BACKGROUND: the co-production of carbapenemases and mcr-genes represents a worrisome event in the treatment of Enterobacteriaceae infections. The aim of the study was to characterize the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy. METHODS: species identification and antibiotic susceptibility profiling were performed using MALDI-TOF and broth microdilution methods, respectively. Transferability of the bla VIM- and mcr- type genes was verified through conjugation experiment. Extracted DNA was sequenced using long reads sequencing technology on the Sequel I platform (PacBio). RESULTS: the first isolate showed clinical resistance against ertapenem yet was colistin susceptible (EUCAST 2020 breakpoints). The mcr-9.2 gene was harbored on a conjugative IncHI2 plasmid, while the bla VIM-1 determinant was harbored on a conjugative IncN plasmid. The second isolate, resistant to both carbapenems and colistin, harbored: mcr-9 gene and its two component regulatory genes for increased expression on the chromosome, mcr-4.3 on non-conjugative (yet co-transferable) ColE plasmid, and bla VIM-1 on a non-conjugative IncA plasmid. CONCLUSIONS: to our knowledge, this is the first report of co-production of VIM and MCR in ECC isolates in Italy.

Biomedical Center Faculty of Medicine Charles University 323 00 Pilsen Czech Republic

Clinical Microbiology Azienda Ospedaliero Universitaria di Modena 411 25 Modena Italy

Department of Clinical Surgical Diagnostic and Pediatric Sciences Unit of Microbiology and Clinical Microbiology University of Pavia 27100 Pavia Italy

Department of Microbiology Faculty of Medicine and University Hospital in Pilsen Charles University 306 05 Pilsen Czech Republic

Laboratory of Clinical Microbiology ASP Golgi Redaelli 201 46 Milan Italy

Risk Analysis and Genomic Epidemiology Unit Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna 43126 Parma Italy

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Mustafa A., Ibrahim M., Rasheed M.A., Kanwal S., Hussain A., Sami A., Ahmed R., Bo Z. Genome-wide analysis of four enterobacter cloacae complex type strains: Insights into virulence and niche adaptation. Sci. Rep. 2020;10:8150. doi: 10.1038/s41598-020-65001-4. PubMed DOI PMC

Annavajhala M.K., Gomez-Simmonds A., Uhlemann A.C. Multidrug-resistant enterobacter cloacae complex emerging as a global, diversifying threat. Front. Microbiol. 2019;10:44. doi: 10.3389/fmicb.2019.00044. PubMed DOI PMC

Petrosillo N., Taglietti F., Granata G. Treatment options for colistin resistant Klebsiella pneumoniae: Present and future. J. Clin. Med. 2019;8:934. doi: 10.3390/jcm8070934. PubMed DOI PMC

Wang C., Feng Y., Liu L., Wei L., Kang M., Zong Z. Identification of novel mobile colistin resistance gene mcr-10. Emerg. Microbes Infect. 2020;9:508–516. doi: 10.1080/22221751.2020.1732231. PubMed DOI PMC

Carattoli A., Villa L., Feudi C., Curcio L., Orsini S., Luppi A., Pezzotti G., Magistrali C.F. Novel plasmid-mediated colistin resistance mcr-4 gene in Salmonella and Escherichia coli, Italy 2013, Spain and Belgium, 2015 to 2016. Euro Surveill. 2017;22:30589. doi: 10.2807/1560-7917.ES.2017.22.31.30589. PubMed DOI PMC

Carroll L.M., Gaballa A., Guldimann C., Sullivan G., Henderson L.O., Wiedmann M. Identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible Salmonella enterica serotype typhimurium isolate. mBio. 2019;10:e00853-19. doi: 10.1128/mBio.00853-19. PubMed DOI PMC

Lauretti L., Riccio M.L., Mazzariol A., Cornaglia G., Amicosante G., Fontana R., Rossolini G.M. Cloning and characterization of blaVIM, a new integron-borne metallo-β-lactamase gene from a pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 1999;43:1584–1590. doi: 10.1128/AAC.43.7.1584. PubMed DOI PMC

Piazza A., Comandatore F., Romeri F., Brilli M., Dichirico B., Ridolfo A., Antona C., Bandi C., Gismondo M.R., Rimoldi S.G. Identification of blaVIM-1 gene in ST307 and ST661 Klebsiella pneumoniae clones in Italy: Old acquaintances for new combinations. Microb. Drug Resist. 2019;25:787–790. doi: 10.1089/mdr.2018.0327. PubMed DOI

Di Tella D., Tamburro M., Guerrizio G., Fanelli I., Sammarco M.L., Ripabelli G. Molecular epidemiological insights into colistin-resistant and carbapenemases-producing clinical Klebsiella pneumoniae isolates. Infect. Drug Resist. 2019;12:3783–3795. doi: 10.2147/IDR.S226416. PubMed DOI PMC

Simoni S., Caucci S., Brenciani A., Morroni G., Giovanetti E., Menzo S., Facinelli B., Mingoia M. Increase and diversity of carbapenemase-producing Escherichia coli isolates, Italy. Future Microbiol. 2019;14:1035–1042. doi: 10.2217/fmb-2019-0069. PubMed DOI

Arcari G., Di Lella F.M., Bibbolino G., Mengoni F., Beccaccioli M., Antonelli G., Faino L., Carattoli A. A multispecies cluster of VIM-1 carbapenemase-producing Enterobacterales linked by a novel, highly conjugative, and broad-host-range IncA plasmid forebodes the reemergence of VIM-1. Antimicrob. Agents Chemother. 2020;64:e02435-19. doi: 10.1128/AAC.02435-19. PubMed DOI PMC

Giani T., Antonelli A., Sennati S., Di Pilato V., Chiarelli A., Cannatelli A., Gatsch C., Luzzaro F., Spanu T., Stefani S., et al. Results of the Italian infection-carbapenem resistance evaluation surveillance trial (iCREST-IT): Activity of ceftazidime/avibactam against Enterobacterales isolated from urine. J. Antimicrob. Chemother. 2020;75:979–983. doi: 10.1093/jac/dkz547. PubMed DOI

Calia C., Pazzani C., Oliva M., Scrascia M., Lovreglio P., Capolongo C., Dionisi A.M., Chiarelli A., Monno R. Carbapenemases-producing Klebsiella pneumoniae in hospitals of two regions of Southern Italy. APMIS. 2017;125:491–498. doi: 10.1111/apm.12666. PubMed DOI

Principe L., Piazza A., Mauri C., Anesi A., Bracco S., Brigante G., Casari E., Agrappi C., Caltagirone M., Novazzi F., et al. Multicenter prospective study on the prevalence of colistin resistance in Escherichia coli: Relevance of mcr-1-positive clinical isolates in Lombardy, Northern Italy. Infect. Drug Resist. 2018;11:377–385. doi: 10.2147/IDR.S160489. PubMed DOI PMC

Del Bianco F., Morotti M., Pedna M.F., Farabegoli P., Sambri V. Microbiological surveillance of plasmid mediated colistin resistance in human Enterobacteriaceae isolates in Romagna (Northern Italy): August 2016–July 2017. Int. J. Infect. Dis. 2018;69:96–98. doi: 10.1016/j.ijid.2018.02.006. PubMed DOI

Carretto E., Brovarone F., Nardini P., Russello G., Barbarini D., Pongolini S., Gagliotti C., Carattoli A., Sarti M. Detection of mcr-4 positive Salmonella enterica serovar Typhimurium in clinical isolates of human origin, Italy, October to November 2016. Euro Surveill. 2018;23:17-00821. doi: 10.2807/1560-7917.ES.2018.23.2.17-00821. PubMed DOI PMC

Lee K., Lim Y.S., Yong D., Yum J.H., Chong Y. Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp. J. Clin. Microbiol. 2003;41:4623–4629. doi: 10.1128/JCM.41.10.4623-4629.2003. PubMed DOI PMC

Doi Y., Potoski B.A., Adams-Haduch J.M., Sidjabat H.E., Pasculle A.W., Paterson D.L. Simple disk-based method for detection of Klebsiella pneumoniae carbapenemase-type-lactamase by use of a boronic acid compound. J. Clin. Microbiol. 2008;46:4083–4086. doi: 10.1128/JCM.01408-08. PubMed DOI PMC

Glupczynski Y., Huang T.-D., Bouchahrouf W., De Castro R.R., Bauraing C., Gérard M., Verbruggen A.-M., Deplano A., Denis O., Bogaerts P. Rapid emergence and spread of OXA-48-producing carbapenem-resistant Enterobacteriaceae isolates in Belgian hospitals. Int. J. Antimicrob. Agents. 2012;39:168–172. doi: 10.1016/j.ijantimicag.2011.10.005. PubMed DOI

Ataei B., Shirani K., Roshandel F. Antibiotic resistance pattern and evaluation of metallo-beta lactamase genes (VIM and IMP) in Pseudomonas aeruginosa strains producing MBL enzyme, isolated from patients with secondary immunodeficiency. Adv. Biomed. Res. 2016;5:124. doi: 10.4103/2277-9175.186986. PubMed DOI PMC

Jolley K.A., Bray J.E., Maiden M.C.J. Open-access bacterial population genomics: BIGSdb software, the PubMLST. org website and their applications. Wellcome Open Res. 2018;3:124. doi: 10.12688/wellcomeopenres.14826.1. PubMed DOI PMC

Bortolaia V., Kaas R.S., Ruppe E., Roberts M.C., Schwarz S., Cattoir V., Philippon A., Allesoe R.L., Rebelo A.R., Florensa A.F., et al. ResFinder 4.0 for predictions of phenotypes from genotypes. J. Antimicrob. Chemother. 2020;75:3491–3500. doi: 10.1093/jac/dkaa345. PubMed DOI PMC

Alcock B.P., Raphenya A.R., Lau T.T., Tsang K.K., Bouchard M., Edalatmand A., Huynh W., Nguyen A.L., Cheng A.A., Liu S., et al. CARD 2020: Antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res. 2020;48:D517–D525. doi: 10.1093/nar/gkz935. PubMed DOI PMC

Carattoli A., Zankari E., García-Fernández A., Voldby Larsen M., Lund O., Villa L., Møller Aarestrup F., Hasman H. In silico detection and typing of plasmids using PlasmidFinder and Plasmid multilocus sequence typing. Antimicrob. Agents Chemother. 2014;58:3895–3903. doi: 10.1128/AAC.02412-14. PubMed DOI PMC

Moura A., Soares M., Pereira C., Leitao N., Henriques I., Correia A. INTEGRALL: A database and search engine for integrons, integrases and gene cassettes. Bioinformatics. 2009;25:1096–1098. doi: 10.1093/bioinformatics/btp105. PubMed DOI

Breland E.J., Zhang E.W., Bermudez T., Martinez C.R., Hadjifrangiskou M. The histidine residue of QseC is required for canonical signaling between QseB and PmrB in uropathogenic Escherichia coli. J. Bacteriol. 2017;199:e00060-17. doi: 10.1128/JB.00060-17. PubMed DOI PMC

Rahube T.O., Yost C.K. Characterization of a mobile and multiple resistance plasmid isolated from swine manure and its detection in soil after manure application. J. Appl. Microbiol. 2012;112:1123–1133. doi: 10.1111/j.1365-2672.2012.05301.x. PubMed DOI

Matsumura Y., Peirano G., Devinney R., Bradford P.A., Motyl M.R., Adams M.D., Chen L., Kreiswirth B., Pitout J.D.D. Genomic epidemiology of global VIM-producing Enterobacteriaceae. J. Antimicrob. Chemother. 2017;72:2249–2258. doi: 10.1093/jac/dkx148. PubMed DOI PMC

Mattioni Marchetti V., Bitar I., Piazza A., Mercato A., Fogato E., Hrabak J., Migliavacca R. Genomic insight of VIM-harboring IncA plasmid from a clinical ST69 Escherichia coli strain in Italy. Microorganisms. 2020;8:1232. doi: 10.3390/microorganisms8081232. PubMed DOI PMC

Chavda K.D., Westblade L.F., Satlin M.J., Hemmert A.C., Castanheira M., Jenkins S.G., Chen L., Kreiswirth B.N. First report of blaVIM-4- and mcr-9-coharboring enterobacter species isolated from a pediatric patient. mSphere. 2019;4:e00629-19. doi: 10.1128/mSphere.00629-19. PubMed DOI PMC

Soliman A.M., Maruyama F., Zarad H.O., Ota A., Nariya H., Shimamoto T., Shimamoto T. Emergence of a multidrug-resistant enterobacter hormaechei clinical isolate from Egypt co-harboring mcr-9 and blaVIM-4. Microorganisms. 2020;8:595. doi: 10.3390/microorganisms8040595. PubMed DOI PMC

Sadek M., Nariya H., Shimamoto T., Kayama S., Yu L., Hisatsune J., Sugai M., Nordmann P., Poirel L., Shimamoto T. First genomic characterization of blaVIM-1 and mcr-9-coharbouring Enterobacter hormaechei isolated from food of animal origin. Pathogens. 2020;9:687. doi: 10.3390/pathogens9090687. PubMed DOI PMC

Bitar I., Papagiannitsis C.C., Kraftova L., Chudejova K., Marchetti V.M., Hrabak J. Detection of five mcr-9-carrying Enterobacterales isolates in four Czech hospitals. mSphere. 2020;5:e01008-20. doi: 10.1128/mSphere.01008-20. PubMed DOI PMC

Karlowsky J.A., Kazmierczak K.M., de Jonge B.L.M., Hackel M.A., Sahm D.F., Bradford P.A. In vitro activity of aztreonam-avibactam against Enterobacteriaceae and Pseudomonas aeruginosa isolated by clinical laboratories in 40 countries from 2012 to 2015. Antimicrob. Agents Chemother. 2017;61:e00472-17. doi: 10.1128/AAC.00472-17. PubMed DOI PMC

Zhanel G.G., Golden A.R., Zelenitsky S., Wiebe K., Lawrence C.K., Adam H.J., Idowu T., Domalaon R., Schweizer F., Zhanel M.A., et al. Cefiderocol: A siderophore cephalosporin with activity against carbapenem-resistant and multidrug-resistant gram-negative bacilli. Drugs. 2019;79:271–289. doi: 10.1007/s40265-019-1055-2. PubMed DOI

García-Fernández A., Villa L., Moodley A., Hasman H., Miriagou V., Guardabassi L., Carattoli A. Multilocus sequence typing of IncN plasmids. J. Antimicrob. Chemother. 2011;66:1987–1991. doi: 10.1093/jac/dkr225. PubMed DOI

Hancock S.J., Phan M.D., Peters K.M., Forde B.M., Chong T.M., Yin W.F., Chan K.G., Paterson D.L., Walsh T.R., Beatson S.A., et al. Identification of IncA/C plasmid replication and maintenance genes and development of a plasmid multilocus sequence typing scheme. Antimicrob. Agents Chemother. 2016;61:e01740-16. doi: 10.1128/AAC.01740-16. PubMed DOI PMC

Marchetti V.M., Bitar I., Mercato A., Nucleo E., Bonomini A., Pedroni P., Hrabak J., Migliavacca R. Complete nucleotide sequence of plasmids of two Escherichia coli strains carrying blaNDM-5 and blaNDM-5 and blaOXA-181 from the same patient. Front. Microbiol. 2020;10:3095. doi: 10.3389/fmicb.2019.03095. PubMed DOI PMC

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