Publication: Loss of Function in Escherichia coli Exposed to Environmentally Relevant Concentrations of Benzalkonium Chloride

Assessing the risk of resistance associated with biocide exposure commonly involves exposing microorganisms to test compounds at concentrations close to minimum inhibitory concentrations. With the aim of representing exposure to environmental residues, Escherichia coli MG1655 was grown for 20 passages in the presence or absence of benzalkonium chloride (BAC) at 100ng/L and 1000ng/L (0.0002% and 0.002% of the MIC, respectively). BAC susceptibility, planktonic growth rates, motility and biofilm-formation were assessed, and differentially expressed genes determined via RNA-sequencing. Following BAC exposure, growth-rate, productivity and biofilm-formation were significantly reduced (p<0.001), whilst BAC susceptibility decreased 2-fold. Transcriptomic analysis identified that 289 genes were upregulated and 391 were downregulated when the BAC-adapted isolates were grown in the presence of BAC. When the BAC adapted isolates were grown in biocide-free medium, 1052 genes were upregulated and 753 were down regulated. Repeated (n=20) passage in biocide-free medium alone resulted in 460 upregulated and 476 down-regulated genes compared to unexposed bacteria. Long-term exposure to environmentally relevant BAC concentrations induced increased transcription of efflux proteins and a reduction in outer-membrane porins and genes associated with motility and chemotaxis, which was manifested phenotypically through loss-of-function (motility). Repeated passage of control cultures in a BAC-free-environment resulted in the up-regulation of multiple respiration-associated proteins, which was reflected by increases in planktonic growth rates. Thus, repeated exposure of E. coli to BAC residues resulted in significant alterations in global gene expression that were associated with marginal decreases in biocide susceptibility, reductions in growth-rate and biofilm-formation, and loss of function for motility.

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