Diversified antibiotic resistance and sensitivity patterns induced by quaternary ammonium compounds

Abstract

The extensive application of quaternary ammonium compounds (QACs) has led to a significant increase in their mass load in wastewater treatment plants (WWTPs). However, little is known about how QACs contribute to the emergence of resistance against different antibiotics. This study aims to reveal the structure- and dose–response relationships between various QACs and their induced antibiotic resistance and sensitivity patterns. 16 QACs were tested for their capacity in inducing multidrug resistance in activated sludge (AS) and AS-isolated E. coli. For AS, the exposure to three representative QACs resulted in up to a 32-fold increase in resistance against norfloxacin, tetracycline, kanamycin and gentamicin. However, the resistance to cefepime (CEF), azithromycin, and rifampicin remained largely unchanged. As a representative strain isolated from AS, the exposure of E. coli to QACs at 1 mg/L exhibited no induced resistance, whereas the MIC for CEF showed a decrease by 62.5%–87.5%. This was likely attributed to downregulation of proton motive force and efflux pump, which promoted the intracellular accumulation of CEF. Additionally, we identified the high-risk structural characteristics of QACs and emphasized the critical importance of determining the threshold concentrations for QACs risk management. This study provides valuable guidance for the assessment and management of environmental resistance risks caused by increased QACs in diverse environments.

Graphical abstract

Keywords

Quaternary ammonium compounds / Antibiotic resistance / Wastewater treatment plants / Pharma RQSAR analysis / Dose-effect relationship / Proton motive force

Highlight

	● QACs could induce diversified antibiotic resistance patterns in activated sludge.
	● QACs with shorter alkyl chains have greater capacity to induce antibiotic resistance.
	● QACs enhance cefepime sensitivity through the downregulation of PMF and efflux.
	● The importance of risk management threshold concentrations for QACs is emphasized.

References

[1] Al Jalali V, Zeitlinger M. (2018). Clinical pharmacokinetics and pharmacodynamics of telavancin compared with the other glycopeptides. Clinical Pharmacokinetics, 57(7): 797–816

[2] Alygizakis N, Galani A, Rousis N I, Aalizadeh R, Dimopoulos M A, Thomaidis N S. (2021). Change in the chemical content of untreated wastewater of Athens, Greece under COVID-19 pandemic. Science of the Total Environment, 799: 149230

[3] An Y, Sherman W, Dixon S L. (2013). Kernel-based partial least squares: Application to fingerprint-based qsar with model visualization. Journal of Chemical Information and Modeling, 53(9): 2312–2321

[4] Arnold W A, Blum A, Branyan J, Bruton T A, Carignan C C, Cortopassi G, Datta S, Dewitt J, Doherty A C, Halden R U. . (2023). Quaternary ammonium compounds: a chemical class of emerging concern. Environmental Science & Technology, 57(20): 7645–7665

[5] Blattner F R, Plunkett G III, Bloch C A, Perna N T, Burland V, Riley M, Collado Vides J, Glasner J D, Rode C K, Mayhew G F. . (1997). The complete genome sequence of Escherichia coli K-12. Science, 277(5331): 1453–1462

[6] Brock M, Maerker C, Schütz A, Völker U, Buckel W. (2002). Oxidation of propionate to pyruvate in Escherichia coli. European Journal of Biochemistry, 269(24): 6184–6194

[7] Clara M, Scharf S, Scheffknecht C, Gans O. (2007). Occurrence of selected surfactants in untreated and treated sewage. Water Research, 41(19): 4339–4348

[8] Cochrane S A, Findlay B, Bakhtiary A, Acedo J Z, Rodriguez Lopez E M, Mercier P, Vederas J C. (2016). Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II. Proceedings of the National Academy of Sciences of the United States of America, 113(41): 11561–11566

[9] De Araújo M J G, Barbosa R C, Fook M V L, Canedo E L, Silva S M L, Medeiros E S, Leite I F. (2018). HDPE/Chitosan blends modified with organobentonite synthesized with quaternary ammonium salt impregnated chitosan. Materials (Basel), 11(2): 291

[10] Diaz-Diaz S, Recacha E, Machuca J, García-Duque A, Docobo Pérez F, Blázquez J, Pascual A, Rodríguez Martínez J M. (2021). Synergistic quinolone sensitization by targeting the recA SOS response gene and oxidative stress. Antimicrobial Agents and Chemotherapy, 65(4): e02004–20

[11] Dwyer D J, Kohanski M A, Collins J J. (2009). Role of reactive oxygen species in antibiotic action and resistance. Current Opinion in Microbiology, 12(5): 482–489

[12] El Meouche I, Dunlop M J. (2018). Heterogeneity in efflux pump expression predisposes antibiotic-resistant cells to mutation. Science, 362(6415): 686–690

[13] Ferreira C, Pereira A M, Pereira M C, Melo L F, Simoes M. (2011). Physiological changes induced by the quaternary ammonium compound benzyldimethyldodecylammonium chloride on Pseudomonas fluorescens. Journal of Antimicrobial Chemotherapy, 66(5): 1036–1043

[14] Frolov N A, Fedoseeva K A, Hansford K A, Vereshchagin A N. (2021). Novel phenyl-based bis-quaternary ammonium compounds as broad-spectrum biocides. ChemMedChem, 16(19): 2954–2959

[15] Fuentes D E, Navarro C A, Tantaleán J C, Araya M A, Saavedra C P, Pérez J M, Calderón I L, Youderian P A, Mora G C, Vásquez C C. (2005). The product of the qacC gene of Staphylococcus epidermidis CH mediates resistance to β-lactam antibiotics in Gram-positive and Gram-negative bacteria. Research in Microbiology, 156(4): 472–477

[16] Gao W, Dang Z C, Liu F S, Wang S, Zhang D W, Yan M X. (2020). Preparation of antistatic epoxy resin coatings based on double comb-like quaternary ammonium salt polymers. RSC Advances, 10(71): 43523–43532

[17] Gaurav A, Bakht P, Saini M, Pandey S, Pathania R. (2023). Role of bacterial efflux pumps in antibiotic resistance, virulence, and strategies to discover novel efflux pump inhibitors. Microbiology, 169(5): 001333

[18] Goosen N, Moolenaar G F. (2001). Role of ATP hydrolysis by UvrA and UvrB during nucleotide excision repair. Research in Microbiology, 152(3−4): 401–409

[19] Griffith J M, Basting P J, Bischof K M, Wrona E P, Kunka K S, Tancredi A C, Moore J P, Hyman M R L, Slonczewski J L. (2019). Experimental evolution of Escherichia coli K-12 in the presence of proton motive force (PMF) uncoupler carbonyl cyanide m-chlorophenylhydrazone selects for mutations affecting PMF-driven drug efflux pumps. Applied and Environmental Microbiology, 85(5): e02792–18

[20] Grimm C, Evers A, Brock M, Maerker C, Klebe G, Buckel W, Reuter K. (2003). Crystal structure of 2-Methylisocitrate Lyase (PrpB) from Escherichia coli and modelling of its ligand bound active centre. Journal of Molecular Biology, 328(3): 609–621

[21] Halliwell B, Adhikary A, Dingfelder M, Dizdaroglu M. (2021). Hydroxyl radical is a significant player in oxidative DNA damage in vivo. Chemical Society Reviews, 50(15): 8355–8360

[22] He K, Xue B, Yang X, Wang S, Li C, Zhang X, Zhao C, Wang X, Qiu Z, Shen Z. . (2022). Low-concentration of trichloromethane and dichloroacetonitrile promote the plasmid-mediated horizontal transfer of antibiotic resistance genes. Journal of Hazardous Materials, 425: 128030

[23] Hong Y, Li L, Luan G, Drlica K, Zhao X. (2017). Contribution of reactive oxygen species to thymineless death in Escherichia coli. Nature Microbiology, 2(12): 1667–1675

[24] Hora P I, Pati S G, Mcnamara P J, Arnold W A. (2020). Increased use of quaternary ammonium compounds during the SARS-CoV-2 pandemic and beyond: consideration of environmental implications. Environmental Science & Technology Letters, 7(9): 622–631

[25] Hostetler Z M, Cory M B, Jones C M, Petersson E J, Kohli R M. (2020). The kinetic and molecular basis for the interaction of LexA and activated RecA revealed by a fluorescent amino acid probe. ACS Chemical Biology, 15(5): 1127–1133

[26] Hu Z, Yang L, Liu Z, Han J, Zhao Y, Jin Y, Sheng Y, Zhu L, Hu B. (2023). Excessive disinfection aggravated the environmental prevalence of antimicrobial resistance during COVID-19 pandemic. Science of the Total Environment, 882: 163598

[27] Inigo M, Deja S, Burgess S C. (2021). Ins and outs of the TCA cycle: the central role of anaplerosis. Annual Review of Nutrition, 41(1): 19–47

[28] Iredell J, Brown J, Tagg K. (2016). Antibiotic resistance in Enterobacteriaceae: mechanisms and clinical implications. BMJ, 352: h6420

[29] Jia Y, Lu H J, Zhu L Z. (2022). Molecular mechanism of antibiotic resistance induced by mono- and twin-chained quaternary ammonium compounds. Science of the Total Environment, 832: 155090

[30] Jones E C, Uphoff S. (2021). Single-molecule imaging of LexA degradation in Escherichia coli elucidates regulatory mechanisms and heterogeneity of the SOS response. Nature Microbiology, 6(8): 981–990

[31] Kaushik V, Tiwari M, Tiwari V. (2022). Interaction of RecA mediated SOS response with bacterial persistence, biofilm formation, and host response. International Journal of Biological Macromolecules, 217: 931–943

[32] Kim K, Kim M, Chung J, Ahn J H, Rhee S. (2009). Crystal structure of metal-dependent allantoinase from Escherichia coli. Journal of Molecular Biology, 387(5): 1067–1074

[33] Kim K S, Pelton J G, Inwood W B, Andersen U, Kustu S, Wemmer D E. (2011). The Rut pathway for pyrimidine degradation: Novel chemistry and toxicity problems. Journal of Bacteriology, 193(1): 326

[34] Kravchuk V, Petrova O, Kampjut D, Wojciechowska Bason A, Breese Z, Sazanov L. (2022). Author correction: a universal coupling mechanism of respiratory complex I. Nature, 611(7936): E9

[35] Larsson D G J, Flach C F. (2022). Antibiotic resistance in the environment. Nature Reviews. Microbiology, 20(5): 257–269

[36] Liu C, Goh S G, You L, Yuan Q, Mohapatra S, Gin K Y H, Chen B. (2023). Low concentration quaternary ammonium compounds promoted antibiotic resistance gene transfer via plasmid conjugation. Science of the Total Environment, 887: 163781

[37] Liu S, Li Z, Shen Y, Jia S, Liu P, Zhang X X. (2024). Prevalence of class 1 integron and its gene cassettes carrying antibiotic resistance genes in drinking water treatment and distribution systems. Frontiers of Environmental Science & Engineering, 18(10): 126

[38] Liu Y, Tong Z, Shi J, Jia Y, Deng T, Wang Z. (2021). Reversion of antibiotic resistance in multidrug-resistant pathogens using non-antibiotic pharmaceutical benzydamine. Communications Biology, 4(1): 1328

[39] Lopatkin A J, Bening S C, Manson A L, Stokes J M, Kohanski M A, Badran A H, Earl A M, Cheney N J, Yang J H, Collins J J. (2021). Clinically relevant mutations in core metabolic genes confer antibiotic resistance. Science, 371(6531): eaba0862

[40] Lu J, Jin M, Nguyen S H, Mao L K, Li J, Coin L J M, Yuan Z G, Guo J H. (2018). Non-antibiotic antimicrobial triclosan induces multiple antibiotic resistance through genetic mutation. Environment International, 118: 257–265

[41] Lu J, Wu J, Zhang C, Wang J, He X. (2024). Occurrence and possible sources of antibiotic resistance genes in seawater of the South China Sea. Frontiers of Environmental Science & Engineering, 18(9): 108

[42] Lusetti S L, Drees J C, Stohl E A, Seifert H S, Cox M M. (2004). The DinI and RecX proteins are competing modulators of RecA function. Journal of Biological Chemistry, 279(53): 55073–55079

[43] Ma W, Zhang D, Li G, Liu J, He G, Zhang P, Yang L, Zhu H, Xu N, Liang S. (2017). Antibacterial mechanism of daptomycin antibiotic against Staphylococcus aureus based on a quantitative bacterial proteome analysis. Journal of Proteomics, 150: 242–251

[44] MacLean A, Legendre F, Appanna V D. (2023). The tricarboxylic acid (TCA) cycle: a malleable metabolic network to counter cellular stress. Critical Reviews in Biochemistry and Molecular Biology, 58(1): 81–97

[45] Maillard J Y, Pascoe M. (2024). Disinfectants and antiseptics: mechanisms of action and resistance. Nature Reviews. Microbiology, 22(1): 4–17

[46] Méchin L, Dubois Brissonnet F, Heyd B, Leveau J Y. (1999). Adaptation of Pseudomonas aeruginosa ATCC 15442 to didecyldimethylammonium bromide induces changes in membrane fatty acid composition and in resistance of cells. Journal of Applied Microbiology, 86(5): 859–866

[47] Merchel Piovesan Pereira B, Wang X, Tagkopoulos I. (2021). Biocide-induced emergence of antibiotic resistance in Escherichia coli. Frontiers in Microbiology, 12: 640923

[48] Meylan S, Porter C B M, Yang J H, Belenky P, Gutierrez A, Lobritz M A, Park J, Kim S H, Moskowitz S M, Collins J J. (2017). Carbon sources tune antibiotic susceptibility in Pseudomonas aeruginosa via tricarboxylic acid cycle control. Cell Chemical Biology, 24(2): 195–206

[49] Minamino T, Namba K. (2008). Distinct roles of the FliI ATPase and proton motive force in bacterial flagellar protein export. Nature, 451(7177): 485–488

[50] Mohapatra S, Yutao L, Goh S G, Ng C, Luhua Y, Tran N H, Gin K Y H. (2023). Quaternary ammonium compounds of emerging concern: Classification, occurrence, fate, toxicity and antimicrobial resistance. Journal of Hazardous Materials, 445: 130393

[51] Mohiuddin S G, Ghosh S, Kavousi P, Orman M A. (2022). Proton motive force inhibitors are detrimental to methicillin-resistant Staphylococcus aureus strains. Microbiology Spectrum, 10(4): e0202422

[52] Moparthi V K, Hägerhäll C. (2011). The evolution of respiratory chain complex I from a smaller last common ancestor consisting of 11 protein subunits. Journal of Molecular Evolution, 72(5−6): 484–497

[53] Mulder I, Siemens J, Sentek V, Amelung W, Smalla K, Jechalke S. (2018). Quaternary ammonium compounds in soil: implications for antibiotic resistance development. Reviews in Environmental Science and Biotechnology, 17(1): 159–185

[54] Naghavi M, Vollset S E, Ikuta K S, Swetschinski L R, Gray A P, Wool E E, Robles Aguilar G, Mestrovic T, Smith G, Han C. (2024). Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050. Lancet, 404(10459): 1199–1226

[55] Nishino K, Yamaguchi A. (2001). Analysis of a complete library of putative drug transporter genes in Escherichia coli. Journal of Bacteriology, 183(20): 5803–5812

[56] Odsbu I, Skarstad K. (2014). DNA compaction in the early part of the SOS response is dependent on RecN and RecA. Microbiology, 160(5): 872–882

[57] ÖstmanM, FickJ, Tysklind M (2018). Detailed mass flows and removal efficiencies for biocides and antibiotics in Swedish sewage treatment plants. Science of the Total Environment, 640–641: 327–336

[58] Qiu Z, Yu Y, Chen Z, Jin M, Yang D, Zhao Z, Wang J, Shen Z, Wang X, Qian D. . (2012). Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera. Proceedings of the National Academy of Sciences of the United States of America, 109(13): 4944–4949

[59] Recacha E, Machuca J, Díaz De Alba P, Ramos-Güelfo M, Docobo Pérez F, Rodriguez Beltrán J, Blázquez J, Pascual A, Rodríguez Martínez J M. (2017). Quinolone resistance reversion by targeting the SOS response. mBio, 8(5): e00971–17

[60] Renzette N, Gumlaw N, Sandler S J. (2007). DinI and RecX modulate RecA-DNA structures in Escherichia coli K-12. Molecular Microbiology, 63(1): 103–115

[61] Roedel A, Dieckmann R, Brendebach H, Hammerl J A, Kleta S, Noll M, Al Dahouk S, Vincze S. (2019). Biocide-tolerant Listeria monocytogenes isolates from german food production plants do not show cross-resistance to clinically relevant antibiotics. Applied and Environmental Microbiology, 85(20): e01253–19

[62] Ruan T, Song S, Wang T, Liu R, Lin Y, Jiang G. (2014). Identification and composition of emerging quaternary ammonium compounds in municipal sewage sludge in China. Environmental Science & Technology, 48(8): 4289–4297

[63] Shi D, Hao H, Wei Z, Yang D, Yin J, Li H, Chen Z, Yang Z, Chen T, Zhou S. . (2022a). Combined exposure to non-antibiotic pharmaceutics and antibiotics in the gut synergistically promote the development of multi-drug-resistance in Escherichia coli. Gut Microbes, 14(1): 2018901

[64] Shi X, Xia Y, Wei W, Ni B J. (2022b). Accelerated spread of antibiotic resistance genes (ARGs) induced by non-antibiotic conditions: roles and mechanisms. Water Research, 224: 119060

[65] Smolders G J F, Van Der Meij J, Van Loosdrecht M C M, Heijnen J J. (1994). Stoichiometric model of the aerobic metabolism of the biological phosphorus removal process. Biotechnology and Bioengineering, 44(7): 837–848

[66] Song M, Liu Y, Li T, Liu X, Hao Z, Ding S, Panichayupakaranant P, Zhu K, Shen J. (2021). Plant natural flavonoids against multidrug resistant pathogens. Advanced Science, 8(15): 2100749

[67] Steinhilper R, Höff G, Heider J, Murphy B J. (2022). Publisher correction: structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli. Nature Communications, 13(1): 7258

[68] Su Z, Chen L, Wen D. (2024). Impact of wastewater treatment plant effluent discharge on the antibiotic resistome in downstream aquatic environments: a mini review. Frontiers of Environmental Science & Engineering, 18(3): 36

[69] Tacconelli E, Pezzani M D. (2019). Public health burden of antimicrobial resistance in Europe. Lancet. Infectious Diseases, 19(1): 4–6

[70] Wang C, Lu H, Li X, Zhu Y, Ji Y, Lu W, Wang G, Dong W, Liu M, Wang X. . (2022). Identification of an anti-virulence drug that reverses antibiotic resistance in multidrug resistant bacteria. Biomedicine and Pharmacotherapy, 153: 113334

[71] Wang P L, Wang H Q, Qi S C, Wang W, Lu H J. (2025). Synergistic effects of quaternary ammonium compounds and antibiotics on the evolution of antibiotic resistance. Water Research, 275: 123206

[72] Wang S Y, Chen J, Zhu L Z. (2024). Understanding the phytotoxic effects of organic contaminants on rice through predictive modeling with molecular descriptors: a data-driven analysis. Journal of Hazardous Materials, 476: 134953

[73] Webber M A, Whitehead R N, Mount M, Loman N J, Pallen M J, Piddock L J V. (2015). Parallel evolutionary pathways to antibiotic resistance selected by biocide exposure. Journal of Antimicrobial Chemotherapy, 70(8): 2241–2248

[74] Wei Z, Wei Y, Li H, Shi D, Yang D, Yin J, Zhou S, Chen T, Li J, Jin M. (2022). Emerging pollutant metformin in water promotes the development of multiple-antibiotic resistance in Escherichia coli via chromosome mutagenesis. Journal of Hazardous Materials, 430: 128474

[75] Xia Y, Wei X, Gao P, Wang C, De Jong A, Chen J H K, Rodríguez Sánchez M J, Rodríguez Nogales A, Diez Echave P, Gálvez J. . (2024). Bismuth-based drugs sensitize Pseudomonas aeruginosa to multiple antibiotics by disrupting iron homeostasis. Nature Microbiology, 9(10): 2600–2613

[76] Xu H, Gao J, Cui Y, Wang Z, Zhao Y, Yuan Y, Zeng L, Fu X. (2024). The combination of ciprofloxacin and dialkyldimethyl ammonium compound synergistically proliferated intracellular resistance genes in nitrifying system. Science of the Total Environment, 930: 172715

[77] Xu Y, Tan L, Li Q, Zheng X, Liu W. (2022). Sublethal concentrations of heavy metals Cu²⁺ and Zn²⁺ can induce the emergence of bacterial multidrug resistance. Environmental Technology & Innovation, 27: 102379

[78] Yang B, Tong Z, Shi J, Wang Z, Liu Y. (2023). Bacterial proton motive force as an unprecedented target to control antimicrobial resistance. Medicinal Research Reviews, 43(4): 1068–1090

[79] Yu J X, Lu H J, Zhu L Z. (2024). Mutation-driven resistance development in wastewater E. coli upon low-level cephalosporins: pharmacophore contribution and novel mechanism. Water Research, 252: 121235

[80] ZhangC, Cui F, ZengG M, JiangM, YangZ Z, YuZ G, Zhu M Y, ShenL Q (2015). Quaternary ammonium compounds (QACs): a review on occurrence, fate and toxicity in the environment. Science of the Total Environment, 518–519: 352–362

[81] Zhang K, Gao J, Lu T, Wang Y, Zhang J, An J, Xu H. (2024). Evolution of microbial community and resistance genes in denitrification system under single and combined exposure to benzethonium chloride and methylparaben. Journal of Hazardous Materials, 480: 136010

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