A multipronged approach for systematic in vitro quantification of catheter-associated biofilms

Abstract Biofilms are a leading cause of infections, especially those initiated on medical tubing. Quantification of pathogenic biofilms is critical for systematic determination of infections, antibiotics prescription, and implant replacement. By the time infectious pathogens are detected in fluids such as blood or urine, substantial and potentially life-threatening biofilms are likely to have already formed. These biofilms can have broad ranging patient care and cost implications. Here we propose a non-strain-specific protocol combining four microbiological assays, which are rarely used together in clinical biofilm assessment, to accurately quantify cellular and extracellular components attached to medical surfaces. Our results demonstrate that the shortcomings of standard approaches can be overcome through the conjunct analysis of total protein (modified Lowry), total biomass (quantitative polymerase chain reaction), cellular activity (ATP luminescence), and extracellular polymeric substances (Periodic Acid-Schiff assay). The efficacy of this multipronged approach was verified using four pathogenic, clinical isolates (Pseudomonas aeruginosa, Staphylococcus aureus, methicillin resistant-Staphylococcus aureus, and Candida albicans) and two types of silicone catheters in vitro. Despite the variation of biofilm matrices among infectious agents, this approach comprehensively quantified the pathogenic load as well as fouling resistance in indwelling catheters obtained from patients. The sensitivity, reproducibility, multi-species specificity, and high-throughput potential makes this approach valuable for quality assessment of catheters, implants, and ventilators in hospital settings.