PROKARYOTES
crossm Complete Genome Sequence of a Multidrug-Resistant, blaNDM-1-Expressing Klebsiella pneumoniae K66-45 Clinical Isolate from Norway Adam Heikal,a,b Ørjan Samuelsen,c,d Tom Kristensen,e
Ole Andreas Økstada,b
Centre for Integrative Microbial Evolution (CIME), Faculty of Mathematics and Natural Sciences, University of Oslo, Blindern, Oslo, Norwaya; Laboratory for Microbial Dynamics (LaMDa), Section for Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Blindern, Oslo, Norwayb; Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norwayc; Department of Pharmacy, Faculty of Health Sciences, UiT–The Arctic University of Norway, Tromsø, Norwayd; Department of Biosciences, University of Oslo, Blindern, Oslo, Norwaye
ABSTRACT Multidrug-resistant Klebsiella pneumoniae is a major cause of hospitalacquired infections. Here, we report the complete genome sequence of the multidrugresistant, blaNDM-1-positive strain K. pneumoniae K66-45, isolated from a hospitalized Norwegian patient.
C
arbapenem-resistant Enterobacteriaceae (CRE), such as Klebsiella pneumoniae, are classified as critical on the World Health Organization’s list of priority pathogens (1). Resistance to carbapenems, the traditional last-resort treatment option for severe community- and hospital-acquired infections, has been reported at proportions up to 54% (2). While CRE remain rare in Scandinavia, their arrival and subsequent local expansion from high-prevalence locations, such as through the return of patients hospitalized overseas, is a concern (3, 4). In 2010, the carbapenem-resistant strain Klebsiella pneumoniae K66-45, belonging to sequence type 11 (part of CG258), was isolated and cultured from a catheter urine sample following the return of a Norwegian patient previously hospitalized in India (5). Antimicrobial susceptibility testing and PCR analysis revealed resistance to a wide range of antibiotics, including meropenem and ertapenem, and the presence (but not location) of blaNDM-1, which encodes the New Delhi metallo--lactamase-1 (NDM-1), as well as genes encoding several other -lactamases (5). To facilitate further investigation into the genetic basis of CRE and the dissemination of carbapenemases among Gram-negative bacteria, the genome sequence of K. pneumoniae K66-45 was determined using next-generation sequencing technology. Genomic DNA was prepared from an overnight culture of K. pneumoniae K66-45 using the MO BIO DNeasy UltraClean Microbial kit (Qiagen, USA). The DNA libraries for single-molecule real-time (SMRT) sequencing were constructed using the BluePippin preparative electrophoresis system (Sage Science, USA) with a 9-kb cutoff. Sequencing was performed using one SMRT cell of the Pacific Biosciences RS II instrument, using P6-C4 chemistry with a 360-min movie time. A total of 104,622 reads were assembled using HGAPv3 (SMRT Analysis version 2.3.0, Pacific Biosciences, USA). Artifact frameshifts in the PacBio sequence were corrected using Pilon (6), and Bowtie2 (7) was subsequently used to map 99.4% of available Illumina sequence reads (⬎300⫻ coverage, GenBank accession no. NZ_LNGZ01000000) back to the Pilon-corrected PacBio consensus sequence. Annotation was performed with the NCBI Prokaryotic Genome Annotation Pipeline. Volume 5 Issue 27 e00601-17
Received 9 May 2017 Accepted 12 May 2017 Published 6 July 2017 Citation Heikal A, Samuelsen Ø, Kristensen T, Økstad OA. 2017. Complete genome sequence of a multidrug-resistant, blaNDM-1-expressing Klebsiella pneumoniae K66-45 clinical isolate from Norway. Genome Announc 5:e00601-17. https://doi.org/10.1128/genomeA.00601-17. Copyright © 2017 Heikal et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Adam Heikal, [email protected], or Ole Andreas Økstad, [email protected].
genomea.asm.org 1
Heikal et al.
The K. pneumoniae K66-45 genome contains a single ~5.4-Mb chromosome, encoding the SHV-11 -lactamase, and four plasmids, pK66-45-1 to pK66-45-4, which encode the multidrug resistance basis of the strain. The plasmid-based resistance determinants include blaNDM-1, blaCTX-M-15, armA, aadA2, qnrS, dfrA12, and sul1 identified on pK6645-1 (338,512 bp); blaOXA-1, aac(6=)-Ib-cr, and aph(3=)-Ia on pK66-45-2 (200,356 bp); and blaOXA-1, blaCTX-M-15, blaOXA-9, blaTEM-1A, aac(6=)-Ib-cr, aac(6=)-Ib, and aadA1 on pK66-45-3 (120,533 bp). No resistance determinants were identified on pK66-45-4 (41,111 bp). As shown previously, the bleomycin resistance gene bleMBL was located downstream of blaNDM-1 (8). This repertoire of genes illustrates the immense reservoir of resistance determinants associated with mobile genetic elements such as plasmids. The acquisition of these plasmids by pathogens such as K. pneumoniae is a major driver in the increase in multidrug resistance that now threatens modern medicine. As CRE isolates remain rare in Scandinavia, this complete genome sequence will provide a valuable reference for investigations into the dissemination and evolution of multidrug resistance, as well as for improved infection control measures for CRE. Accession number(s). This complete genome project has been deposited at GenBank under the accession numbers CP020901 to CP020905. ACKNOWLEDGMENTS The sequencing service was provided by the Norwegian Sequencing Centre (http:// www.sequencing.uio.no), a national technology platform hosted by the University of Oslo and supported by the “Functional Genomics” and “Infrastructure” programs of the Research Council of Norway and the Southeastern Regional Health Authorities.
REFERENCES 1. Tacconelli E, Margini N. 2017. Global priority list of antibiotic-resistant bacteria to guide Research, discovery, and development of new antibiotics. World Health Organization, Geneva. 2. World Health Organization. 2014. Antimicrobial resistance: global report on surveillance (in IRIS). World Health Organization, Geneva. 3. Löfmark S, Sjöström K, Mäkitalo B, Edquist P, Tegmark Wisell K, Giske CG. 2015. Carbapenemase-producing Enterobacteriaceae in Sweden 2007–2013: experiences from seven years of systematic surveillance and mandatory reporting. Drug Resist Updat 20:29 –38. https://doi.org/10.1016/j.drup .2015.05.001. 4. Tofteland S, Naseer U, Lislevand JH, Sundsfjord A, Samuelsen O. 2013. A long-term low-frequency hospital outbreak of KPC-producing Klebsiella pneumoniae involving intergenus plasmid diffusion and a persisting environmental reservoir. PLoS One 8:e59015. https://doi.org/10.1371/ journal.pone.0059015.
Volume 5 Issue 27 e00601-17
5. Samuelsen Ø, Thilesen CM, Heggelund L, Vada AN, Kümmel A, Sundsfjord A. 2011. Identification of NDM-1-producing enterobacteriaceae in Norway. J Antimicrob Chemother 66:670 – 672. https://doi.org/10.1093/jac/ dkq483. 6. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. https://doi.org/10.1371/journal.pone .0112963. 7. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. https://doi.org/10.1038/nmeth.1923. 8. Dortet L, Nordmann P, Poirel L. 2012. Association of the emerging carbapenemase NDM-1 with a bleomycin resistance protein in Enterobacteriaceae and Acinetobacter baumannii. Antimicrob Agents Chemother 56: 1693–1697. https://doi.org/10.1128/AAC.05583-11.
genomea.asm.org 2
crossm Complete Genome Sequence of a Multidrug-Resistant, blaNDM-1-Expressing Klebsiella pneumoniae K66-45 Clinical Isolate from Norway Adam Heikal,a,b Ørjan Samuelsen,c,d Tom Kristensen,e
Ole Andreas Økstada,b
Centre for Integrative Microbial Evolution (CIME), Faculty of Mathematics and Natural Sciences, University of Oslo, Blindern, Oslo, Norwaya; Laboratory for Microbial Dynamics (LaMDa), Section for Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Blindern, Oslo, Norwayb; Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norwayc; Department of Pharmacy, Faculty of Health Sciences, UiT–The Arctic University of Norway, Tromsø, Norwayd; Department of Biosciences, University of Oslo, Blindern, Oslo, Norwaye
ABSTRACT Multidrug-resistant Klebsiella pneumoniae is a major cause of hospitalacquired infections. Here, we report the complete genome sequence of the multidrugresistant, blaNDM-1-positive strain K. pneumoniae K66-45, isolated from a hospitalized Norwegian patient.
C
arbapenem-resistant Enterobacteriaceae (CRE), such as Klebsiella pneumoniae, are classified as critical on the World Health Organization’s list of priority pathogens (1). Resistance to carbapenems, the traditional last-resort treatment option for severe community- and hospital-acquired infections, has been reported at proportions up to 54% (2). While CRE remain rare in Scandinavia, their arrival and subsequent local expansion from high-prevalence locations, such as through the return of patients hospitalized overseas, is a concern (3, 4). In 2010, the carbapenem-resistant strain Klebsiella pneumoniae K66-45, belonging to sequence type 11 (part of CG258), was isolated and cultured from a catheter urine sample following the return of a Norwegian patient previously hospitalized in India (5). Antimicrobial susceptibility testing and PCR analysis revealed resistance to a wide range of antibiotics, including meropenem and ertapenem, and the presence (but not location) of blaNDM-1, which encodes the New Delhi metallo--lactamase-1 (NDM-1), as well as genes encoding several other -lactamases (5). To facilitate further investigation into the genetic basis of CRE and the dissemination of carbapenemases among Gram-negative bacteria, the genome sequence of K. pneumoniae K66-45 was determined using next-generation sequencing technology. Genomic DNA was prepared from an overnight culture of K. pneumoniae K66-45 using the MO BIO DNeasy UltraClean Microbial kit (Qiagen, USA). The DNA libraries for single-molecule real-time (SMRT) sequencing were constructed using the BluePippin preparative electrophoresis system (Sage Science, USA) with a 9-kb cutoff. Sequencing was performed using one SMRT cell of the Pacific Biosciences RS II instrument, using P6-C4 chemistry with a 360-min movie time. A total of 104,622 reads were assembled using HGAPv3 (SMRT Analysis version 2.3.0, Pacific Biosciences, USA). Artifact frameshifts in the PacBio sequence were corrected using Pilon (6), and Bowtie2 (7) was subsequently used to map 99.4% of available Illumina sequence reads (⬎300⫻ coverage, GenBank accession no. NZ_LNGZ01000000) back to the Pilon-corrected PacBio consensus sequence. Annotation was performed with the NCBI Prokaryotic Genome Annotation Pipeline. Volume 5 Issue 27 e00601-17
Received 9 May 2017 Accepted 12 May 2017 Published 6 July 2017 Citation Heikal A, Samuelsen Ø, Kristensen T, Økstad OA. 2017. Complete genome sequence of a multidrug-resistant, blaNDM-1-expressing Klebsiella pneumoniae K66-45 clinical isolate from Norway. Genome Announc 5:e00601-17. https://doi.org/10.1128/genomeA.00601-17. Copyright © 2017 Heikal et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Adam Heikal, [email protected], or Ole Andreas Økstad, [email protected].
genomea.asm.org 1
Heikal et al.
The K. pneumoniae K66-45 genome contains a single ~5.4-Mb chromosome, encoding the SHV-11 -lactamase, and four plasmids, pK66-45-1 to pK66-45-4, which encode the multidrug resistance basis of the strain. The plasmid-based resistance determinants include blaNDM-1, blaCTX-M-15, armA, aadA2, qnrS, dfrA12, and sul1 identified on pK6645-1 (338,512 bp); blaOXA-1, aac(6=)-Ib-cr, and aph(3=)-Ia on pK66-45-2 (200,356 bp); and blaOXA-1, blaCTX-M-15, blaOXA-9, blaTEM-1A, aac(6=)-Ib-cr, aac(6=)-Ib, and aadA1 on pK66-45-3 (120,533 bp). No resistance determinants were identified on pK66-45-4 (41,111 bp). As shown previously, the bleomycin resistance gene bleMBL was located downstream of blaNDM-1 (8). This repertoire of genes illustrates the immense reservoir of resistance determinants associated with mobile genetic elements such as plasmids. The acquisition of these plasmids by pathogens such as K. pneumoniae is a major driver in the increase in multidrug resistance that now threatens modern medicine. As CRE isolates remain rare in Scandinavia, this complete genome sequence will provide a valuable reference for investigations into the dissemination and evolution of multidrug resistance, as well as for improved infection control measures for CRE. Accession number(s). This complete genome project has been deposited at GenBank under the accession numbers CP020901 to CP020905. ACKNOWLEDGMENTS The sequencing service was provided by the Norwegian Sequencing Centre (http:// www.sequencing.uio.no), a national technology platform hosted by the University of Oslo and supported by the “Functional Genomics” and “Infrastructure” programs of the Research Council of Norway and the Southeastern Regional Health Authorities.
REFERENCES 1. Tacconelli E, Margini N. 2017. Global priority list of antibiotic-resistant bacteria to guide Research, discovery, and development of new antibiotics. World Health Organization, Geneva. 2. World Health Organization. 2014. Antimicrobial resistance: global report on surveillance (in IRIS). World Health Organization, Geneva. 3. Löfmark S, Sjöström K, Mäkitalo B, Edquist P, Tegmark Wisell K, Giske CG. 2015. Carbapenemase-producing Enterobacteriaceae in Sweden 2007–2013: experiences from seven years of systematic surveillance and mandatory reporting. Drug Resist Updat 20:29 –38. https://doi.org/10.1016/j.drup .2015.05.001. 4. Tofteland S, Naseer U, Lislevand JH, Sundsfjord A, Samuelsen O. 2013. A long-term low-frequency hospital outbreak of KPC-producing Klebsiella pneumoniae involving intergenus plasmid diffusion and a persisting environmental reservoir. PLoS One 8:e59015. https://doi.org/10.1371/ journal.pone.0059015.
Volume 5 Issue 27 e00601-17
5. Samuelsen Ø, Thilesen CM, Heggelund L, Vada AN, Kümmel A, Sundsfjord A. 2011. Identification of NDM-1-producing enterobacteriaceae in Norway. J Antimicrob Chemother 66:670 – 672. https://doi.org/10.1093/jac/ dkq483. 6. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. https://doi.org/10.1371/journal.pone .0112963. 7. Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. https://doi.org/10.1038/nmeth.1923. 8. Dortet L, Nordmann P, Poirel L. 2012. Association of the emerging carbapenemase NDM-1 with a bleomycin resistance protein in Enterobacteriaceae and Acinetobacter baumannii. Antimicrob Agents Chemother 56: 1693–1697. https://doi.org/10.1128/AAC.05583-11.
genomea.asm.org 2
