Whole-Genome Assembly of Klebsiella pneumoniae Coproducing NDM-1 and OXA-232 Carbapenemases Using Single-Molecule, Real-Time Sequencing

Abstract

The whole-genome sequence of a carbapenem-resistant Klebsiella pneumoniae strain, PittNDM01, which coproduces NDM-1 and OXA-232 carbapenemases, was determined in this study. The use of single-molecule, real-time (SMRT) sequencing provided a closed genome in a single sequencing run. K. pneumoniae PittNDM01 has a single chromosome of 5,348,284 bp and four plasmids: pPKPN1 (283,371 bp), pPKPN2 (103,694 bp), pPKPN3 (70,814 bp), and pPKPN4 (6,141 bp). The contents of the chromosome were similar to that of the K. pneumoniae reference genome strain MGH 78578, with the exception of a large inversion spanning 23.3% of the chromosome. In contrast, three of the four plasmids are unique. The plasmid pPKPN1, an IncHI1B-like plasmid, carries the bla NDM-1 , armA, and qnrB1 genes, along with tellurium and mercury resistance operons. bla NDM-1 is carried on a unique structure in which Tn125 is further bracketed by IS26 downstream of a class 1 integron. The IncFIA-like plasmid pPKPN3 also carries an array of resistance elements, including bla CTX-M-15 and a mercury resistance operon. The ColE-type plasmid pPKPN4 carrying bla OXA-232 is identical to a plasmid previously reported from France. SMRT sequencing was useful in resolving the complex bacterial genomic structures in the de novo assemblies.

Genomic Characteristics of NDM-Producing Enterobacteriaceae Isolates in Australia and Their bla NDM Genetic Contexts

“…The aminoglycoside resistance determinant rmtC was also identified upstream of the ⌬Tn125 structure of NGC type FII-C and is very similar to IncFII pKOX_NDM1 (GenBank accession no. JQ314407) of Klebsiella oxytoca isolated in Taiwan (26 (42).…”

Aminoglycoside Resistance

“…22 Some of these plasmids additionally carry plasmid-mediated fluoroquinolone-resistance genes, such as qnrB1 . 23 Acquisition of these MDR plasmids would therefore simultaneously confer resistance to most β-lactams including carbapenems, aminoglycosides, and fluoroquinolones, the three key groups of agents with activity against gram-negative bacteria. armA has also been found in P aeruginosa carrying another metallo-β-lactamase gene bla IMP-1 in Korea, 24 and K pneumoniae or other Enterobacteriaceae carrying a Klebsiella pneumoniae carbapenemase (KPC)-type carbapenemase gene bla KPC-2 in Italy and China.…”

Microbial taxonomy in the post-genomic era: Rebuilding from scratch?

“…Third-generation sequencing technologies, such as Pacific Biosciences and Oxford Nanopore, have the advantage of long reads (10,000 bp or longer), although currently their throughput and base calling accuracy is lower than the second-generation machines. However, many bacterial genomes have been sequenced and assembled with a single run on Pacific Biosciences machines (Doi et al 2014;Forde et al 2014;Shiwa et al 2014).…”