The aim of this study was to report the characterization of the first mcr-positive Enterobacterales isolated from Czech hospitals. In 2019, one Citrobacter freundii and four Enterobacter isolates were recovered from Czech hospitals. The production of carbapenemases was examined by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay. Additionally, bacteria were screened for the presence of carbapenemase-encoding genes and plasmid-mediated colistin resistance genes by PCR. To define the genetic units carrying mcr genes, the genomic DNAs of mcr-carrying clinical isolates were sequenced on the PacBio Sequel I platform. Results showed that all isolates carried blaVIM- and mcr-like genes. Analysis of whole-genome sequencing (WGS) data revealed that all isolates carried mcr-9-like alleles. Furthermore, the three sequence type 106 (ST106) Enterobacter hormaechei isolates harbored the blaVIM-1 gene, while the ST764 E. hormaechei and ST95 C. freundii included blaVIM-4 Analysis of plasmid sequences showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. Additionally, at least one multidrug resistance (MDR) region was identified in each mcr-9-carrying IncHI2 plasmid. The blaVIM-4 gene was found in the MDR regions of p48880_MCR_VIM and p51929_MCR_VIM. In the three remaining isolates, blaVIM-1 was localized on plasmids (∼55 kb) exhibiting repA-like sequences 99% identical to the respective gene of pKPC-CAV1193. In conclusion, to the best of our knowledge, these 5 isolates were the first mcr-9-positive bacteria of clinical origin identified in the Czech Republic. Additionally, the carriage of the blaVIM-1 on pKPC-CAV1193-like plasmids is described for the first time. Thus, our findings underline the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants.IMPORTANCE Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the "last-resort" antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr, in different host species and geographic areas were published. Here, we report the first detection of Enterobacterales carrying mcr-9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 Enterobacter hormaechei isolates harbored blaVIM-1, while the ST764 E. hormaechei and ST95 Citrobacter freundii isolates included blaVIM-4 Analysis of WGS data showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. blaVIM-4 was found in the MDR regions of IncHI2 plasmids, while blaVIM-1 was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms.

Biomedical Center Faculty of Medicine Charles University Pilsen Czech Republic

Department of Microbiology Faculty of Medicine and University Hospital in Pilsen Charles University Pilsen Czech Republic

Department of Microbiology University Hospital of Larissa Larissa Greece

Zobrazit více v PubMed

Biswas S, Brunel JM, Dubus JC, Reynaud-Gaubert M, Rolain JM. 2012. Colistin: an update on the antibiotic of the 21st century. Expert Rev Anti Infect Ther 10:917–934. doi:10.1586/eri.12.78. PubMed DOI

Gurjar M. 2015. Colistin for lung infection: an update. J Intensive Care 3:3. doi:10.1186/s40560-015-0072-9. PubMed DOI PMC

Yilmaz GR, Dizbay M, Guven T, Pullukcu H, Tasbakan M, Guzel OT, Tekce YT, Ozden M, Turhan O, Guner R, Cag Y, Bozkurt F, Karadag FY, Kartal ED, Gozel G, Bulut C, Erdinc S, Keske S, Acikgoz ZC, Tasyaran MA. 2016. Risk factors for infection with colistin-resistant gram-negative microorganisms: a multicenter study. Ann Saudi Med 36:216–222. doi:10.5144/0256-4947.2016.216. PubMed DOI PMC

Poudyal A, Howden BP, Bell JM, Gao W, Owen RJ, Turnidge JD, Nation RL, Li J. 2008. In vitro pharmacodynamics of colistin against multidrug-resistant Klebsiella pneumoniae. J Antimicrob Chemother 62:1311–1318. doi:10.1093/jac/dkn425. PubMed DOI

Olaitan AO, Morand S, Rolain JM. 2014. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol 5:643. doi:10.3389/fmicb.2014.00643. PubMed DOI PMC

Liu Y-Y, Wang Y, Walsh TR, Yi L-X, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, Yu L-F, Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu J-H, Shen J. 2016. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 16:161–168. doi:10.1016/S1473-3099(15)00424-7. PubMed DOI

Skov RL, Monnet DL. 2016. Plasmid-mediated colistin resistance (mcr-1 gene): three months later, the story unfolds. Euro Surveill 21:30155. doi:10.2807/1560-7917.ES.2016.21.9.30155. PubMed DOI

Al-Tawfiq JA, Laxminarayan R, Mendelson M. 2017. How should we respond to the emergence of plasmid-mediated colistin resistance in humans and animals? Int J Infect Dis 54:77–84. doi:10.1016/j.ijid.2016.11.415. PubMed DOI

Li Y, Dai X, Zeng J, Gao Y, Zhang Z, Zhang L. 2020. Characterization of the global distribution and diversified plasmid reservoirs of the colistin resistance gene mcr-9. Sci Rep 10:8113. doi:10.1038/s41598-020-65106-w. PubMed DOI PMC

European Committee on Antimicrobial Susceptibility Testing. 2012. EUCAST guidelines for detection of resistance mechanism and specific resistances of clinical and/or epidemiological importance. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Consultation/EUCAST_guidelines_detection_of_resistance_mechanisms_121222.pdf

Rotova V, Papagiannitsis CC, Skalova A, Chudejova K, Hrabak J. 2017. Comparison of imipenem and meropenem antibiotics for the MALDI-TOF MS detection of carbapenemase activity. J Microbiol Methods 137:30–33. doi:10.1016/j.mimet.2017.04.003. PubMed DOI

Papagiannitsis CC, Izdebski R, Baraniak A, Fiett J, Herda M, Hrabak J, Derde LPG, Bonten MJM, Carmeli Y, Goossens H, Hryniewicz W, Brun-Buisson C, Gniadkowski M, MOSAR WP2, WP3 and WP5 study groups . 2015. Survey of metallo-β-lactamase-producing Enterobacteriaceae colonizing patients in European ICUs and rehabilitation units, 2008–11. J Antimicrob Chemother 70:1981–1988. doi:10.1093/jac/dkv055. PubMed DOI

Franceschini N, Caravelli B, Docquier JD, Galleni M, Frère JM, Amicosante G, Rossolini GM. 2000. Purification and biochemical characterization of the VIM-1 metallo-β-lactamase. Antimicrob Agents Chemother 44:3003–3007. doi:10.1128/aac.44.11.3003-3007.2000. PubMed DOI PMC

Borowiak M, Baumann B, Fischer J, Thomas K, Deneke C, Hammerl JA, Szabo I, Malorny B. 2020. Development of a novel mcr-6 to mcr-9 multiplex PCR and assessment of mcr-1 to mcr-9 occurrence in colistin-resistant Salmonella enterica isolates from environment, feed, animals and food (2011–2018) in Germany. Frontiers Microbiol 11:80. doi:10.3389/fmicb.2020.00080. PubMed DOI PMC

European Committee on Antimicrobial Susceptibility Testing. 2003. Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution. Clin Microbiol Infect 9:1–7. PubMed

Darling AE, Mau B, Perna NT. 2010. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 5:e11147. doi:10.1371/journal.pone.0011147. PubMed DOI PMC

Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. 2011. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12:402. doi:10.1186/1471-2164-12-402. PubMed DOI PMC

Hoffmann H, Roggenkamp A. 2003. Population genetics of the nomenspecies Enterobacter cloacae. Appl Environ Microbiol 69:5306–5318. doi:10.1128/aem.69.9.5306-5318.2003. PubMed DOI PMC

García-Fernández A, Carattoli A. 2010. Plasmid double locus sequence typing for IncHI2 plasmids, a subtyping scheme for the characterization of IncHI2 plasmids carrying extended-spectrum beta-lactamase and quinolone resistance genes. J Antimicrob Chemother 65:1155–1161. doi:10.1093/jac/dkq101. PubMed DOI

Zingali T, Chapman TA, Webster J, Chowdhury PR, Djordjevic SP. 2020. Genomic characterisation of a multiple drug resistant IncHI2 ST4 plasmid in Escherichia coli ST744 in Australia. Microorganisms 8:896. doi:10.3390/microorganisms8060896. PubMed DOI PMC

Chavda KD, Westblade LF, Satlin MJ, Hemmert AC, Castanheira M, Jenkins SG, Chen L, Kreiswirth BN. 2019. First report of blaVIM-4- and mcr-9-coharboring Enterobacter species isolated from a pediatric patient. mSphere 4:e00629-19. doi:10.1128/mSphere.00629-19. PubMed DOI PMC

Kieffer N, Royer G, Decousser JW, Bourrel AS, Palmieri M, Ortiz De La Rosa JM, Jacquier H, Denamur E, Nordmann P, Poirel L. 2019. mcr-9, an inducible gene encoding an acquired phosphoethanolamine transferase in Escherichia coli, and its origin. Antimicrob Agents Chemother 63:e00965-19. doi:10.1128/AAC.00965-19. PubMed DOI PMC

Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. 2005. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63:219–228. doi:10.1016/j.mimet.2005.03.018. PubMed DOI

Lombardi G, Luzzaro F, Docquier JD, Riccio ML, Perilli M, Colì A, Amicosante G, Rossolini GM, Toniolo A. 2002. Nosocomial infections caused by multidrug-resistant isolates of Pseudomonas putida producing VIM-1 metallo-beta-lactamase. J Clin Microbiol 40:4051–4055. doi:10.1128/jcm.40.11.4051-4055.2002. PubMed DOI PMC

Papousek I, Papagiannitsis CC, Medvecky M, Hrabak J, Dolejska M. 2017. Complete nucleotide sequences of two VIM-1-encoding plasmids from Klebsiella pneumoniae and Leclercia adecarboxylata isolates of Czech origin. Antimicrob Agents Chemother 61:e02648-16. doi:10.1128/AAC.02648-16. PubMed DOI PMC

Gelbíčová T, Barakova A, Florianová M, Jamborova I, Zelendova M, Pospisilova L, Koláčková I, Karpíšková R. 2019. Dissemination and comparison of genetic determinants of mcr-mediated colistin resistance in Enterobacteriaceae via retailed raw meat products. Front Microbiol 10:2824. doi:10.3389/fmicb.2019.02824. PubMed DOI PMC

Bitar I, Medvecky M, Gelbicova T, Jakubu V, Hrabak J, Zemlickova H, Karpiskova R, Dolejska M. 2019. Complete nucleotide sequences of mcr-4.3-carrying plasmids in Acinetobacter baumannii sequence type 345 of human and food origin from the Czech Republic, the first case in Europe. Antimicrob Agents Chemother 63:e01166-19. doi:10.1128/AAC.01166-19. PubMed DOI PMC

Matamoros S, van Hattem JM, Arcilla MS, Willemse N, Melles DC, Penders J, Vinh TN, Thi Hoa N, Bootsma MCJ, van Genderen PJ, Goorhuis A, Grobusch M, Molhoek N, Oude Lashof AML, Stobberingh EE, Verbrugh HA, de Jong MD, Schultsz C. 2017. Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction. Sci Rep 7:1–10. doi:10.1038/s41598-017-15539-7. PubMed DOI PMC

VIM-1-producing Enterobacter asburiae with mobile colistin resistance genes from wastewaters

Plasmid-mediated colistin resistance among human clinical Enterobacterales isolates: national surveillance in the Czech Republic

Implication of different replicons in the spread of the VIM-1-encoding integron, In110, in Enterobacterales from Czech hospitals

Genomic Characterization of VIM and MCR Co-Producers: The First Two Clinical Cases, in Italy