. The gene for the broad-spectrum and carbapenem-active metallo-b-lactamase NDM-1 is rapidly spreading. We present the complete genome of Klebsiella pneumoniae ATCC BAA-2146, the first U.S. isolate found to encode NDM-1, and describe its repertoire of antibiotic-resistance genes and mutations, including genes for eight b-lactamases and 15 additional antibiotic-resistance enzymes. To elucidate the evolution of this rich repertoire, the mobile elements of the genome were characterized, including four plasmids with varying degrees of conservation and mosaicism and eleven chromosomal genomic islands. One island was identified by a novel phylogenomic approach, that further indicated the cps-lps polysaccharide synthesis locus, where operon translocation and fusion was noted. Unique plasmid segments and mosaic junctions were identified. Plasmid-borne blaCTX-M-15 was transposed recently to the chromosome by ISEcp1. None of the eleven full copies of IS26, the most frequent IS element in the genome, had the expected 8-bp direct repeat of the integration target sequence, suggesting that each copy underwent homologous recombination subsequent to its last transposition event. Comparative analysis likewise indicates IS26 as a frequent recombinational junction between plasmid ancestors, and also indicates a resolvase site. In one novel use of high-throughput sequencing, homologously recombinant subpopulations of the bacterial culture were detected. In a second novel use, circular transposition intermediates were detected for the novel insertion sequence ISKpn21 of the ISNCY family, suggesting that it uses the two-step transposition mechanism of IS3. Robust genome-based phylogeny showed that a unified Klebsiella cluster contains Enterobacter aerogenes and Raoultella, suggesting the latter genus should be abandoned.
Carbapenems are one of few antimicrobials that have been effective against multidrug-resistant bacteria, but their utility is threatened by the emergence of carbapenem-resistant Enterobacte-riaceae (CRE). Klebsiella pneumoniae is the most common CRE species in the United States, typically encountered as a hospital-acquired infection with high morbidity and mortality, and resistant to nearly all available antibiotics [1–4]. Enzymes that inactivate carbape-nems are a major mechanism of resistance. The serine b-lactamase KPC, known since 2001, has become the most common carbapenemase in the U.S. and other countries . A more recent concern is the carbapenem-active metallo-b-lactamase NDM-1, first identified in a K. pneumoniae isolate from 2008 . Alarmingly, blaNDM-1 is often found on large conjugative plasmids along with additional antibiotic resistance determinants . In some settings the gene region can form tandem repeats, elevating copy number . The recent spread of blaNDM-1 both among different species and across a large geographic area has been remarkable and well documented
K. pneumoniae strain ATCC BAA-2146 (Kpn2146) was the first U.S isolate found to encode NDM-1 together with a wide variety of additional antibiotic resistance determinants . Susceptibility testing performed at ATCC found Kpn2146 to be resistant to every one of the 34 antimicrobial and antimicrobial/inhibitor combinations tested. While Kpn2146 resistance genes have been analyzed by both microarray  and (incomplete) genome sequencing [16,17], neither approach fully elucidated the complex Kpn2146 antibiotic resistance gene repertoire. For example some Kpn2146 antibiotic resistance genes were unrecognized in the previous work, and duplicated genes were counted only once by microarray and on one contig in the incomplete genome. Even when an incomplete genome does deliver the complete gene list, the question of how a pathogen accumulates such large collections of resistance genes requires the contextual information that comes from completing the genome. The complete genome is required to reveal gene duplication events, to determine plasmid vs. chromo-somal gene location, and to apply phylogenomic methods to understand the evolution of the genome. In this study, we present the completed Kpn2146 genome, identifying four plasmids, and enabling a detailed survey of its antibiotic-resistance determinants that fully explains its resistance profile. These determinants include 23 primarily plasmid-borne genes encoding antibiotic-resistance enzymes, eight of which are b-lactamase genes. It is crucial to understand how such richly endowed pathogens arise, which requires analysis of the mobile fraction of the genome. Accord-ingly, we surveyed genomic islands in the chromosome, mosaicism in the plasmids, and transposable elements throughout the genome.
DNA preparation and sequencing Klebsiella pneumoniae ATCC BAA-2146 (Kpn2146) was isolated in 2010 from the urine of a U.S. hospital patient who had recently received medical care in India . Genomic DNA was obtained from American Type Culture Collection (ATCC), and re-suspended in water. A previously described Illumina paired-end genomic sequence dataset from a single MiSeq run, after quality and primer sequence trimming, consisted of 3,023,757 read pairs, with reads averaging 88.3 bp . A Pacific Biosciences sequence dataset (PacBio) was generated from 2 mg genomic DNA at the Yale Genome Sequencing Center, which performed the 5 kb template preparation and sequenced the library on two SMRT cells, yielding 88,073 direct reads and 1744 circular consensus sequences (size distribution: mean 2408; median 1948; range 50-18951; N50 3254 bp).