A Standardised Combinational Method for Evaluating Antimicrobial Compounds Against Biofilm Attachment, Development...

Biofilm-mediated antimicrobial resistance remains a significant challenge for healthcare and patient safety. Currently, there are gaps in standardised methods for assessing antimicrobials against biofilm formations such as (1) assessment of initial bacterial attachment inhibition, as well as (2) assessment of antimicrobial compounds against both the external biofilm mass and biofilm-embedded metabolically active bacteria. The aim of this study is to address these gaps by combining several anti-biofilm techniques. In the procedure96-well anti-biofilm assessments were performed using plate well and lid peg growth surfaces so as to determine the effects of bioactive compounds (silver nitrate (AgNO₃), nisin, chitosan and zinc oxide nanopowder (ZnO)) on biofilm growth inhibition, formed biofilm reduction and bacterial attachment inhibition. These studies focused on the initial attachment stage against in vitro biofilms of P. aeruginosa and S. aureus. Effects were measured against biofilm mass using Crystal Violet (CV) staining, while embedded bacteria metabolic activity was measured using Resazurin. AgNO₃ exhibited significant inhibition and reduction against P. aeruginosa at all stages of biofilm development (p < 0.0001). AgNO₃ showed significant results against S. aureus during biofilm development and against the embedded, metabolically active population of established biofilms (p < 0.0001). Nisin showed significant inhibition against S. aureus biofilm populations (p < 0.0001). Chitosan showed significant increases in S. aureus biofilm formations following exposure, during initial attachment (p < 0.02), during biofilm growth (p < 0.0001) and against formed biofilm populations (p < 0.0001). ZnO showed significant increases during initial attachment exposure (p < 0.0001), but also exhibited growth inhibition (p < 0.0001) and biofilm reduction (p < 0.0001). Although variance in anti-biofilm efficacy was evident depending upon treatment used, Gram-staining phenotype and test growth surfaces, this combinational method offers potential for high throughput screening and for evaluating pipeline bioactives isolated from different environments for biofilm prevention, inhibition and removal. Additionally, this approach will help elucidate the relationship between bacteria of interest and biofilm mitigation.