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Antimicrob Agents Chemother. 2016 January; 60(1): 646–649.
Published online 2015 December 31. Prepublished online 2015 November 2. doi:  10.1128/AAC.01886-15
PMCID: PMC4704230

Double Copies of blaKPC-3::Tn4401a on an IncX3 Plasmid in Klebsiella pneumoniae Successful Clone ST512 from Italy

Abstract

A carbapenem-resistant sequence type 512 (ST512) Klebsiella pneumoniae carbapenemase 3 (KPC-3)-producing K. pneumoniae strain showing a novel variant plasmid content was isolated in Palermo, Italy, in 2014. ST512 is a worldwide successful clone associated with the spread of blaKPC genes located on the IncFIIk pKpQIL plasmid. In our ST512 strain, the blaKPC-3 gene was unusually located on an IncX3 plasmid, whose complete sequence was determined. Two copies of blaKPC-3::Tn4401a caused by intramolecular transposition events were detected in the plasmid.

TEXT

Extensively drug-resistant (XDR) and pandrug-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains of the hyperepidemic clonal complex 258 (CC258) are detected worldwide as hospital-acquired pathogens and are frequently responsible for outbreaks. In particular, sequence type 258 (ST258), ST512, ST11, and ST340 are the most frequently detected variants of KPC-producing K. pneumoniae isolates (1, 2).

During May and June 2011, a countrywide Italian survey focusing on the diffusion of carbapenem-nonsusceptible K. pneumoniae isolates showed that the most frequent lineages belonged to CC258 (ST258 or ST512) (3). The epidemiology of KPC-producing K. pneumoniae in Palermo, Italy, also confirmed the emergence of ST258 beginning in 2008 (4). More recently, a 6-month surveillance performed in Sicily suggested that a major epidemiological change is likely ongoing in this geographic area, with ST258 still being prevalent but circulating along with several additional STs, including ST307 and ST273 (4). In particular, only one isolate of ST512 (i.e., K. pneumoniae strain 45) was identified on a total of 94 KPC-producing K. pneumoniae strains (4). This unique isolate of ST512 was further investigated and described in this study.

As shown in Table 1, the ST512 K. pneumoniae 45 strain showed an XDR phenotype (4). It was screened by PCR for the following plasmid-mediated quinolone resistance and β-lactamase genes: qnrA, qnrB, qnrC, qnrD, qnrS, aac(6′)-Ib-cr, qepA, oqxAB, blaKPC, blaVIM, blaNDM, blaOXA-48, blaOXA, blaSHV, blaTEM, blaLAP, blaCTX-M, and blaCMY (5,8). Positive amplicons underwent Sanger DNA sequencing to identify the variant genes. K. pneumoniae 45 was positive for the blaKPC-3, blaOXA-1, blaCTX-M-15, blaTEM-1, aac(6′)-Ib-cr, blaSHV-11, and oqxAB genes. The implementation of the PCR-based replicon typing (PBRT) kit (Diatheva) indicated that plasmids carried by strain 45 were not typeable. Therefore, the blaKPC-3-carrying plasmid was transformed in Escherichia coli DH5α competent cells (Invitrogen) and selected on Luria-Bertani agar plates (Sigma) containing ampicillin (50 μg/ml). The transformants were then screened by PCR for the presence of blaKPC genes. Plasmid DNA of one representative blaKPC-3-positive transformant (named p45) was purified using an Invitrogen plasmid midi kit (Invitrogen) and fully sequenced. A shotgun library was obtained, and sequencing was performed with the 454-GS Junior platform, according to the standard sequencing procedure (Roche Diagnostics). Plasmid coverage was >80×. The reads were aligned and assembled using the Newbler assembler software version 2.0.01.14 (Roche Diagnostics). We found that plasmid p45 was split into three contigs, and the complete sequence was reconstructed by a PCR-based gap closure method. Open reading frames (ORFs) were predicted and annotated using the Artemis software (Wellcome Trust Sanger Institute, United Kingdom). Each predicted protein was compared against the protein database using BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The gene sequences were compared and aligned with GenBank data using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The IncX3 plasmids pKpS90 and pIncX-SHV (GenBank accession no. JX461340 and JN247852, respectively) were used as references for annotation and comparative analyses.

TABLE 1
Antimicrobial susceptibility profiles of the original KPC-producing K. pneumoniae isolates and transformantsa

As shown in Fig. 1, our results indicated that p45 is 63,203 bp in size and shows the typical IncX3 scaffold, including the replicase gene, tax, and pilX gene clusters (9). The blaKPC-3 gene is located in the Tn3-like element Tn4401a (10). However, we observed that two copies of Tn4401a were present at a distance of 8,658 bp within the IncX3 plasmid scaffold of p45.

FIG 1
Major structural features of plasmids p45, pKpS90, and pIncX-SHV. Predicted open reading frames (ORFs) on plasmids are represented by white arrows. The ORFs of p45 were identified in this study; the ORFs of pKpS90 and pIncX-SHV were deduced from GenBank ...

One copy of Tn4401a was integrated within an IS3000-a1 element, which was interrupted by the blaKPC-3::Tn4401a insertion. The duplication of a 5-bp sequence was identified at the site of integration, immediately flanking the inverted repeat right (IRR) and inverted repeat left (IRL) of Tn4401a. The blaSHV-11 gene was identified 4 kb from the blaKPC-3::Tn4401a integration site, followed by an IS26 element. The same structure carrying IS26-blaSHV-11-IS3000 was previously detected in plasmid pIncX-SHV (11; GenBank accession no. JN247852). In a similar plasmid (i.e., pKpS90), blaKPC-2::Tn4401a was integrated in the same region as p45 but within a different site, causing the interruption of the ygbK gene (12). The progenitor of p45 was therefore a pIncX-SHV-like plasmid carrying the blaSHV-11 gene (11).

The second copy of Tn4401a in p45 was integrated in topB (a gene constantly present in all IncX3 plasmids) and in opposite orientation compared to that of the first transposon (Fig. 1). The target site duplication was also identified in this site, adjacent to the inverted repeats of the transposon. It is plausible that the acquisition of the second Tn4401a in p45 occurred by intramolecular transposition. In the literature, the simultaneous presence of two blaKPC copies located in trans on different plasmids in the same bacterial host was previously reported in several collections (13,16). However, only two examples of in cis blaKPC genes were previously described and differed from the arrangement described for p45. Two blaKPC-3::Tn4401a copies were identified in MNCRE44, an extraintestinal pathogenic E. coli strain belonging to the ST131 H30R subclade and found in the United States (13). Its plasmid (pMNCRE44_5) was a 116-kb hybrid of IncX3 and IncFIA(HI1) plasmids, and the two transposon copies were located as one copy for each of the two fused plasmid portions (13). The duplication of the similar transposon blaKPC-2::Tn4401b was described for the IncN plasmid S9 from K. pneumoniae in the United States (17).

The MICs for several antibiotics for KPC-producing K. pneumoniae strains of ST512 and ST258 carrying blaKPC-3 on pKpQIL (18), those of KPC-producing K. pneumoniae 45 (ST512 and with IncX3) and those of their corresponding E. coli DH5α transformants, were determined implementing the microdilution ESB1F and GNX2F plates (Trek Diagnostics) (Table 1). As result, we noted that the E. coli DH5α transformant carrying the IncX3 plasmid with two copies of blaKPC-3::Tn4401a showed significantly increased MICs for carbapenems, cephalosporins, and β-lactam–β-lactamase inhibitor combinations compared to those of the transformant carrying the classical pKpQIL plasmid. This phenomenon was not observed for the original K. pneumoniae strains of different STs and possessing different blaKPC genetic backgrounds. The difference in MICs may be due not only to the double copy of the blaKPC genes and their levels of gene expression (14) but also to different copy numbers of IncX3 and pKpQIL.

In conclusion, our study describes the change in the typical plasmid content of ST512 K. pneumoniae: the worldwide-described pKpQIL plasmid carrying blaKPC (19; GenBank accession no. GU595196) was replaced by an IncX3 plasmid carrying two copies of blaKPC-3::Tn4401a. The change in plasmid type in K. pneumoniae strain 45 might represent an important evolution of the ST512 lineage.

Nucleotide sequence accession number. The GenBank file for K. pneumoniae strain ST512 plasmid p45-IncX3 was compiled using Sequin (http://www.ncbi.nlm.nih.gov/Sequin/) and deposited at the NCBI GenBank under accession no. KT362706.

Funding Statement

The Italian Flagship “InterOmics” project (PB.P05) funded by MIUR and coordinated by the Italian Council of National Research (CNR) (A.C.) provided funding.

REFERENCES

1. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL 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. doi:.10.1111/j.1469-0691.2011.03570.x [PubMed] [Cross Ref]
2. Pitout JD, Nordmann P, Poirel L 2015. Carbapenemase-producing Klebsiella pneumoniae: a key pathogen set for global nosocomial dominance. Antimicrob Agents Chemother 59:5873–5884. doi:.10.1128/AAC.01019-15 [PMC free article] [PubMed] [Cross Ref]
3. Giani T, Pini B, Arena F, Conte V, Bracco S, Migliavacca R, AMCLI-CRE Survey Participants, Pantosti A, Pagani L, Luzzaro F, Rossolini GM. 2013. Epidemic diffusion of KPC carbapenemase-producing Klebsiella pneumoniae in Italy: results of the first countrywide survey, 15 May to 30 June 2011. Euro Surveill 18:pii=20489 http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20489. [PubMed]
4. Bonura C, Giuffrè M, Aleo A, Fasciana T, Di Bernardo F, Stampone T, Giammanco A, MDR-GN Working Group, Palma DM, Mammina C 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. doi:.10.1371/journal.pone.0132936 [PMC free article] [PubMed] [Cross Ref]
5. Poirel L, Walsh TR, Cuvillier V, Nordmann P 2011. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 70:119–123. doi:.10.1016/j.diagmicrobio.2010.12.002 [PubMed] [Cross Ref]
6. Pérez-Pérez FJ, Hanson ND 2002. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153–2162. doi:.10.1128/JCM.40.6.2153-2162.2002 [PMC free article] [PubMed] [Cross Ref]
7. 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. doi:.10.1093/jac/dkn470 [PubMed] [Cross Ref]
8. Mabilat C, Goussard S 1995. PCR detection and identification of genes for extended-spectrum beta-lactamases, p 553–559. In Persing DH, Smith TF, Tenover FC, White TJ (ed), Diagnostic molecular microbiology: principles and applications. American Society for Microbiology, Washington, DC.
9. Johnson TJ, Bielak EM, Fortini D, Hansen LH, Hasman H, Debroy C, Nolan LK, Carattoli A 2012. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae. Plasmid 68:43–50. doi:.10.1016/j.plasmid.2012.03.001 [PubMed] [Cross Ref]
10. Naas T, Cuzon G, Villegas MV, Lartigue MF, Quinn JP, Nordmann P 2008. Genetic structures at the origin of acquisition of the beta-lactamase blaKPC gene. Antimicrob Agents Chemother 52:1257–1263. doi:.10.1128/AAC.01451-07 [PMC free article] [PubMed] [Cross Ref]
11. García-Fernández A, Villa L, Carta C, Venditti C, Giordano A, Venditti M, Mancini C, Carattoli A 2012. Klebsiella pneumoniae ST258 producing KPC-3 identified in Italy carries novel plasmids and OmpK36/OmpK35 porin variants. Antimicrob Agents Chemother 56:2143–2145. doi:.10.1128/AAC.05308-11 [PMC free article] [PubMed] [Cross Ref]
12. Kassis-Chikhani N, Frangeul L, Drieux L, Sengelin C, Jarlier V, Brisse S, Arlet G, Decré D 2013. Complete nucleotide sequence of the first KPC-2- and SHV-12-encoding IncX plasmid, pKpS90, from Klebsiella pneumoniae. Antimicrob Agents Chemother 57:618–620. doi:.10.1128/AAC.01712-12 [PMC free article] [PubMed] [Cross Ref]
13. Johnson TJ, Hargreaves M, Shaw K, Snippes P, Lynfield R, Aziz M, Price LB 2015. Complete genome sequence of a carbapenem-resistant extraintestinal pathogenic Escherichia coli strain belonging to the sequence type 131 H30R subclade. Genome Announc 3(2):e00272-15. doi:.10.1128/genomeA.00272-15 [PMC free article] [PubMed] [Cross Ref]
14. Kitchel B, Rasheed JK, Endimiani A, Hujer AM, Anderson KF, Bonomo R, Patel B 2010. Genetic factors associated with elevated carbapenem resistance in KPC-producing Klebsiella pneumoniae. Antimicrob Agents Chemother 54:4201–4207. doi:.10.1128/AAC.00008-10 [PMC free article] [PubMed] [Cross Ref]
15. Baraniak A, Grabowska A, Izdebski R, Fiett J, Herda M, Bojarska K, Zabicka D, Kania-Pudlo M, Mlynarczyk G, Žak-Pulawska Z, Hryniewicz W, Gniadkowski M, KPC-PL Study Group 2011. Molecular characteristics of KPC-producing Enterobacteriaceae at the early stage of their dissemination in Poland, 2008–2009. Antimicrob Agents Chemother 55:5493–5499. doi:.10.1128/AAC.05118-11 [PMC free article] [PubMed] [Cross Ref]
16. Cuzon GC, Naas T, Truong H, Villegas MV, Wisell K, Carmeli Y, Gales AC, Navon-Venezia S, Quinn JP, Nordmann P 2010. Worldwide diversity of Klebsiella pneumoniae that produces β-lactamase blaKPC-2 gene. Emerg Infect Dis 16:1349–1356. doi:.10.3201/eid1609.091389 [PMC free article] [PubMed] [Cross Ref]
17. Gootz TD, Lescoe MK, Dib-Hajj F, Dougherty BA, He W, Della-Latta P, Huard R 2009. Genetic organization of transposase regions surrounding blaKPC carbapenemase genes on plasmids from Klebsiella strains isolated in a New York City hospital. Antimicrob Agents Chemother 53:1998–2004. doi:.10.1128/AAC.01355-08 [PMC free article] [PubMed] [Cross Ref]
18. Capone A, Giannella M, Fortini D, Giordano A, Meledandri M, Ballardini M, Venditti M, Bordi E, Capozzi D, Balice MP, Tarasi A, Parisi G, Lappa A, Carattoli A, Petrosillo N, SEERBIO-GRAB Network 2013. High rate of colistin resistance among patients with carbapenem-resistant Klebsiella pneumoniae infection accounts for an excess of mortality. Clin Microbiol Infect 19:E23–E30. doi:.10.1111/1469-0691.12070 [PubMed] [Cross Ref]
19. Leavitt A, Chmelnitsky I, Carmeli Y, Navon-Venezia S 2010. Complete nucleotide sequence of KPC-3-encoding plasmid pKpQIL in the epidemic Klebsiella pneumoniae sequence type 258. Antimicrob Agents Chemother 54:4493–4496. doi:.10.1128/AAC.00175-10 [PMC free article] [PubMed] [Cross Ref]

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