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Resistance determinants and mobile genetic elements of an NDM-1-encoding Klebsiella pneumoniae strain.

Hudson CM, Bent ZW, Meagher RJ, Williams KP - PLoS ONE (2014)

Bottom Line: 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 β-lactamases and 15 additional antibiotic-resistance enzymes.In one novel use of high-throughput sequencing, homologously recombinant subpopulations of the bacterial culture were detected.Robust genome-based phylogeny showed that a unified Klebsiella cluster contains Enterobacter aerogenes and Raoultella, suggesting the latter genus should be abandoned.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Sandia National Laboratories, Livermore, California, United States of America.

ABSTRACT
Multidrug-resistant Enterobacteriaceae are emerging as a serious infectious disease challenge. These strains can accumulate many antibiotic resistance genes though horizontal transfer of genetic elements, those for β-lactamases being of particular concern. Some β-lactamases are active on a broad spectrum of β-lactams including the last-resort carbapenems. The gene for the broad-spectrum and carbapenem-active metallo-β-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 β-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.

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Operon translocation and fusion at the cps-lps polysaccharide synthesis locus.The cps P1, P2 and P3 promoters are taken from [68], while a promoter (Plps) has been mapped in K. pneumoniae MGH 78578 to the intergenic space between uge and the first lps gene [69]. A) The cps-lps region of K. pneumoniae 342, which is typical of Klebsiella. Genes of cps are in yellow (common in most strains) or blue (varying in gene identity, count, and order); genes of lps are in red. The manCB unit (orange arrows) is occasionally found in cps, and occasionally in lps, and here unusually in both. The diamond represents the JUMPstart DNA/RNA motif at whose ops sequence RfaH is loaded onto the elongating RNA polymerase in place of NusG, preventing Rho-based termination for the small number of long transcription units that are controlled by ops-RfaH, and physically coupling the elongating RNA polymerase to the trailing ribosome [70]. B) Kpn2146 cps-lps. The boxed cps P3 unit has been deleted from its usual site, and moreover translocated to a nearby position, apparently by transposition and/or homologous recombination mechanisms; note the complex pattern of surrounding IS insertions and the directly repeated flanking sequence copies (gray arrows).ΔIS, incomplete IS copy; dotted lines, gene or IS interrupted by ISs; GT, glucosyl transferase, Hyp, hypothetical.
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pone-0099209-g006: Operon translocation and fusion at the cps-lps polysaccharide synthesis locus.The cps P1, P2 and P3 promoters are taken from [68], while a promoter (Plps) has been mapped in K. pneumoniae MGH 78578 to the intergenic space between uge and the first lps gene [69]. A) The cps-lps region of K. pneumoniae 342, which is typical of Klebsiella. Genes of cps are in yellow (common in most strains) or blue (varying in gene identity, count, and order); genes of lps are in red. The manCB unit (orange arrows) is occasionally found in cps, and occasionally in lps, and here unusually in both. The diamond represents the JUMPstart DNA/RNA motif at whose ops sequence RfaH is loaded onto the elongating RNA polymerase in place of NusG, preventing Rho-based termination for the small number of long transcription units that are controlled by ops-RfaH, and physically coupling the elongating RNA polymerase to the trailing ribosome [70]. B) Kpn2146 cps-lps. The boxed cps P3 unit has been deleted from its usual site, and moreover translocated to a nearby position, apparently by transposition and/or homologous recombination mechanisms; note the complex pattern of surrounding IS insertions and the directly repeated flanking sequence copies (gray arrows).ΔIS, incomplete IS copy; dotted lines, gene or IS interrupted by ISs; GT, glucosyl transferase, Hyp, hypothetical.

Mentions: Learned phyloblocks indicated, in addition to a new island, the genomic locus of capsular polysaccharide (cps) and lipopolysaccharide (lps) synthesis genes (Fig. 6). This region is not an integrase-mobilized genomic island, yet the cps cluster is known to be so highly varied as to suggest horizontal transfer of genes within the array [62]. The capsule is the outermost cell surface, a key Klebsiella pathogenicity determinant subject to immune surveillance. In other Enterobacteriaceae, the large cps and lps gene clusters are typically separate, but in Klebsiella, lps is found immediately downstream of cps. Nevertheless there normally appears to be transcriptional separation between Klebsiella cps and lps; cps terminates with the reverse-oriented gene uge, and an lps promoter has been found in the large intergenic space between uge and lps (Fig. 6A) [62]. The Kpn2146 cps-lps region has undergone a major rearrangement with gene-regulatory consequences (Fig. 6B). The terminal cps P3 transcription unit is deleted from its usual site, fusing the lps operon to the main cps operon. Morever this cps P3 unit has translocated to a nearby location, within a complex array of insertion sequences. In this new location the P3 unit is transcriptionally isolated, whereas at the usual location transcription could be supplemented by the upstream P2. Deletion of a polysaccharide synthesis gene cluster by homologous recombination between repeated manCB units has been noted before [63], but in our case the translocation has preserved the deleted cps subcluster.


Resistance determinants and mobile genetic elements of an NDM-1-encoding Klebsiella pneumoniae strain.

Hudson CM, Bent ZW, Meagher RJ, Williams KP - PLoS ONE (2014)

Operon translocation and fusion at the cps-lps polysaccharide synthesis locus.The cps P1, P2 and P3 promoters are taken from [68], while a promoter (Plps) has been mapped in K. pneumoniae MGH 78578 to the intergenic space between uge and the first lps gene [69]. A) The cps-lps region of K. pneumoniae 342, which is typical of Klebsiella. Genes of cps are in yellow (common in most strains) or blue (varying in gene identity, count, and order); genes of lps are in red. The manCB unit (orange arrows) is occasionally found in cps, and occasionally in lps, and here unusually in both. The diamond represents the JUMPstart DNA/RNA motif at whose ops sequence RfaH is loaded onto the elongating RNA polymerase in place of NusG, preventing Rho-based termination for the small number of long transcription units that are controlled by ops-RfaH, and physically coupling the elongating RNA polymerase to the trailing ribosome [70]. B) Kpn2146 cps-lps. The boxed cps P3 unit has been deleted from its usual site, and moreover translocated to a nearby position, apparently by transposition and/or homologous recombination mechanisms; note the complex pattern of surrounding IS insertions and the directly repeated flanking sequence copies (gray arrows).ΔIS, incomplete IS copy; dotted lines, gene or IS interrupted by ISs; GT, glucosyl transferase, Hyp, hypothetical.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048246&req=5

pone-0099209-g006: Operon translocation and fusion at the cps-lps polysaccharide synthesis locus.The cps P1, P2 and P3 promoters are taken from [68], while a promoter (Plps) has been mapped in K. pneumoniae MGH 78578 to the intergenic space between uge and the first lps gene [69]. A) The cps-lps region of K. pneumoniae 342, which is typical of Klebsiella. Genes of cps are in yellow (common in most strains) or blue (varying in gene identity, count, and order); genes of lps are in red. The manCB unit (orange arrows) is occasionally found in cps, and occasionally in lps, and here unusually in both. The diamond represents the JUMPstart DNA/RNA motif at whose ops sequence RfaH is loaded onto the elongating RNA polymerase in place of NusG, preventing Rho-based termination for the small number of long transcription units that are controlled by ops-RfaH, and physically coupling the elongating RNA polymerase to the trailing ribosome [70]. B) Kpn2146 cps-lps. The boxed cps P3 unit has been deleted from its usual site, and moreover translocated to a nearby position, apparently by transposition and/or homologous recombination mechanisms; note the complex pattern of surrounding IS insertions and the directly repeated flanking sequence copies (gray arrows).ΔIS, incomplete IS copy; dotted lines, gene or IS interrupted by ISs; GT, glucosyl transferase, Hyp, hypothetical.
Mentions: Learned phyloblocks indicated, in addition to a new island, the genomic locus of capsular polysaccharide (cps) and lipopolysaccharide (lps) synthesis genes (Fig. 6). This region is not an integrase-mobilized genomic island, yet the cps cluster is known to be so highly varied as to suggest horizontal transfer of genes within the array [62]. The capsule is the outermost cell surface, a key Klebsiella pathogenicity determinant subject to immune surveillance. In other Enterobacteriaceae, the large cps and lps gene clusters are typically separate, but in Klebsiella, lps is found immediately downstream of cps. Nevertheless there normally appears to be transcriptional separation between Klebsiella cps and lps; cps terminates with the reverse-oriented gene uge, and an lps promoter has been found in the large intergenic space between uge and lps (Fig. 6A) [62]. The Kpn2146 cps-lps region has undergone a major rearrangement with gene-regulatory consequences (Fig. 6B). The terminal cps P3 transcription unit is deleted from its usual site, fusing the lps operon to the main cps operon. Morever this cps P3 unit has translocated to a nearby location, within a complex array of insertion sequences. In this new location the P3 unit is transcriptionally isolated, whereas at the usual location transcription could be supplemented by the upstream P2. Deletion of a polysaccharide synthesis gene cluster by homologous recombination between repeated manCB units has been noted before [63], but in our case the translocation has preserved the deleted cps subcluster.

Bottom Line: 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 β-lactamases and 15 additional antibiotic-resistance enzymes.In one novel use of high-throughput sequencing, homologously recombinant subpopulations of the bacterial culture were detected.Robust genome-based phylogeny showed that a unified Klebsiella cluster contains Enterobacter aerogenes and Raoultella, suggesting the latter genus should be abandoned.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Sandia National Laboratories, Livermore, California, United States of America.

ABSTRACT
Multidrug-resistant Enterobacteriaceae are emerging as a serious infectious disease challenge. These strains can accumulate many antibiotic resistance genes though horizontal transfer of genetic elements, those for β-lactamases being of particular concern. Some β-lactamases are active on a broad spectrum of β-lactams including the last-resort carbapenems. The gene for the broad-spectrum and carbapenem-active metallo-β-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 β-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.

Show MeSH
Related in: MedlinePlus