BACKGROUND: Wastewaters are considered as important players in the spread of antimicrobial resistance, thus affecting the health of humans and animals. Here, we focused on wastewaters as a possible source of carbapenemase-producing Enterobacterales for the environment. METHODS: A total of 180 presumptive coliforms from hospital and municipal wastewaters, and a river in the Czech Republic were obtained by selective cultivation on meropenem-supplemented media and tested for presence of carbapenemase-encoding genes by PCR. Strains carrying genes of interest were characterized by testing antimicrobial susceptibility, carbapenemase production and combination of short- and long- read whole-genome sequencing. The phylogenetic tree including publicly available genomes of Enterobacter asburiae was conducted using Prokka, Roary and RAxML. RESULTS: Three VIM-producing Enterobacter asburiae isolates, members of the Enterobacter cloacae complex, were detected from hospital and municipal wastewaters, and the river. The blaVIM-1 gene was located within a class 1 integron that was carried by different F-type plasmids and one non-typeable plasmid. Furthermore, one of the isolates carried plasmid-borne colistin-resistance gene mcr-10, while in another isolate chromosomally located mcr-9 without colistin resistance phenotype was detected. In addition, the analysis of 685 publicly available E. asburiae genomes showed they frequently carry carbapenemase genes, highlighting the importance of this species in the emergence of resistance to last-line antibiotics. CONCLUSION: Our findings pointed out the important contribution of hospital and community wastewaters in transmission of multi-drug resistant pathogens.

Central European Institute of Technology University of Veterinary Sciences Brno Brno Czech Republic

Department of Biology and Wildlife Diseases Faculty of Veterinary Hygiene and Ecology University of Veterinary Sciences Brno Brno Czech Republic

Division of Clinical Microbiology and Immunology Department of Laboratory Medicine The University Hospital Brno Brno Czech Republic

Faculty of Medicine Charles University Pilsen Czech Republic

Microbial Resistance and Drug Discovery VIB VUB Center for Structural Biology VIB Flanders Institute for Biotechnology Brussels Belgium

Structural Biology Brussels Vrije Universiteit Brussel Brussels Belgium

See more in PubMed

Davin-Regli A, Lavigne JP, Pagès JM. PubMed PMC

Wu W, Feng Y, Zong Z. Precise species identification for Enterobacter: a genome sequence-based study with reporting of two novel species, Enterobacter quasiroggenkampii sp. nov. and Enterobacter quasimori sp. nov. MSystems. 2020;5(5):e00527–20. 10.1128/msystems.00527-20. PubMed PMC

Annavajhala MK, Gomez-Simmonds A, Uhlemann AC. Multidrug-resistant PubMed PMC

Bitar I, Papagiannitsis CC, Kraftova L, Chudejova K, Mattioni Marchetti V, Hrabak J. Detection of five mcr-9 -carrying Enterobacterales isolates in four Czech hospitals. MSphere. 2020;5. 10.1128/msphere.01008-20. PubMed PMC

Wang C, Feng Y, Liu L, Wei L, Kang M, Zong Z. Identification of novel mobile colistin resistance gene PubMed PMC

Manaia CM, Rocha J, Scaccia N, Marano R, Radu E, Biancullo F, et al. Antibiotic resistance in wastewater treatment plants: tackling the black box. Environ Int. 2018;115:312–24. 10.1016/j.envint.2018.03.044. PubMed

Kutilova I, Medvecky M, Leekitcharoenphon P, Munk P, Masarikova M, Davidova-Gerzova L, et al. Extended-spectrum beta-lactamase-producing PubMed

Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis. 2011;70:119–23. 10.1016/j.diagmicrobio.2010.12.002. PubMed

The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 13.0, 2023. http://www.eucast.org

CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2020.

Dolejska M, Masarikova M, Dobiasova H, Jamborova I, Karpiskova R, Havlicek M, et al. High prevalence of PubMed PMC

Gilchrist CLM, Chooi YH. Clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics. 2021;37:2473–5. 10.1093/bioinformatics/btab007. PubMed

Zelendova M, Papagiannitsis CC, Sismova P, Medvecky M, Pomorska K, Palkovicova J, et al. Plasmid-mediated colistin resistance among human clinical PubMed PMC

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30:2068–9. 10.1093/bioinformatics/btu153. PubMed

Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MTG, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015;31:3691–3. 10.1093/bioinformatics/btv421. PubMed PMC

Page AJ, Taylor B, Delaney AJ, Soares J, Seemann T, Keane JA, et al. SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments. Microb Genom. 2016;2:e000056. 10.1099/mgen.0.000056. PubMed PMC

Stamatakis A. Bioinformatics. 2014;30:1312–3. 10.1093/bioinformatics/btu033. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. PubMed PMC

Letunic I, Bork P. Interactive tree of life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 2019;47:W256–9. 10.1093/nar/gkz239. PubMed PMC

Botelho J, Grosso F, Peixe L. Characterization of the pJB12 plasmid from PubMed PMC

Xu T, Zhang C, Ji Y, Song J, Liu Y, Guo Y, et al. Identification of PubMed

Hrabak J, Studentova V, Jakubu V, Adamkova V, Dvorakova L, Balejova M, et al. Prevalence study on carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolates in Czech hospitals-results from Czech Part of European Survey on Carbapenemase–Producing Enterobacteriaceae (EuSCAPE). Epidemiol Mikrobiol Imunol. 2015;64:87–91. PubMed

Mattioni VM, Kraftova L, Finianos M, Sourenian T, Hrabak J, Bitar I. Polyclonal spread of fosfomycin resistance among carbapenemase-producing members of the PubMed PMC

Surveillance Atlas of Infectious Diseases, European Centre for Disease Prevention and, Control. https://atlas.ecdc.europa.eu/public/index.aspx. Accessed 15 January 2024.

Chudejova K, Rotova V, Skalova A, Medvecky M, Adamkova V, Papagiannitsis CC, et al. Emergence of sequence type 252 PubMed

Rotova V, Papagiannitsis CC, Chudejova K, Medvecky M, Skalova A, Adamkova V, et al. First description of the emergence of PubMed

Finianos M, Kraftova L, Papagiannitsis CC, Adamkova V, Hrabak J, Bitar I. Genomic characterisation of three GES-producing enterobacterales isolated in the Czech Republic. J Glob Antimicrob Resist. 2022;29:116–9. 10.1016/j.jgar.2022.02.016. PubMed

Bleichenbacher S, Stevens MJA, Zurfluh K, Perreten V, Endimiani A, Stephan R, et al. Environmental dissemination of carbapenemase-producing Enterobacteriaceae in rivers in Switzerland. Environ Pollut. 2020;265. 10.1016/j.envpol.2020.115081. PubMed

Mills MC, Lee J. The threat of carbapenem-resistant bacteria in the environment: evidence of widespread contamination of reservoirs at a global scale. Environ Pollut. 2019;255. 10.1016/j.envpol.2019.113143. PubMed

Davidova-Gerzova L, Lausova J, Sukkar I, Nesporova K, Nechutna L, Vlkova K, et al. Hospital and community wastewater as a source of multidrug-resistant ESBL-producing PubMed PMC

Sismova P, Sukkar I, Kolidentsev N, Palkovicova J, Chytilova I, Bardon J, et al. Plasmid-mediated colistin resistance from fresh meat and slaughtered animals in the Czech Republic: nation-wide surveillance 2020–2021. Microbiol Spectr. 2023;11. 10.1128/spectrum.00609-23. PubMed PMC

Rada AM, De E, Cadena L, Agudelo C, Capataz C, Orozco N, et al. Dynamics of PubMed PMC

Li X, Jiang T, Wu C, Kong Y, Ma Y, Wu J, et al. Molecular epidemiology and genomic characterization of a plasmid-mediated PubMed PMC

Kananizadeh P, Oshiro S, Watanabe S, Iwata S, Kuwahara-Arai K, Shimojima M, et al. Emergence of carbapenem-resistant and colistin-susceptible PubMed PMC