title: Formulation of Biocides Increases Antimicrobial Potency and Mitigates the Enrichment of Nonsusceptible Bacteria in Multispecies Biofilms.
abstract: The current investigation aimed to generate data to inform the development of risk assessments of biocide usage. Stabilized domestic drain biofilm microcosms were exposed daily over 6 months to increasing concentrations (0.01% to 1%) of the biocide benzalkonium chloride (BAC) in a simple aqueous solution (BAC-s) or in a complex formulation (BAC-f) representative of a domestic cleaning agent. Biofilms were analyzed 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 levels of viable bacteria than exposure to BAC-s, while 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 populations of organisms with lower antimicrobial susceptibility, and the effects were subsequently suppressed by exposure to 1% BAC-f, the concentration most closely reflecting deployment in formulated products.IMPORTANCEAssessment of the risks of biocide use has been based mainly on the exposure of axenic cultures of bacteria to biocides in simple aqueous solutions. The current investigation aimed to assess the effects of formulation on the outcome of biocide exposure in multispecies biofilms. Formulation of the cationic biocide BAC significantly increased antimicrobial potency. Bacteria with lower antimicrobial susceptibility whose populations were enriched after low-level biocide exposure were more effectively suppressed by the biocide at in-use concentrations (1% [wt/vol]) in a formulation than in a simple aqueous solution. These observations underline the importance of simulating normal deployment conditions in considering the risks and benefits of biocide use.
authors.affiliation: Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Forbes
authors.affiliation: School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, United Kingdom.
authors.forename: Nicola
authors.initials: N
authors.lastname: Cowley
authors.affiliation: School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, United Kingdom.
authors.forename: Gavin
authors.identifier: http://orcid.org/0000-0002-9138-0859
authors.initials: G
authors.lastname: Humphreys
authors.affiliation: School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, United Kingdom.
authors.forename: Hitesh
authors.initials: H
authors.lastname: Mistry
authors.affiliation: Unilever, Safety and Environmental Assurance Centre, Colworth Science Park, Sharnbrook, Bedfordshire, United Kingdom.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Amézquita
authors.affiliation: School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, United Kingdom andrew.mcbain@manchester.ac.uk.
authors.forename: Andrew J
authors.identifier: http://orcid.org/0000-0002-5255-5425
authors.initials: AJ
authors.lastname: McBain
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D001548:Benzalkonium Compounds / Q000737:chemistry / Q000494:pharmacology; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D004202:Disinfectants / Q000737:chemistry* / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects*; D012336:RNA, Ribosomal, 16S; D018570:Risk Assessment
chemical_list: D000890:Anti-Infective Agents; D001548:Benzalkonium Compounds; D004202:Disinfectants; D012336:RNA, Ribosomal, 16S
keywords: CDFF; antibiotics; biocides; formulation; microbicides; resistance; susceptibility