Formulation of Biocides Increases Antimicrobial Potency and Mitigates the Enrichment of Non-Susceptible Bacteria in Multi-Species Biofilms

The current investigation aimed to generate data to inform the development of risk-assessments of biocide usage. Stabilised domestic drain biofilm microcosms were exposed daily over six months to increasing concentrations (0.01 to 1 %) of the biocide benzalkonium chloride in simple aqueous solution (BAC-s) or in a complex formulation (BAC-f) representative of a domestic cleaning agent. Biofilms were analysed by culture, differentiating by bacterial functional group and by BAC or antibiotic susceptibility. Bacterial isolates were identified by 16S rRNA sequencing and changes in biofilm composition were assessed by high throughput sequencing. Exposure to BAC-f resulted in significantly larger reductions in viable bacteria than BAC-s, whilst bacterial diversity greatly decreased during exposure to both BAC-s and BAC-f, as evidenced by sequencing and viable counts. Increases in the abundance of bacteria exhibiting reduced antibiotic or BAC susceptibility following exposure to BAC at 0.1 % were significantly greater for BAC-s than BAC-f. Bacteria with reduced BAC and antibiotic susceptibility were generally suppressed by higher BAC concentrations and formulation significantly enhanced this effect. Significant decreases in the antimicrobial susceptibility of bacteria isolated from the systems before and after long-term BAC exposure were not detected. In summary, dose-dependent suppression of bacterial viability by BAC was enhanced by formulation. Biocide exposure decreased bacterial diversity and transiently enriched organisms with lower antimicrobial susceptibility that were subsequently supressed by exposure to 1% BAC-f, the concentration most closely reflecting deployment in formulated products.