Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila

Luz Chacón; Benno Kuropka; Enrique González-Tortuero; Frank Schreiber; Keilor Rojas-Jiménez; Alexandro Rodríguez-Rojas

doi:10.3389/fmicb.2023.1180128

Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila

Front Microbiol. 2023 Jun 2:14:1180128. doi: 10.3389/fmicb.2023.1180128. eCollection 2023.

Authors

Luz Chacón^{1

2

3}, Benno Kuropka⁴, Enrique González-Tortuero⁵, Frank Schreiber³, Keilor Rojas-Jiménez⁶, Alexandro Rodríguez-Rojas^{1

7}

Affiliations

¹ Evolutionary Biology, Institut für Biologie, Freie Universität Berlin, Berlin, Germany.
² Health Research Institute, University of Costa Rica, San José, Costa Rica.
³ Division of Biodeterioration and Reference Organisms (4.1), Department of Materials and the Environment, Federal Institute for Materials Research and Testing (BAM), Berlin, Germany.
⁴ Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
⁵ School of Science, Engineering, and Environment (SEE), University of Salford, Manchester, United Kingdom.
⁶ Escuela de Biología, University of Costa Rica, San José, Costa Rica.
⁷ Small Animal Internal Medicine, Clinic for Small Animals, University of Veterinary Medicine (Vetmeduni), Vienna, Austria.

Abstract

Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and characterized mechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 different sequenced A. hydrophila strains, consists of approximately 4.6 Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand how bacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments.

Keywords: benzalkonium chloride; biocide; low-susceptibility; primary response; quaternary ammonium disinfectants; wastewater.

Grants and funding

Deutscher Akademischer Austauschdienst (DAAD) Short-Term Grants, 2021 and the University o Costa Rica for the financial support of the LC internship in Germany. Vicerrectoría de Investigación of University of Costa Rica for the financial funds for the project B7-255. AR-R was supported by SFB973 from Deutsche Forschungsgemeinschaft (http://www.sfb973.de/), project C5 (https://www.sfb973.de/members_staff/c5/index.html).