Whole genome sequencing and phenotypic analysis of carbapenem- and polymyxin-resistant Enterobacter cloacae complex in natural water bodies

Abstract

The presence of the Enterobacter cloacae complex (ECC) in natural water bodies poses potential public health risks because these bacteria can enter humans from the environment within the “One Health” framework. Nevertheless, there is a notable deficiency in the isolation and knowledge of carbapenem- and polymyxin-resistant ECC strains from natural water bodies. In this research, 48 ECC strains were isolated from natural water bodies. Of these, eight strains were resistant to carbapenems (imipenem) and polymyxins (polymyxin B and colistin sulfate) and were classified as multi-locus sequence typing 13. Comparative genomic analysis identified bla_CMY-63, pmrE, pmrF, and various efflux pump genes as key genetic determinants of co-resistance to carbapenems and polymyxins. Notably, these strains exhibited relatively high virulence, with cell death rates ranging from 57% to 84%. They also exhibited virulence against Galleria mellonella. This research sheds light on the potential health risks of carbapenem- and polymyxin-resistant ECC strains displaying virulence in natural water bodies.

Graphical abstract

Keywords

Enterobacter cloacae complex / Natural water bodies / Carbapenem / Polymyxin / Co-resistance

Highlight

	● Carbapenem- and polymyxin-resistant ECC strains were isolated.
	● Carbapenem- and polymyxin-resistant ECC strains exhibited high cytotoxicity.
	● Carbapenem- and polymyxin-resistant ECC strains were classified as ST13.

References

[1] Boyd D A, Mataseje L F, Davidson R, Delport J A, Fuller J, Hoang L, Lefebvre B, Levett P N, Roscoe D L, Willey B M. . (2017). Enterobacter cloacae complex isolates harboring bla_NMC-A or bla_IMI-type class A carbapenemase genes on novel chromosomal integrative elements and plasmids. Antimicrobial Agents and Chemotherapy, 61(5): e02578–16

[2] Bush K, Bradford P A. (2016). β-Lactams and β-Lactamase inhibitors: an overview. Cold Spring Harbor Perspectives in Medicine, 6(8): a025247

[3] Bush K, Fisher J F. (2011). Epidemiological expansion, structural studies, and clinical challenges of new β-lactamases from gram-negative bacteria. Annual Review of Microbiology, 65: 455–478

[4] ChavdaBLv J NHouM YChavdaK DKreiswirth B NFengY JChenLYuF Y (2018). Coidentification of mcr-4.3 and bla_NDM-1 in a clinical Enterobacter cloacae isolate from China. Antimicrobial Agents and Chemotherapy, 62(10): 10.1128/aac.00649–18

[5] Chen J J, Tian S F, Nian H, Wang R X, Li F S, Jiang N, Chu Y Z. (2021). Carbapenem-resistant Enterobacter cloacae complex in a tertiary hospital in Northeast China, 2010–2019. BMC Infectious Diseases, 21(1): 611

[6] Chen L, Wen Y M. (2011). The role of bacterial biofilm in persistent infections and control strategies. International Journal of Oral Science, 3(2): 66–73

[7] Cohen S P, Mcmurry L M, Levy S B. (1988). marA locus causes decreased expression of OmpF porin in multiple-antibiotic-resistant (Mar) mutants of Escherichia coli. Journal of Bacteriology, 170(12): 5416–5422

[8] Crisan C V, Hammer B K. (2020). The Vibrio cholerae type VI secretion system: toxins, regulators and consequences. Environmental Microbiology, 22(10): 4112–4122

[9] Esposito F, Cardoso B, Fontana H, Fuga B, Cardenas-Arias A, Moura Q, Fuentes-Castillo D, Lincopan N. (2021). Genomic analysis of carbapenem-resistant Pseudomonas aeruginosa isolated from urban rivers confirms spread of clone sequence type 277 carrying broad resistome and virulome beyond the hospital. Frontiers in Microbiology, 12: 701921

[10] Ganbold M, Seo J, Wi Y M, Kwon K T, Ko K S. (2023). Species identification, antibiotic resistance, and virulence in Enterobacter cloacae complex clinical isolates from South Korea. Frontiers in Microbiology, 14: 1122691

[11] Gao W H, Li C C, Wang F T, Yang Y L, Zhang L, Wang Z X, Chen X, Tan M X, Cao G X, Zong G L. (2024). An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202. International Microbiology, 27(1): 277–290

[12] Grimsey E M, Weston N, Ricci V, Stone J W, Piddock L J V. (2020). Overexpression of RamA, which regulates production of the multidrug resistance efflux pump AcrAB-TolC, increases mutation rate and influences drug resistance phenotype. Antimicrobial Agents and Chemotherapy, 64(4): e02460–19

[13] Guérin F, Lallement C, Goudergues B, Isnard C, Sanguinetti M, Cacaci M, Torelli R, Cattoir V, Giard J C. (2020). Landscape of in vivo fitness-associated genes of Enterobacter cloacae complex. Frontiers in Microbiology, 11: 1609

[14] Hoffmann H, Roggenkamp A. (2003). Population genetics of the nomenspecies Enterobacter cloacae. Applied and Environmental Microbiology, 69(9): 5306–5318

[15] Ikehata Y, Doukyu N. (2022). Improving the organic solvent tolerance of Escherichia coli with vanillin, and the involvement of an AcrAB-TolC efflux pump in vanillin tolerance. Journal of Bioscience and Bioengineering, 133(4): 347–352

[16] Jeon M, Huh K, Ko J H, Cho S Y, Huh H J, Lee N Y, Kang C I, Chung D R, Peck K R. (2021). Difference in the clinical outcome of bloodstream infections caused by Klebsiella aerogenes and Enterobacter cloacae complex. Open Forum Infectious Diseases, 8(8): ofab390

[17] Jia S Y, Gao X R, Zhang Y Y, Shi P, Wang C, Zhou Q, Ye L, Zhang X X. (2023). Tertiary wastewater treatment processes can be a double-edged sword for water quality improvement in view of mitigating antimicrobial resistance and pathogenicity. Environmental Science & Technology, 57(1): 509–519

[18] Kikuchi T, Mizunoe Y, Takade A, Naito S, Yoshida S. (2005). Curli fibers are required for development of biofilm architecture in Escherichia coli K-12 and enhance bacterial adherence to human uroepithelial cells. Microbiology and Immunology, 49(9): 875–884

[19] Lamari F, Chakroun I, Rtimi S. (2017). Assessment of the correlation among antibiotic resistance, adherence to abiotic and biotic surfaces, invasion and cytotoxicity of Pseudomonas aeruginosa isolated from diseased gilthead sea bream. Colloids and Surfaces B: Biointerfaces, 158: 229–236

[20] Lebov J, Grieger K, Womack D, Zaccaro D, Whitehead N, Kowalcyk B, Macdonald P D M. (2017). A framework for One Health research. One Health, 3: 44–50

[21] Lepuschitz S, Schill S, Stoeger A, Pekard-Amenitsch S, Huhulescu S, Inreiter N, Hartl R, Kerschner H, Sorschag S, Springer B. . (2019). Whole genome sequencing reveals resemblance between ESBL-producing and carbapenem resistant Klebsiella pneumoniae isolates from Austrian rivers and clinical isolates from hospitals. Science of the Total Environment, 662: 227–235

[22] Lin M M, Yang Y Q, Yang Y X, Chen G P, He R W, Wu Y P, Zhong L L, El-Sayed Ahmed M A E, Feng S Y, Shen C. . (2020). Co-occurrence of mcr-9 and bla_NDM-1 in Enterobacter cloacae isolated from a patient with bloodstream infection. Infection and Drug Resistance, 13: 1397–1402

[23] Liu S X, Chen L Q, Wang L B, Zhou B B, Ye D D, Zheng X K, Lin Y S, Zeng W L, Zhou T L, Ye J Z. (2022). Cluster differences in antibiotic resistance, biofilm formation, mobility, and virulence of clinical Enterobacter cloacae complex. Frontiers in Microbiology, 13: 814831

[24] Liu S X, Fang R C, Zhang Y, Chen L J, Huang N, Yu K H, Zhou C, Cao J M, Zhou T L. (2021). Characterization of resistance mechanisms of Enterobacter cloacae complex co-resistant to carbapenem and colistin. BMC Microbiology, 21(1): 208

[25] Long J Y, Yang C, Liu J J, Ma C J, Jiao M, Hu H M, Xiong J, Zhang Y, Wei W, Yang H Z. . (2024). Tannic acid inhibits Escherichia coli biofilm formation and underlying molecular mechanisms: biofilm regulator CsgD. Biomedicine & Pharmacotherapy, 175: 116716

[26] López-Jiménez A T, Mostowy S. (2021). Emerging technologies and infection models in cellular microbiology. Nature Communications, 12(1): 6764

[27] Low K E, Howell P L. (2018). Gram-negative synthase-dependent exopolysaccharide biosynthetic machines. Current Opinion in Structural Biology, 53: 32–44

[28] Manohar P, Shanthini T, Ayyanar R, Bozdogan B, Wilson A, Tamhankar A J, Nachimuthu R, Lopes B S. (2017). The distribution of carbapenem- and colistin-resistance in Gram-negative bacteria from the Tamil Nadu region in India. Journal of Medical Microbiology, 66(7): 874–883

[29] Miltgen G, Bonnin R A, Avril C, Benoit-Cattin T, Martak D, Leclaire A, Traversier N, Roquebert B, Jaffar-Bandjee M C, Lugagne N. . (2018). Outbreak of IMI-1 carbapenemase-producing colistin-resistant Enterobacter cloacae on the French island of Mayotte (Indian Ocean). International Journal of Antimicrobial Agents, 52(3): 416–420

[30] Norgan A P, Freese J M, Tuin P M, Cunningham S A, Jeraldo P R, Patel R. (2016). Carbapenem- and colistin-resistant Enterobacter cloacae from delta, Colorado, in 2015. Antimicrobial Agents and Chemotherapy, 60(5): 3141–3144

[31] Piedra-Carrasco N, Fàbrega A, Calero-Cáceres W, Cornejo-Sánchez T, Brown-Jaque M, Mir-Cros A, Muniesa M, González-López J J. (2017). Carbapenemase-producing Enterobacteriaceae recovered from a Spanish river ecosystem. PLoS One, 12(4): e0175246

[32] Poirel L, Jayol A, Nordmann P. (2017). Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. Clinical Microbiology Reviews, 30(2): 557–596

[33] Sharma P, Haycocks J R J, Middlemiss A D, Kettles R A, Sellars L E, Ricci V, Piddock L J V, Grainger D C. (2017). The multiple antibiotic resistance operon of enteric bacteria controls DNA repair and outer membrane integrity. Nature Communications, 8(1): 1444

[34] Sreekumaran S, Priya V K, Premnath M, Prathiush P R, Anisha M N, Mathew J, Jayachandran K, Radhakrishnan E K. (2024). Novel in-genome based analysis demonstrates the evolution of OmpK37, antimicrobial resistance gene from a potentially pathogenic pandrug resistant Klebsiella pneumoniae MS1 isolated from healthy broiler feces. Science of the Total Environment, 930: 172713

[35] Stepanović S, Vuković D, Hola V, Di Bonaventura G, Djukić S, Cirković I, Ruzicka F. (2007). Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by Staphylococci. Journal of Pathology, Microbiology and Immunology, 115(8): 891–899

[36] Tarumoto N, Kodana M, Watanabe N, Sakai J, Imai K, Yamaguchi T, Taji Y, Ebihara Y, Murakami T, Maeda T. . (2018). First report of the isolation of bla_IMI-1-producing colistin-heteroresistant Enterobacter cloacae in Japan, September 2016. Journal of Infection and Chemotherapy, 24(11): 941–943

[37] Yitayew B, Woldeamanuel Y, Asrat D, Aseffa A, Olsson P E, Mihret A, Jass J. (2022). Carbapenemase-producing Aeromonas species isolated from the urban-impacted Akaki River in Ethiopia. Journal of Water and Health, 20(6): 903–914

[38] Zhang T, Wu H Y, Ma C C, Yang Y D, Li H B, Yang Z W, Zhou S Q, Shi D Y, Chen T J, Yang D. . (2024). Emergence of colistin-resistant Stenotrophomonas maltophilia with high virulence in natural aquatic environments. Science of the Total Environment, 933: 173221

[39] Zhao Y, Yang Q E, Zhou X, Wang F H, Muurinen J, Virta M P, Brandt K K, Zhu Y G. (2021). Antibiotic resistome in the livestock and aquaculture industries: status and solutions. Critical Reviews in Environmental Science and Technology, 51(19): 2159–2196

[40] ZhouS QWang H RLiH BYangY DShiD Y YangZ WYang DChenT JLiJ WJinM (2025). Emergence of polymyxin-resistant Yersinia enterocolitica strains in natural aquatic environments. Environmental Pollution, 364(Pt 2): 125341

RIGHTS & PERMISSIONS

Higher Education Press 2025