Biocide-resistant Klebsiella quasipneumoniae in seawater cooled system: a reservoir of efflux pumps driving cross-resistance to biocides and antibiotics

Abstract

Antimicrobial resistance (AMR) poses a significant threat to public health and is increasingly recognized within the “One Health” framework, which emphasizes the interconnectedness of human, animal, and environmental health. While extensive research has focused on regulating antibiotic use across healthcare and other sectors, the impact of intensive biocide use on AMR development, particularly in seawater-cooled systems, remains underexplored. In this study, we report the isolation and characterization of a multidrug-resistant Klebsiella quasipneumoniae strain from the cooling water system of a coastal power plant, where continuous chlorination at 0.2 ppm is employed for biofouling control. The isolated strain displayed broad-spectrum resistance to multiple biocides and antibiotics. Interestingly, the strain shown enhances biofilm formation in response to biocides and antibiotics, thereby compounding its resistance profile. Efflux assays with ethidium bromide (EtBr) and whole-genome sequencing revealed that efflux pumps are central to the resistance mechanisms. Additionally, the presence of β-lactamase (OKP-A) and FosA genes confers resistance to the β-lactam and epoxide classes of antibiotics. The strain was found to be salt-tolerant and preferred to grow at normal salinity, indicating a non-marine origin of this isolate. These findings highlight the prevalence of biocide and antibiotic-resistant pathogens in marine cooling water systems that primarily rely on biocides for biofouling control. In line with One Health principles, our research advocates for a reassessment of biocide practices in marine cooling water systems and the implementation of proactive measures to mitigate the spread of antimicrobial resistance (AMR).

Graphical abstract

Keywords

Biofilms / Biocide resistance / Efflux pumps / Klebsiella quasipneumoniae

Highlight

	● Biocide resistant Klebsiella quasipneumoniae isolated from marine cooling water.
	● K. quasipneumoniae exhibits enhanced biofilm formation in response to biocide.
	● The strain exhibits cross-resistance to multiple antibiotics and biocides.
	● Efflux pumps were identified as key mechanisms for biocide and antibiotic resistance.
	● An urgent need to rationalize biocide use in cooling water systems was highlighted.

References

[1] Alav I, Sutton J M, Rahman K M. (2018). Role of bacterial efflux pumps in biofilm formation. Journal of Antimicrobial Chemotherapy, 73(8): 2003–2020

[2] Altayb H N, Hosawi S, Baothman O, Kazmi I, Chaieb K, Abu Zeid I M, Elbadawi H S, Lopes B S, Moglad E. (2023). Molecular insights into novel environmental strains of Klebsiella quasipneumoniae harboring different antimicrobial-resistance genes. Frontiers in Public Health, 10: 1068888

[3] Bharatham N, Bhowmik P, Aoki M, Okada U, Sharma S, Yamashita E, Shanbhag A P, Rajagopal S, Thomas T, Sarma M. . (2021). Structure and function relationship of OqxB efflux pump from Klebsiella pneumoniae. Nature Communications, 12(1): 5400

[4] Blair J M A, Piddock L J V. (2016). How to measure export via bacterial multidrug resistance efflux pumps. mBio, 7(4): e00840–16

[5] Blanco P, Hernando-Amado S, Reales-Calderon J A, Corona F, Lira F, Alcalde-Rico M, Bernardini A, Sanchez M B, Martinez J L. (2016). Bacterial multidrug efflux pumps: much more than antibiotic resistance determinants. Microorganisms, 4(1): 14

[6] Calero-Cáceres W, de Almeida Kumlien A C M, Balcázar J L. (2024). Advances and challenges in assessing antimicrobial resistance in environmental settings. Current Opinion in Environmental Science & Health, 41: 100571

[7] Cedeño-Muñoz J S, Aransiola S A, Reddy K V, Ranjit P, Victor-Ekwebelem M O, Oyedele O J, Pérez-Almeida I B, Maddela N R, Rodríguez-Díaz J M. (2024). Antibiotic resistant bacteria and antibiotic resistance genes as contaminants of emerging concern: occurrences, impacts, mitigations and future guidelines. Science of the Total Environment, 952: 175906

[8] Chang R K, Miller M, Shahin K, Batac F, Field C L, Duignan P, Struve C, Byrne B A, Murray M J, Greenwald K. . (2022). Genetics and pathology associated with Klebsiella pneumoniae and Klebsiella spp. isolates from North American pacific coastal marine mammals. Veterinary Microbiology, 265: 109307

[9] Chetri S. (2023). The culmination of multidrug-resistant efflux pumps vs. meager antibiotic arsenal era: urgent need for an improved new generation of EPIs. Frontiers in Microbiology, 14: 1149418

[10] Chew K L, Octavia S, Lai D, Lin R T P, Teo J W P. (2021). Genomic characterization of Klebsiella quasipneumoniae from clinical specimens in Singapore. Antimicrobial Agents and Chemotherapy, 65(8): e0041221

[11] Christensen G D, Simpson W A, Younger J J, Baddour L M, Barrett F F, Melton D M, Beachey E H. (1985). Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. Journal of Clinical Microbiology, 22(6): 996–1006

[12] Coombs K, Rodriguez-Quijada C, Clevenger J O, Sauer-Budge A F. (2023). Current understanding of potential linkages between biocide tolerance and antibiotic cross-resistance. Microorganisms, 11(8): 2000

[13] De Beer D, Srinivasan R, Stewart P S. (1994). Direct measurement of chlorine penetration into biofilms during disinfection. Applied and Environmental Microbiology, 60(12): 4339–4344

[14] Dhakal J, Sharma C S, Nannapaneni R, McDaniel C D, Kim T, Kiess A. (2019). Effect of chlorine-induced sublethal oxidative stress on the biofilm-forming ability of Salmonella at different temperatures, nutrient conditions, and substrates. Journal of Food Protection, 82(1): 78–92

[15] Dong N, Yang X M, Chan E W C, Zhang R, Chen S. (2022). Klebsiella species: taxonomy, hypervirulence and multidrug resistance. eBioMedicine, 79: 103998

[16] Du D J, Wang K X, Neuberger A, van Veen H W, Pos K M, Piddock L J V, Luisi B F. (2018). Multidrug efflux pumps: structure, function and regulation. Nature Reviews Microbiology, 16(9): 523–539

[17] Dulanto Chiang A, Dekker J P. (2024). Efflux pump-mediated resistance to new beta lactam antibiotics in multidrug-resistant gram-negative bacteria. Communications Medicine, 4(1): 170

[18] Elekhnawy E, Sonbol F, Abdelaziz A, Elbanna T. (2020). Potential impact of biocide adaptation on selection of antibiotic resistance in bacterial isolates. Future Journal of Pharmaceutical Sciences, 6(1): 97

[19] Elliott A G, Ganesamoorthy D, Coin L, Cooper M A, Cao M D. (2016). Complete genome sequence of Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603. Genome Announcements, 4(3): e00438–16

[20] Feldgarden M, Brover V, Gonzalez-Escalona N, Frye J G, Haendiges J, Haft D H, Hoffmann M, Pettengill J B, Prasad A B, Tillman G E. . (2021). AMRFinderPlus and the reference gene catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence. Scientific Reports, 11(1): 12728

[21] Fuga B, Cerdeira L, Andrade F, Zaccariotto T, Esposito F, Cardoso B, Rodrigues L, Neves I, Levy C E, Lincopan N. (2020). Genome sequences of clinical isolates of NDM-1-producing Klebsiella quasipneumoniae subsp. similipneumoniae and KPC-2-producing Klebsiella quasipneumoniae subsp. quasipneumoniae from Brazil. Microbiology Resource Announcements, 9(10): e00089–20

[22] 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

[23] Gupta V, Shekhawat S S, Kulshreshtha N M, Gupta A B. (2022). A systematic review on chlorine tolerance among bacteria and standardization of their assessment protocol in wastewater. Water Science and Technology, 86(2): 261–291

[24] Hajiagha M N, Kafil H S. (2023). Efflux pumps and microbial biofilm formation. Infection, Genetics and Evolution, 112: 105459

[25] Håkonsholm F, Hetland M A K, Svanevik C S, Lunestad B T, Löhr I H, Marathe N P. (2022). Insights into the genetic diversity, antibiotic resistance and pathogenic potential of Klebsiella pneumoniae from the Norwegian marine environment using whole-genome analysis. International Journal of Hygiene and Environmental Health, 242: 113967

[26] Heng H, Yang X M, Ye L W, Tang Y, Guo Z H, Li J, Chan E W C, Zhang R, Chen S. (2024). Global genomic profiling of Klebsiella pneumoniae: a spatio-temporal population structure analysis. International Journal of Antimicrobial Agents, 63(2): 107055

[27] Holt K E, Wertheim H, Zadoks R N, Baker S, Whitehouse C A, Dance D, Jenney A, Connor T R, Hsu L Y, Severin J. . (2015). Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proceedings of the National Academy of Sciences of the United States of America, 112(27): E3574–E3581

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

[29] Huang C L, Tao S N, Yuan J L, Li X N. (2022). Effect of sodium hypochlorite on biofilm of Klebsiella pneumoniae with different drug resistance. American Journal of Infection Control, 50(8): 922–928

[30] Jones I A, Joshi L T. (2021). Biocide use in the antimicrobial era: a review. Molecules, 26(8): 2276

[31] Jurelevicius D, Cotta S R, Montezzi L F, Dias A C F, Mason O U, Picão R C, Jansson J K, Seldin L. (2021). Enrichment of potential pathogens in marine microbiomes with different degrees of anthropogenic activity. Environmental Pollution, 268: 115757

[32] JutkinaJMarathe N PFlachC FLarssonD G J (2018). Antibiotics and common antibacterial biocides stimulate horizontal transfer of resistance at low concentrations. Science of the Total Environment, 616–617: 616–617

[33] Khan A, Joshi H M. (2024). Combating chlorine-resistant marine Klebsiella pneumoniae biofilms with chlorine-tolerant bacteriophages. Chemosphere, 368: 143782

[34] Kola A, Piening B, Pape U F, Veltzke-Schlieker W, Kaase M, Geffers C, Wiedenmann B, Gastmeier P. (2015). An outbreak of carbapenem-resistant OXA-48-producing Klebsiella pneumonia associated to duodenoscopy. Antimicrobial Resistance and Infection Control, 4(1): 8

[35] Koren S, Walenz B P, Berlin K, Miller J R, Bergman N H, Phillippy A M. (2017). Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Research, 27(5): 722–736

[36] Lloyd N A, Nazaret S, Barkay T. (2018). Whole genome sequences to assess the link between antibiotic and metal resistance in three coastal marine bacteria isolated from the mummichog gastrointestinal tract. Marine Pollution Bulletin, 135: 514–520

[37] Lukić Bilela L, Matijošytė I, Krutkevičius J, Alexandrino D A M, Safarik I, Burlakovs J, Gaudêncio S P, Carvalho M F. (2023). Impact of per- and polyfluorinated alkyl substances (PFAS) on the marine environment: raising awareness, challenges, legislation, and mitigation approaches under the one health concept. Marine Pollution Bulletin, 194: 115309

[38] MaillardJ Y (2018). Resistance of bacteria to biocides. Microbiology Spectrum, 6(2): 10.1128/microbiolspec.arba-0006–2017

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

[40] Mathers A J, Crook D, Vaughan A, Barry K E, Vegesana K, Stoesser N, Parikh H I, Sebra R, Kotay S, Walker A S. . (2019). Klebsiella quasipneumoniae provides a window into carbapenemase gene transfer, plasmid rearrangements, and patient interactions with the hospital environment. Antimicrobial Agents and Chemotherapy, 63(6): e02513–18

[41] McDonnell G, Russell A D. (1999). Antiseptics and disinfectants: activity, action, and resistance. Clinical Microbiology Reviews, 12(1): 147–179

[42] McEwenS ACollignon P J (2018). Antimicrobial resistance: a one health perspective. Microbiology Spectrum, 6(2): 10.1128/microbiolspec.arba-0009–2017

[43] Nishino K, Yamasaki S, Nakashima R, Zwama M, Hayashi-Nishino M. (2021). Function and inhibitory mechanisms of multidrug efflux pumps. Frontiers in Microbiology, 12: 737288

[44] Niu C, Gilbert E S. (2004). Colorimetric method for identifying plant essential oil components that affect biofilm formation and structure. Applied and Environmental Microbiology, 70(12): 6951–6956

[45] Ntshonga P, Gobe I, Koto G, Strysko J, Paganotti G M. (2024). Biocide resistance in Klebsiella pneumoniae: a narrative review. Infection Prevention in Practice, 6(2): 100360

[46] Olson R D, Assaf R, Brettin T, Conrad N, Cucinell C, Davis J J, Dempsey D M, Dickerman A, Dietrich E M, Kenyon R W. . (2023). Introducing the bacterial and viral bioinformatics resource center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Research, 51(D1): D678–D689

[47] Paixão L, Rodrigues L, Couto I, Martins M, Fernandes P, De Carvalho C C C R, Monteiro G A, Sansonetty F, Amaral L, Viveiros M. (2009). Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. Journal of Biological Engineering, 3(1): 18

[48] Peng R L, Xu Y S, Li R Q, Wang W, Wang H L, Zhang X Y, Yuan Q B. (2024). Marine microplastics enrich antibiotic resistance genes (ARGs), especially extracellular ARGs: an investigation in the East China Sea. Marine Pollution Bulletin, 209: 117260

[49] Qiu L L, Wang Y B, Du W C, Ai F X, Yin Y, Guo H Y. (2023). Efflux pumps activation caused by mercury contamination prompts antibiotic resistance and pathogen’s virulence under ambient and elevated CO₂ concentration. Science of the Total Environment, 863: 160831

[50] Rocha J, Henriques I, Gomila M, Manaia C M. (2022). Common and distinctive genomic features of Klebsiella pneumoniae thriving in the natural environment or in clinical settings. Scientific Reports, 12(1): 10441

[51] Schaffner U, Heimpel G E, Mills N J, Muriithi B W, Thomas M B, Gc Y D, Wyckhuys K A G. (2024). Biological control for one health. Science of the Total Environment, 951: 175800

[52] Sharma A, Gupta V K, Pathania R. (2019). Efflux pump inhibitors for bacterial pathogens: from bench to bedside. Indian Journal of Medical Research, 149(2): 129–145

[53] Shemesh M, Kolter R, Losick R. (2010). The biocide chlorine dioxide stimulates biofilm formation in Bacillus subtilis by activation of the histidine kinase KinC. Journal of Bacteriology, 192(24): 6352–6356

[54] Sousa M, Machado I, Simões L C, Simões M. (2025). Biocides as drivers of antibiotic resistance: a critical review of environmental implications and public health risks. Environmental Science and Ecotechnology, 25: 100557

[55] The Lancet. (2023). One health: a call for ecological equity. The Lancet, 401(10372): 169

[56] Thorpe H A, Booton R, Kallonen T, Gibbon M J, Couto N, Passet V, López-Fernández S, Rodrigues C, Matthews L, Mitchell S. . (2022). A large-scale genomic snapshot of Klebsiella spp. isolates in northern Italy reveals limited transmission between clinical and non-clinical settings. Nature Microbiology, 7(12): 2054–2067

[57] Uzoechi S C, Abu-Lail N I. (2019). The effects of β-lactam antibiotics on surface modifications of multidrug-resistant Escherichia coli: a multiscale approach. Microscopy and Microanalysis, 25(1): 135–150

[58] Vinitha P, E V P. (2010). Chlorination for power plant biofouling control: potential impact on entrained phytoplankton. International Journal of Environmental Studies, 67(4): 515–530

[59] Wand M E, Sutton J M (2022). Efflux-mediated tolerance to cationic biocides, a cause for concern? Microbiology, 168(11): 001263

[60] Wang Y, Zhiming Zhang Z M, Xia M Q, Zhang X M, Lan R X, Wei B Q, Liu Y, Lu Y, Fan G D. (2025). Mechanism and risk control of chlorine-resistant bacteria in drinking water supply systems: a comprehensive bibliometric analysis. Water, 17(7): 956

[61] Wattam A R, Davis J J, Assaf R, Boisvert S, Brettin T, Bun C, Conrad N, Dietrich E M, Disz T, Gabbard J L. . (2017). Improvements to PATRIC, the all-bacterial bioinformatics database and analysis resource center. Nucleic Acids Research, 45(D1): D535–D542

[62] Willmott T, Jadaan L, Humphreys G J, Lu J R, McBain A J, Chapman J. (2024). Sustainable application of biocides to promote hygiene and minimize antimicrobial resistance. Sustainable Microbiology, 1(1): qvae015

[63] Wyres K L, Holt K E. (2018). Klebsiella pneumoniae as a key trafficker of drug resistance genes from environmental to clinically important bacteria. Current Opinion in Microbiology, 45: 131–139

[64] Xu F Q, Jiang M X, Li D, Yu P F, Ma H, Lu H J. (2024). Protective effects of antibiotic resistant bacteria on susceptibles in biofilm: influential factors, mechanism, and modeling. Science of the Total Environment, 930: 172668

[65] Yang L, Wu X, Wu G Q, Wu Y G, Li H, Shao B. (2024). Association analysis of antibiotic and disinfectant resistome in human and foodborne E. coli in Beijing, China. Science of the Total Environment, 944: 173888

[66] Zhou N, Cheng Z L, Zhang X X, Lv C, Guo C Y, Liu H D, Dong K, Zhang Y, Liu C, Chang Y F. . (2022). Global antimicrobial resistance: a system-wide comprehensive investigation using the global one health index. Infectious Diseases of Poverty, 11(1): 92

RIGHTS & PERMISSIONS

Higher Education Press 2025