Development of a Protocol for Predicting Bacterial Resistance to Microbicides

Knapp, Laura; Amézquita, Aléjandro; McClure, Peter; Stewart, Sara E.; Maillard, Jean‐Yves

doi:10.1128/aem.03843-14

Cited by 48 publications

(54 citation statements)

References 19 publications

(20 reference statements)

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“…These include the fact that in vitro studies most commonly involve the exposure of single-species, planktonic cultures of bacteria to biocides delivered in simple aqueous solution at concentrations that have been selected to be sublethal but are relatively close to MICs (20). There is, therefore, the potential to increase the realism in such studies, and this has been done to a limited extent by introducing complex communities (28) and formulation (26,29).…”

Section: Discussionmentioning

confidence: 99%

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

Forbes

Morgan

Humphreys

et al. 2019

Appl Environ Microbiol

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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.

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Section: Discussionmentioning

confidence: 99%

“…The fact that environmentally relevant concentrations of biocides are likely to be considerably below those required to inhibit microbial growth has significance for risk assessment where in vitro testing has an important role (26,27). In such in vitro testing, microorganisms are generally exposed to the biocides at sublethal concentrations.…”

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confidence: 99%

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

Forbes

Morgan

Humphreys

et al. 2019

Appl Environ Microbiol

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“…7 Knapp et al reported on the use of this protocol to determine the effect of exposure to chlorhexidine, benzalkonium chloride, and 3 biocidal products to Pseudomonas aeruginosa, Burkholderia cepacia, B lata, Klebsiella pneumoniae, and 2 Salmonella enterica serovar Typhimurium strains. 10,11 Bacterial resistance to cationic agents (eg, biguanides, quaternary ammonium compounds) and phenolics (eg, TRI) has been widely reported 2,4,6,7,9,[11][12][13][14] and is often perceived to present a higher risk for the development of bacterial resistance to antimicrobials. A number of resistance mechanisms to these biocides have been described, including overexpression of efflux and changes in bacterial surface.…”

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confidence: 99%

Use of a predictive protocol to measure the antimicrobial resistance risks associated with biocidal product usage

Wesgate

Grasha²,

Maillard

2016

American Journal of Infection Control

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Using a decision tree based on the change in antimicrobial susceptibility test results, we were able to provide information on the effect of biocide exposure on the development of bacterial resistance to antimicrobials. Such information should address the call from the U.S. Food and Drug Administration and European Union Biocidal Products Regulation for manufacturers to provide information on antimicrobial resistance and cross-resistance in bacteria after the use of their product.

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“…In the real world, however, microbicides are deployed in complex formulations containing sequestrants, surfactants, and other compounds. Recent investigations indicate that the formulation of microbicides can significantly enhance antibacterial potency and that decreases in microbicide susceptibility after sublethal microbicide exposure may be significantly lower in frequency and extent when the microbicides are incorporated into formulations reflecting application in the real world (24,33). This highlights the value of risk assessments that more accurately reflect the way in which microbicides are deployed.…”

Section: Discussionmentioning

confidence: 95%

Variable Effects of Exposure to Formulated Microbicides on Antibiotic Susceptibility in Firmicutes and Proteobacteria

Forbes

Knight

Cowley

et al. 2016

Appl Environ Microbiol

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Microbicides are broad-spectrum antimicrobial agents that generally interact with multiple pharmacological targets. While they are widely deployed in disinfectant, antiseptic, and preservative formulations, data relating to their potential to select for microbicide or antibiotic resistance have been generated mainly by testing the compounds in much simpler aqueous solutions. In the current investigation, antibiotic susceptibility was determined for bacteria that had previously exhibited decreased microbicide susceptibility following repeated exposure to microbicides either in formulation with sequestrants and surfactants or in simple aqueous solution. Statistically significant increases in antibiotic susceptibility occurred for 12% of bacteria after exposure to microbicides in formulation and 20% of bacteria after exposure to microbicides in aqueous solutions, while 22% became significantly less susceptible to the antibiotics, regardless of formulation. Of the combinations of a bacterium and an antibiotic for which British Society for Antimicrobial Chemotherapy breakpoints are available, none became resistant. Linear modeling taking into account phylogeny, microbicide, antibiotic, and formulation identified small but significant effects of formulation that varied depending on the bacterium and microbicide. Adaptation to formulated benzalkonium chloride in particular was more likely to increase antibiotic susceptibility than adaptation to the simple aqueous solution. In conclusion, bacterial adaptation through repeated microbicide exposure was associated with both increases and decreases in antibiotic susceptibility. Formulation of the microbicide to which the bacteria had previously adapted had an identifiable effect on antibiotic susceptibility, but it effect was typically small relative to the differences observed among microbicides. Susceptibility changes resulting in resistance were not observed. IMPORTANCEThe safety of certain microbicide applications has been questioned due to the possibility that microbicide exposure could select for microbicide and antibiotic resistance. Evidence that this may happen is based mainly on in vitro experiments where bacteria have been exposed to microbicides in aqueous solution. Microbicides are, however, normally deployed in products formulated with surfactants, sequestrants, and other compounds. While this may influence the frequency and extent of susceptibility changes, few studies reported in the literature have assessed this. In the current investigation, therefore, we have investigated changes in antibiotic susceptibility in bacteria which exhibited decreased microbicide susceptibility following repeated exposure to microbicides in simple aqueous solutions and in formulation. We report that the microbicide formulation had an identifiable effect on antibiotic susceptibility, but it was typically small relative to the differences observed among microbicides. We did not observe susceptibility changes resulting in resistance.

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Development of a Protocol for Predicting Bacterial Resistance to Microbicides

Cited by 48 publications

References 19 publications

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

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

Use of a predictive protocol to measure the antimicrobial resistance risks associated with biocidal product usage

Variable Effects of Exposure to Formulated Microbicides on Antibiotic Susceptibility in Firmicutes and Proteobacteria

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