KPC-3-producing bacteria are endemic in many countries but only recently became apparent their wide distribution in different Portuguese hospitals. The aim of this study is to characterize genetic backgrounds associated with bla KPC-3 among Klebsiella pneumoniae isolates recently identified on non-hospitalized patients in Portugal. Twenty KPC-producing K. pneumoniae identified between October 2014 and November 2015 in three different community laboratories were characterized. Isolates were mainly from patients from long-term care facilities (n = 11) or nursing homes (n = 6), most of them (75%) previously hospitalized in different Portuguese hospitals. Standard methods were used for bacterial identification and antibiotic susceptibility testing. Carbapenemase production was assessed by the Blue-Carba test, and identification of bla genes was performed by PCR and sequencing. Epidemiological features of KPC-producing K. pneumoniae included population structure (XbaI-PFGE, MLST and wzi sequencing), genetic context (mapping of Tn4401), and plasmid (replicon typing, S1-PFGE, and hybridization) analysis. All K. pneumoniae isolates produced KPC-3, with two MDR K. pneumoniae epidemic clones representing 75% of the isolates, namely ST147 (wzi64/K14.64, February-November 2015) and ST15 (two lineages exhibiting capsular types wzi19/K19 or wzi93/K60, July-November 2015). Other sporadic clones were detected: ST231 (n = 3; wzi104), ST348 (n = 1; wzi94) and ST109 (n = 1, wzi22/K22.37). bla KPC-3 was identified within Tn4401d in all isolates, located in most cases (80%) on cointegrated plasmids (repA FIA+repA FII+ori ColE1;105-250 kb) or in 50 kb IncN plasmids. In conclusion, this study highlights a polyclonal structure of KPC-3-producing K. pneumoniae and the predominance of the ST147 clone among non-hospitalized patients in Portugal, linked to platforms still unnoticed in Europe (bla KPC-3-Tn4401d-IncFIA) or firstly reported (bla KPC-3-Tn4401d-IncN). This scenario underlines the recent penetration of successful mobile genetic elements in previously circulating MDR K. pneumoniae lineages (mainly ST147 and ST15) in Portugal, rather than the importation of the global lineages from clonal group 258.

Botelho Moniz Análises Clínicas Santo Tirso Portugal

Faculty of Pharmacy in Hradec Králové Charles UniversityPrague Czech Republic; Faculty of Military Health Sciences University of DefenseBrno Czech Republic

UCIBIO REQUIMTE Laboratório de Microbiologia Faculdade de Farmácia Universidade do Porto Porto Portugal

UCIBIO REQUIMTE Laboratório de Microbiologia Faculdade de Farmácia Universidade do PortoPorto Portugal; FP ENAS CEBIMED Faculdade de Ciências da Saúde Universidade Fernando PessoaPorto Portugal

Zobrazit více v PubMed

Albiger B., Glasner C., Struelens M. J., Grundmann H., Monnet D. L., European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) working group (2015). Carbapenemase-producing Enterobacteriaceae in Europe: assessment by national experts from 38 countries, May 2015. Euro Surveill. 20:30062. 10.2807/1560-7917.ES.2015.20.45.30062 PubMed DOI

Baraniak A., Izdebski R., Fiett J., Herda M., Derde L. P. G., Bonten M. J. M., et al. . (2015). KPC-like carbapenemase-producing Enterobacteriaceae colonizing patients in Europe and Israel. Antimicrob. Agents Chemother. 60, 1912–1917. 10.1128/AAC.02756-15 PubMed DOI PMC

Bialek-Davenet S., Criscuolo A., Ailloud F., Passet V., Jones L., Delannoy-Vieillard A.-S., et al. . (2014). Genomic definition of hypervirulent and multidrug-resistant Klebsiella pneumoniae clonal groups. Emerg. Infect. Dis. 20, 1812–1820. 10.3201/eid2011.140206 PubMed DOI PMC

Bogaerts P., Rezende de Castro R., de Mendonça R., Huang T.-D., Denis O., Glupczynski Y. (2013). Validation of carbapenemase and extended-spectrum β-lactamase multiplex endpoint PCR assays according to ISO 15189. J. Antimicrob. CheMother. 68, 1576–1582. 10.1093/jac/dkt065 PubMed DOI

Bonura C., Giuffrè M., Aleo A., Fasciana T., Di Bernardo F., Stampone T., et al. . (2015). An update of the evolving epidemic of blaKPC carrying Klebsiella pneumoniae in Sicily, Italy, 2014: emergence of multiple Non-ST258 clones. PLoS ONE 10:e0132936. 10.1371/journal.pone.0132936 PubMed DOI PMC

Bowers J. R., Kitchel B., Driebe E. M., MacCannell D. R., Roe C., Lemmer D., et al. . (2015). Genomic analysis of the emergence and rapid global dissemination of the clonal group 258 Klebsiella pneumoniae pandemic. PLoS ONE 10:e0133727. 10.1371/journal.pone.0133727 PubMed DOI PMC

Brisse S., Passet V., Haugaard A. B., Babosan A., Kassis-Chikhani N., Struve C., et al. . (2013). wzi Gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J. Clin. Microbiol. 51, 4073–4078. 10.1128/JCM.01924-13 PubMed DOI PMC

Calisto F., Caneiras C., Serqueira S., Lito L., Melo-Cristino J., Duarte A. (2012). Klebsiella pneumoniae producing carbapenemase KPC-3 identifed in hospital wards. Rev. Port. Doenças Infecc. 8, 127–134.

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

Chavda K. D., Chen L., Jacobs M. R., Rojtman A. D., Bonomo R. A., Kreiswirth B. N. (2015). Complete sequence of a bla(KPC)-harboring cointegrate plasmid isolated from Escherichia coli. Antimicrob. Agents Chemother. 59, 2956–2959. 10.1128/AAC.00041-15 PubMed DOI PMC

Chen L., Chavda K. D., Melano R. G., Hong T., Rojtman A. D., Jacobs M. R., et al. . (2014a). Molecular survey of the dissemination of two blaKPC-harboring IncFIA plasmids in New Jersey and New York hospitals. Antimicrob. Agents Chemother. 58, 2289–2294. 10.1128/AAC.02749-13 PubMed DOI PMC

Chen L., Mathema B., Chavda K. D., DeLeo F. R., Bonomo R. A., Kreiswirth B. N. (2014b). Carbapenemase-producing Klebsiella pneumoniae: molecular and genetic decoding. Trends Microbiol. 22, 686–696. 10.1016/j.tim.2014.09.003 PubMed DOI PMC

Chmelnitsky I., Shklyar M., Leavitt A., Sadovsky E., Navon-Venezia S., Ben Dalak M., et al. . (2014). Mix and match of KPC-2 encoding plasmids in Enterobacteriaceae-comparative genomics. Diagn. Microbiol. Infect. Dis. 79, 255–260. 10.1016/j.diagmicrobio.2014.03.008 PubMed DOI

Curiao T., Morosini M. I., Ruiz-Garbajosa P., Robustillo A., Baquero F., Coque T. M., et al. (2010). Emergence of blaKPC−3-Tn4401a associated with a pKPN3/4-like plasmid within ST384 and ST388 Klebsiella pneumoniae clones in Spain. J. Antimicrob. Chemother. 65, 1608–1614. 10.1093/jac/dkq174 PubMed DOI

Cuzon G., Naas T., Nordmann P. (2011). Functional characterization of Tn4401, a Tn3-based transposon involved in blaKPC gene mobilization. Antimicrob. Agents Chemother. 55, 5370–5373. 10.1128/AAC.05202-11 PubMed DOI PMC

Deleo F. R., Chen L., Porcella S. F., Martens C. A., Kobayashi S. D., Porter A. R., et al. . (2014). Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae. Proc. Natl. Acad. Sci. U.S.A. 111, 4988–4993. 10.1073/pnas.1321364111 PubMed DOI PMC

Feldman N., Adler A., Molshatzki N., Navon-Venezia S., Khabra E., Cohen D., et al. . (2013). Gastrointestinal colonization by KPC-producing Klebsiella pneumoniae following hospital discharge: duration of carriage and risk factors for persistent carriage. Clin. Microbiol. Infect. 19, E190–E196. 10.1111/1469-0691.12099 PubMed DOI

Garbari L., Busetti M., Dolzani L., Petix V., Knezevich A., Bressan R., et al. . (2015). pKBuS13, a KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 833, carrying Tn4401b inserted into an Xer site-specific recombination locus. Antimicrob. Agents Chemother. 59, 5226–5231. 10.1128/AAC.04543-14 PubMed DOI PMC

García-Fernández A., Fortini D., Veldman K., Mevius D., Carattoli A. (2009). Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella. J. Antimicrob. Chemother. 63, 274–281. 10.1093/jac/dkn470 PubMed DOI

Holt K. E., Wertheim H., Zadoks R. N., Baker S., Whitehouse C. A., Dance D., et al. . (2015). Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc. Natl. Acad. Sci. U.S.A. 112, E3574–E3581. 10.1073/pnas.1501049112 PubMed DOI PMC

Machado P., Silva A., Lito L., Melo-Cristino J., Duarte A. (2010). Emergence of Klebsiella pneumoniae ST11-producing KPC-3 carbapenemase at a Lisbon hospital. [Abstract]. Clin. Microbiol. Infect. 16(Suppl. 2), S28 (O129).

Magiorakos A. P., Srinivasan A., Carey R. B., Carmeli Y., Falagas M. E., Giske C. G., et al. . (2012). Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18, 268–281. 10.1111/j.1469-0691.2011.03570.x PubMed DOI

Manageiro V., Ferreira E., Almeida J., Barbosa S., Simões C., Bonomo R. A., et al. . (2015). Predominance of KPC-3 in a survey for carbapenemase-producing Enterobacteriaceae in Portugal. Antimicrob. Agents Chemother. 59, 3588–3592. 10.1128/AAC.05065-14 PubMed DOI PMC

Munoz-Price L. S., Poirel L., Bonomo R. A., Schwaber M. J., Daikos G. L., Cormican M., et al. . (2013). Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet. Infect. Dis. 13, 785–796. 10.1016/S1473-3099(13)70190-7 PubMed DOI PMC

Naas T., Cuzon G., Truong H.-V., Nordmann P. (2012). Role of ISKpn7 and deletions in blaKPC gene expression. Antimicrob. Agents Chemother. 56, 4753–4759. 10.1128/AAC.00334-12 PubMed DOI PMC

Nordmann P., Cuzon G., Naas T. (2009). The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet. Infect. Dis. 9, 228–236. 10.1016/S1473-3099(09)70054-4 PubMed DOI

Nordmann P., Poirel L. (2014). The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clin. Microbiol. Infect. 20, 821–830. 10.1111/1469-0691.12719 PubMed DOI

Ocampo A. M., Chen L., Cienfuegos A. V., Roncancio G., Chavda K. D., Kreiswirth B. N., et al. . (2015). A two-year surveillance in five Colombian Tertiary Care Hospitals reveals high frequency of Non-CG258 clones of carbapenem-resistant Klebsiella pneumoniae with distinct clinical characteristics. Antimicrob. Agents Chemother. 60, 332–342. 10.1128/AAC.01775-15 PubMed DOI PMC

Pires J., Novais Â., Peixe L. (2013). Blue-carba, an easy biochemical test for detection of diverse carbapenemase producers directly from bacterial cultures. J. Clin. Microbiol. 51, 4281–4283. 10.1128/JCM.01634-13 PubMed DOI PMC

Poirel L., Barbosa-Vasconcelos A., Simões R. R., Da Costa P. M., Liu W., Nordmann P. (2012). Environmental KPC-producing Escherichia coli isolates in Portugal. Antimicrob. Agents Chemother. 56, 1662–1663. 10.1128/AAC.05850-11 PubMed DOI PMC

Rodrigues C., Machado E., Ramos H., Peixe L., Novais Â. (2014). Expansion of ESBL-producing Klebsiella pneumoniae in hospitalized patients: a successful story of international clones (ST15, ST147, ST336) and epidemic plasmids (IncR, IncFIIK). Int. J. Med. Microbiol. 304, 1100–1108. 10.1016/j.ijmm.2014.08.003 PubMed DOI

Rodrigues C., Sousa C., Machado E., Novais Â., Peixe L. (2015). Multidrug resistant (MDR) Klebsiella pneumoniae ST15 isolates: is their genomic plasticity the reason for its success?, in Abstract retrieved from Abstracts in 25th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) (Copenhagen: ), (P0991).

Villa L., García-Fernández A., Fortini D., Carattoli A. (2010). Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J. Antimicrob. Chemother. 65, 2518–2529. 10.1093/jac/dkq347 PubMed DOI

WHO (2014). Antimicrobial Resistance: Global Report on Surveillance. Geneva: WHO Press, World Health Organization.

Yigit H., Queenan A. M., Anderson G. J., Domenech-Sanchez A., Biddle J. W., Steward C. D., et al. . (2001). Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45, 1151–61. 10.1128/AAC.45.4.1151-1161.2001 PubMed DOI PMC

Zhou K., Lokate M., Deurenberg R. H., Tepper M., Arends J. P., Raangs E. G. C., et al. (2015). Use of whole-genome sequencing to trace, control and characterize the regional expansion of extended-spectrum β-lactamase producing ST15 Klebsiella pneumoniae. Sci Rep. 11:6:20840 10.1038/srep20840 PubMed DOI PMC