Complete Nucleotide Sequences of Two bla _KPC-2 -Bearing IncN Plasmids Isolated from Sequence Type 442 Klebsiella pneumoniae Clinical Strains Four Years Apart

Abstract

We sequenced the oldest bla KPC-2 -bearing plasmid isolated in Brazil and another plasmid also carried by a Klebsiella pneumoniae strain of sequence type 442 (ST442) , Even though the first description of KPC-producing Klebsiella pneumoniae in Brazil was published in 2009, describing a strain isolated from a clinical sample in 2006 (1), a later publication reported the presence of a KPC-bearing strain isolated in May 2005 (2). The latter strain, FCF1305, was isolated in the state of São Paulo, Brazil, from a blood culture and belongs to sequence type 442 (ST442). We completely sequenced the bla KPC-2 -bearing plasmid isolated from FCF1305 and also another bla KPC-2 -bearing plasmid isolated from another K. pneumoniae strain, FCF3SP, also belonging to ST442 and also isolated from a blood culture from a patient in the state of São Paulo, but 52 months later, in September 2009. We chose to sequence the bla KPC-2 -bearing plasmids of these strains because FCF1305 was the oldest strain-bearing KPC in Brazil and strain FCF3SP was the strain most geographically, clinically, and epidemiologically similar to FCF1305 that we could find, but isolated 52 months later, so that differences accumulated in the wild during this period could be evaluated.Because strain FCF1305 bore three plasmids, whereas strain FCF3SP bore five, isolation of the bla KPC-2 -bearing plasmids of each strain was carried out by transformation into Escherichia coli TOP10 (Invitrogen) as described previously (3). Plasmid DNA isolated from E. coli TOP10(pFCF1305) and E. coli TOP10 (pFCF3SP) by alkaline lysis miniprep (3) was used to construct genomic fragment libraries (one for each plasmid) which were sequenced using the Roche GS-FLX sequencer (Roche Life Sciences, Branford, CT, USA) according to the manufacturer's instructions.Sequencing adapters were clipped from the reads and used for de novo assembly using Mira 4.0 (4). Plasmid de novo contigs were then merged by assembling de novo using Geneious (5) with stringent parameters (maximum gap size ϭ 1; minimum overlap identity ϭ 95%). Circular (with large overlapping ends) contigs of ϳ50 kbp were considered to be the plasmids. All other contigs generated which had no identity to the circular contigs were then subjected to a BLAST search (6) against GenBank's nonredundant (nr) database, and 100% of them had as top hits Enterobacteriaceae chromosomes or phages and so were discarded. The plasmid sequences were automatically annotated using the RAST server (7) and manually curated. Repeat regions were identified using UGENE (8) and insertion sequences were identified by online analysis using IS finder (9). The complete sequences of both plasmids were successfully obtained by de novo assembly yielding a 53,081-bp sequence for pFCF1305 (mean coverage of 261ϫ Ϯ 48ϫ; minimum coverage of 39ϫ; maximum coverage of 445ϫ) and a 54,605-bp sequence for pFCF3SP (mean coverage of 1,013ϫ Ϯ 374ϫ; minimum coverage of 67ϫ; maximum coverage of 2,383ϫ).Both plasmids were found to have IncN plasmid backbones and carry a versi...

Complete sequences of KPC-2-encoding plasmid p628-KPC and CTX-M-55-encoding p628-CTXM coexisted in Klebsiella pneumoniae

“…At least eight isoforms of Tn 4401 have been named, i.e., Tn 4401a to Tn 4401g and a separate Tn 4401d (Table S1 in Supplementary Material). Several unnamed Tn 4401 isoforms have been also reported recently (Cuzon et al, 2011 ; Li et al, 2011 ; Ho et al, 2013b ; Naas et al, 2013 ; Perez-Chaparro et al, 2014 ). Tn 4401b is considered as the prototype one, and the other isoforms result from occurrence of distinct deletion or insertion events at different sites.…”

Coproduction of NDM-1 and KPC-2 in Enterobacter hormaechei from Brazil

“…3 The 47,755 bp contig carrying bla KPC-2 shared 100% identity with the 54.6 kb plasmid pKPC_FCF3/SP belonging to IncN recovered in Brazil from K. pneumoniae isolated in September, 2009, from a blood culture (GenBank accession number CP004367.2). 11 Using the sequence of pKPC_FCF3/SP as a reference to map the reads of our isolate with Geneious software R6.1 (Biomatters), we succeeded to recover the whole plasmid sequence. In this plasmid, bla KPC-2 was found associated to the transposon Tn4401 isoform b.…”

Comprehensive Genome Analysis of Carbapenemase-Producing Enterobacter spp.: New Insights into Phylogeny, Population Structure, and Resistance Mechanisms

“…In addition, bla KPC in 15 isolates was located on Tn 4401b on the pKPC_UVA01-like plasmids with identical Tn 4401b isoforms and TSDs. The bla KPC genes in the remaining 16 isolates were associated with IncN-like (e.g., pKPC_FCF/3SP-like) (50) ( n = 8), pKpQIL-like ( n = 2), IncL/M-like ( n = 1), IncA/C (p34998-239kb, n = 1), and pBK28610-like ( n = 1) plasmids and a novel IncX (X7) plasmid ( n = 3) (Fig. 6).…”