Copy Number Change of the NDM-1 sequence in a multidrug-resistant Klebsiella pneumoniae clinical isolate

PLoS One. 2013 Apr 29;8(4):e62774. doi: 10.1371/journal.pone.0062774. Print 2013.

Abstract

The genetic features of the antimicrobial resistance of a multidrug resistant Klebsiella pneumoniae strain harboring bla NDM-1 were investigated to increase our understanding of the evolution of NDM-1. The strain, KPX, came from a Taiwanese patient with a hospitalization history in New Delhi. Complete DNA sequencing was performed; and the genes responsible for antimicrobial resistance were systematically examined and isolated by library screening. KPX harbored two resistance plasmids, pKPX-1 and pKPX-2, which are 250-kb and 141-kb in size, respectively, with bla NDM-1 present on pKPX-1. The plasmid pKPX-1 contained genes associated with the IncR and IncF groups, while pKPX-2 belonged to the IncF family. Each plasmid carried multiple antimicrobial resistance genetic determinants. The gene responsible for resistance to carbapenems was found on pKPX-1 and that for resistance to aztreonam was found on pKPX-2. To our surprise, we discovered that bla NDM-1 exists on pKPX-1 as multiple copies in the form of tandem repeats. Amplification of bla NDM-1 was found to occur by duplication of an 8.6-kb unit, with the copy number of the repeat varying from colony to colony. This repeat sequence is identical to that of the pNDM-MAR except for two base substitutions. The copy number of bla NDM-1 of colonies under different conditions was assessed by Southern blotting and quantitative PCR. The bla NDM-1 sequence was maintained in the presence of the antimicrobial selection; however, removal of antimicrobial selection led to the emergence of susceptible bacterial populations with a reduced copy number or even the complete loss of the bla NDM-1 sequence. The dynamic nature of the NDM-1 sequence provides a strong argument for judicious use of the broad-spectrum antimicrobials in order to reduce the development and spread of antimicrobial resistance among pathogens.

Publication types

  • Case Reports
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Anti-Bacterial Agents / pharmacology
  • Carbapenems / pharmacology
  • Colony Count, Microbial
  • DNA Copy Number Variations*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics*
  • Drug Resistance, Multiple, Bacterial / drug effects
  • Drug Resistance, Multiple, Bacterial / genetics*
  • Humans
  • Klebsiella Infections / drug therapy
  • Klebsiella Infections / microbiology
  • Klebsiella pneumoniae / drug effects
  • Klebsiella pneumoniae / enzymology
  • Klebsiella pneumoniae / genetics*
  • Male
  • Plasmids / chemistry*
  • Sequence Analysis, DNA
  • Tandem Repeat Sequences
  • beta-Lactamases / chemistry
  • beta-Lactamases / genetics*
  • beta-Lactamases / metabolism

Substances

  • Anti-Bacterial Agents
  • Carbapenems
  • DNA, Bacterial
  • beta-Lactamases
  • beta-lactamase NDM-1

Grants and funding

This work is supported in part by National Science Council grants to TLC (NSC 98-2314-B-010-010-MY3), SFT (NSC 99-2320-B-400 -013-MY3) and YTC (NSC 100-2311-B-005-001). SFT, TLL, CMC have received long-term support from the National Health Research Institutes. TLC is supported by Immunology Research Center at Taipei Veterans General Hospital (V100E4-005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.