Nested Russian Doll-Like Genetic Mobility Drives Rapid Dissemination of the Carbapenem Resistance Gene blaKPC

ABSTRACT The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.

[1]  N. Datta,et al.  Conjugative plasmids in bacteria of the ‘pre-antibiotic’ era , 1983, Nature.

[2]  R. Hall,et al.  Distribution of the blaTEM gene and blaTEM-containing transposons in commensal Escherichia coli. , 2011, The Journal of antimicrobial chemotherapy.

[3]  R. Eng,et al.  Virulence and plasmid transferability of KPC Klebsiella pneumoniae at the Veterans Affairs Healthcare System of New Jersey. , 2012, Microbial drug resistance.

[4]  J. Patel,et al.  Fatal cross infection by carbapenem‐resistant Klebsiella in two liver transplant recipients , 2009, Transplant infectious disease : an official journal of the Transplantation Society.

[5]  Derrick E. Wood,et al.  Kraken: ultrafast metagenomic sequence classification using exact alignments , 2014, Genome Biology.

[6]  R. Bonomo,et al.  First Clinical Cases of OXA-48-Producing Carbapenem-Resistant Klebsiella pneumoniae in the United States: the “Menace” Arrives in the New World , 2013, Journal of Clinical Microbiology.

[7]  V. Miriagou,et al.  KPC-Producing, Multidrug-Resistant Klebsiella pneumoniae Sequence Type 258 as a Typical Opportunistic Pathogen , 2013, Antimicrobial Agents and Chemotherapy.

[8]  F. Baquero,et al.  Multiclonal dispersal of KPC genes following the emergence of non-ST258 KPC-producing Klebsiella pneumoniae clones in Madrid, Spain. , 2013, The Journal of antimicrobial chemotherapy.

[9]  A. Mathers,et al.  Molecular Dissection of an Outbreak of Carbapenem-Resistant Enterobacteriaceae Reveals Intergenus KPC Carbapenemase Transmission through a Promiscuous Plasmid , 2011, mBio.

[10]  B. Kreiswirth,et al.  A Two-Year Surveillance in Five Colombian Tertiary Care Hospitals Reveals High Frequency of Non-CG258 Clones of Carbapenem-Resistant Klebsiella pneumoniae with Distinct Clinical Characteristics , 2015, Antimicrobial Agents and Chemotherapy.

[11]  O. Gascuel,et al.  A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. , 2003, Systematic biology.

[12]  H. Barrios,et al.  Characteristics of KPC-2-producing Klebsiella pneumoniae (ST258) clinical isolates from outbreaks in 2 Mexican medical centers. , 2014, Diagnostic microbiology and infectious disease.

[13]  A. Mathers,et al.  Modified Hodge Test versus Indirect Carbapenemase Test: Prospective Evaluation of a Phenotypic Assay for Detection of Klebsiella pneumoniae Carbapenemase (KPC) in Enterobacteriaceae , 2013, Journal of Clinical Microbiology.

[14]  S. Solomon,et al.  Antibiotic resistance threats in the United States: stepping back from the brink. , 2014, American family physician.

[15]  Samir N. Patel,et al.  Lateral dissemination and inter-patient transmission of blaKPC-3: role of a conjugative plasmid in spreading carbapenem resistance. , 2016, The Journal of antimicrobial chemotherapy.

[16]  M. Asensi,et al.  Molecular epidemiology of KPC-2-producing Enterobacteriaceae (non-Klebsiella pneumoniae) isolated from Brazil. , 2015, Diagnostic microbiology and infectious disease.

[17]  Y. Carmeli,et al.  Persistence of Klebsiella pneumoniae ST258 as the predominant clone of carbapenemase-producing Enterobacteriaceae in post-acute-care hospitals in Israel, 2008-13. , 2015, The Journal of antimicrobial chemotherapy.

[18]  A. Mathers,et al.  Admission Surveillance for Carbapenamase-Producing Enterobacteriaceae at a Long-Term Acute Care Hospital , 2013, Infection Control & Hospital Epidemiology.

[19]  R. Hall,et al.  Evolution of Transposons Containing blaTEM Genes , 2005, Antimicrobial Agents and Chemotherapy.

[20]  S. Partridge,et al.  Analysis of antibiotic resistance regions in Gram-negative bacteria. , 2011, FEMS microbiology reviews.

[21]  C. Donskey,et al.  Intestinal Carriage of Carbapenemase-Producing Organisms: Current Status of Surveillance Methods , 2015, Clinical Microbiology Reviews.

[22]  Hui Wang,et al.  Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. , 2013, The Lancet. Infectious diseases.

[23]  A. Mathers,et al.  The Limits of Serial Surveillance Cultures in Predicting Clearance of Colonization with Carbapenemase-Producing Enterobacteriaceae , 2015, Infection Control & Hospital Epidemiology.

[24]  R. Bonomo,et al.  Comparative Genomic Analysis of KPC-Encoding pKpQIL-Like Plasmids and Their Distribution in New Jersey and New York Hospitals , 2014, Antimicrobial Agents and Chemotherapy.

[25]  J. Quinn,et al.  Genetic Structures at the Origin of Acquisition of the β-Lactamase blaKPC Gene , 2008, Antimicrobial Agents and Chemotherapy.

[26]  James K. Bonfield,et al.  Genome analysis Advance Access publication May 30, 2010 Gap5—editing , 2010 .

[27]  P. Nordmann,et al.  Functional Characterization of Tn4401, a Tn3-Based Transposon Involved in blaKPC Gene Mobilization , 2011, Antimicrobial Agents and Chemotherapy.

[28]  D. Toney,et al.  Control of Simultaneous Outbreaks of Carbapenemase-Producing Enterobacteriaceae and Extensively Drug-Resistant Acinetobacter baumannii Infection in an Intensive Care Unit Using Interventions Promoted in the Centers for Disease Control and Prevention 2012 Carbapenemase-Resistant Enterobacteriaceae To , 2014, Infection Control & Hospital Epidemiology.

[29]  Heng Li Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM , 2013, 1303.3997.

[30]  Alessandra Carattoli,et al.  Plasmids and the spread of resistance. , 2013, International journal of medical microbiology : IJMM.

[31]  A. Sarah Walker,et al.  Characterisation of Clostridium difficile Hospital Ward–Based Transmission Using Extensive Epidemiological Data and Molecular Typing , 2012, PLoS medicine.

[32]  A. Mathers,et al.  Clinical Microbiology Costs for Methods of Active Surveillance for Klebsiella pneumoniae Carbapenemase–Producing Enterobacteriaceae , 2014, Infection Control & Hospital Epidemiology.

[33]  Iraida E. Robledo,et al.  ISEcp1-mediated transposition of blaKPC into the chromosome of a clinical isolate of Acinetobacter baumannii from Puerto Rico. , 2014, Journal of medical microbiology.

[34]  K. Bush,et al.  Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae , 2008, Antimicrobial Agents and Chemotherapy.

[35]  B. Liu,et al.  Rates of Mutation and Host Transmission for an Escherichia coli Clone over 3 Years , 2011, PloS one.

[36]  Y. Carmeli,et al.  Molecular Epidemiology, Sequence Types, and Plasmid Analyses of KPC-Producing Klebsiella pneumoniae Strains in Israel , 2010, Antimicrobial Agents and Chemotherapy.

[37]  Daniel J. Wilson,et al.  Genome Sequencing of an Extended Series of NDM-Producing Klebsiella pneumoniae Isolates from Neonatal Infections in a Nepali Hospital Characterizes the Extent of Community- versus Hospital-Associated Transmission in an Endemic Setting , 2014, Antimicrobial Agents and Chemotherapy.

[38]  N. Datta,et al.  Plasmids of the same Inc groups in Enterobacteria before and after the medical use of antibiotics , 1983, Nature.

[39]  L. Bry,et al.  Genomically Informed Surveillance for Carbapenem-Resistant Enterobacteriaceae in a Health Care System , 2015, mBio.

[40]  G. B. Golding,et al.  Antibiotic resistance is ancient , 2011, Nature.

[41]  Y. Carmeli,et al.  Molecular Epidemiology of KPC-Producing Klebsiella pneumoniae Isolates in the United States: Clonal Expansion of Multilocus Sequence Type 258 , 2009, Antimicrobial Agents and Chemotherapy.

[42]  P. Nordmann,et al.  Functional Characterization of Tn 4401 , a Tn 3 -Based Transposon Involved in bla KPC Gene Mobilization (cid:1) , 2011 .

[43]  Anna E. Sheppard,et al.  Klebsiella pneumoniae Carbapenemase (KPC)-Producing K. pneumoniae at a Single Institution: Insights into Endemicity from Whole-Genome Sequencing , 2015, Antimicrobial Agents and Chemotherapy.

[44]  S. Partridge Resistance mechanisms in Enterobacteriaceae. , 2015, Pathology.

[45]  J. Iredell,et al.  Different IncI1 plasmids from Escherichia coli carry ISEcp1-blaCTX-M-15 associated with different Tn2-derived elements. , 2015, Plasmid.

[46]  R. Bonomo,et al.  Carbapenemase-producing Klebsiella pneumoniae: molecular and genetic decoding. , 2014, Trends in microbiology.

[47]  Aaron E. Darling,et al.  A5-miseq: an updated pipeline to assemble microbial genomes from Illumina MiSeq data , 2014, Bioinform..

[48]  P. Nordmann,et al.  Outbreak Caused by NDM-1- and RmtB-Producing Escherichia coli in Bulgaria , 2014, Antimicrobial Agents and Chemotherapy.

[49]  J. Iredell,et al.  IncI shufflons: Assembly issues in the next-generation sequencing era. , 2015, Plasmid.

[50]  R. Bonomo,et al.  Complete Nucleotide Sequence of a blaKPC-Harboring IncI2 Plasmid and Its Dissemination in New Jersey and New York Hospitals , 2013, Antimicrobial Agents and Chemotherapy.

[51]  F. di Bernardo,et al.  An Update of the Evolving Epidemic of bla KPC Carrying Klebsiella pneumoniae in Sicily, Italy, 2014: Emergence of Multiple Non-ST258 Clones , 2015, PloS one.

[52]  Jiachang Cai,et al.  Emergence of Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli Isolates Possessing the Plasmid-Mediated Carbapenem-Hydrolyzing β-Lactamase KPC-2 in Intensive Care Units of a Chinese Hospital , 2008, Antimicrobial Agents and Chemotherapy.

[53]  K. Bush,et al.  Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae , 2001, Antimicrobial Agents and Chemotherapy.

[54]  Fernando de la Cruz,et al.  Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences , 2014, PLoS genetics.

[55]  Iraida E. Robledo,et al.  Guidance for control of infections with carbapenem-resistant or carbapenemase-producing Enterobacteriaceae in acute care facilities. , 2009, MMWR. Morbidity and mortality weekly report.

[56]  L. Rice,et al.  The KQ Element, a Complex Genetic Region Conferring Transferable Resistance to Carbapenems, Aminoglycosides, and Fluoroquinolones in Klebsiella pneumoniae , 2008, Antimicrobial Agents and Chemotherapy.

[57]  J. Patel,et al.  Regional Dissemination of KPC-Producing Klebsiella pneumoniae , 2009, Antimicrobial Agents and Chemotherapy.

[58]  R. Olsen,et al.  Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae , 2014, Proceedings of the National Academy of Sciences.

[59]  Gopi Patel,et al.  Outcomes of Carbapenem-Resistant Klebsiella pneumoniae Infection and the Impact of Antimicrobial and Adjunctive Therapies , 2008, Infection Control & Hospital Epidemiology.

[60]  M. Falagas,et al.  Deaths Attributable to Carbapenem-Resistant Enterobacteriaceae Infections , 2014, Emerging infectious diseases.

[61]  Jonas Korlach,et al.  Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae , 2014, Science Translational Medicine.