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

Abstract

Assessing the risk of resistance associated with biocide exposure commonly involves exposing microorganisms to biocides at concentrations close to the MIC. With the aim of representing exposure to environmental biocide residues, Escherichia coli MG1655 was grown for 20 passages in the presence or absence of benzalkonium chloride (BAC) at 100 ng/liter and 1,000 ng/liter (0.0002% and 0.002% of the MIC, respectively). BAC susceptibility, planktonic growth rates, motility, and biofilm formation were assessed, and differentially expressed genes were determined via transcriptome sequencing. Planktonic growth rate and biofilm formation were significantly reduced (P < 0.001) following BAC adaptation, while BAC minimum bactericidal concentration increased 2-fold. Transcriptomic analysis identified 289 upregulated and 391 downregulated genes after long-term BAC adaptation compared with the respective control organism passaged in BAC-free medium. When the BAC-adapted bacterium was grown in BAC-free medium, 1,052 genes were upregulated and 753 were downregulated. Repeated passage solely in biocide-free medium resulted in 460 upregulated and 476 downregulated genes compared with unexposed bacteria. Long-term exposure to environmentally relevant BAC concentrations increased the expression of genes associated with efflux and reduced the expression of genes associated with outer-membrane porins, motility, and chemotaxis. This was manifested phenotypically through the loss of function (motility). Repeated passage in a BAC-free environment resulted in the upregulation of multiple respiration-associated genes, which was reflected by increased growth rate. In summary, repeated exposure of E. coli to BAC residues resulted in significant alterations in global gene expression that were associated with minor decreases in biocide susceptibility, reductions in growth rate and biofilm formation, and loss of motility. IMPORTANCE Exposure to very low concentrations of biocides in the environment is a poorly understood risk factor for antimicrobial resistance. Repeated exposure to trace levels of the biocide benzalkonium chloride (BAC) resulted in loss of function (motility) and a general reduction in bacterial fitness but relatively minor decreases in susceptibility. These changes were accompanied by widespread changes in the Escherichia coli transcriptome. These results demonstrate the importance of including phenotypic characterization in studies designed to assess the risks of biocide exposure.

Influence of surface copper content onStenotrophomonas maltophiliabiofilm control using chlorine and mechanical stress

“…The use of copper alloys, with reduced copper content, could help to reduce the costs from material acquisition while having the potential advantage of exerting antimicrobial activity. Several studies describing disinfection strategies were performed using bacteria in the planktonic state but these are inadequate to represent the behavior of those found adhered on surfaces (Mir et al 1997;Khan et al 2017;K€ ohler et al 2018;Forbes et al 2019;Samir et al 2019). Several other works studied DW biofilms formed using reactors operating under conditions far from those found in DWDS, particularly the annular reactor (Chang and Craik 2012), the rotating disc reactor (Murga et al 2001;Pelleïeux et al 2012), and the CDC biofilm reactor (Park and Hu 2010;Abe et al 2011;Armbruster et al 2012).…”

Mitochondrial Dysfunctions May Be One of the Major Causative Factors Underlying Detrimental Effects of Benzalkonium Chloride

“…At the cellular level, in neuroblastoma cells [42] and the mouse neonatal brain [43], BAC exposure significantly modified lipid homeostasis changing several lipid classes. Longterm exposure of Escherichia coli to environmentally relevant BAC concentrations resulted in significant alterations in global gene expression with increasing the expression of genes associated with efflux and reducing the expression of genes associated with outer-membrane porins, motility, and chemotaxis [44]. In cultured human alveolar epithelial cells, BAC induced a dose-and concentration-dependent oxidative stress [45].…”

Antimicrobial Adaptation in Uropathogenic Escherichia coli

“…Exposure to BAC has previously lead to a decrease in motility of E.coli [184], which RNA-sequencing revealed was due to a decrease in the expression of genes associated with flagella synthesis. A reduction in motility associated genes may explain the decreased biofilm formation in our BAC exposed isolates.…”

“…It is important, then, that new coating agents should be assessed using long-term exposure to elucidate the effects of these antimicrobials over an extended amount of time. Research into the lo ng-term effects of biocides has shown multiple phenotypic and genotypic changes occurring as bacteria respond to the antimicrobial challenge, which has been correlated with phenotypic effects such as changes in antimicrobial susceptibility, biofilm formation and relative pathogenicity [80,184,193]. , and F-DPD.…”

“…BAC exposure induced increases in HUEPC invasion for 2/8 isolates. BAC exposure has been shown to increase expression of efflux pumps[100] and decrease expression of motility associated genes[184]. But to our knowledge there are no studies that have investigated the effects of BAC on E.coli cell invasion or have identified genes associated with invasion/adhesion that are associated with BAC exposure.When exposed to silver nitrate, 1/8 isolates showed increases in HUEPC invasion.…”