CV and the other techniques indicates an increase in biofilm biomass without an increase in bacterial growth. As expected, sub-MICs did not reduce the viable cell count, as shown by the SPM. The CV staining method revealed that sub-MICs of imipenem and ciprofloxacin had the highest significance rate (80%) showing an inductive effect on the biofilm development. On the other hand, doxycycline, azithromycin, and gentamicin displayed lower significance rates of 73%, 53%, and 47%, respectively. CONCLUSION: Exposure to sub-MIC doses of antimicrobial agents induces the biofilm-forming capacity of S. aureus via increasing the total biomass without significantly affecting the bacterial growth of viable count.
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DOI: 10.1186/s12879-024-09790-3
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