title: Transcriptional analysis ofblaNDM-1and copy number alteration under carbapenem stress.
abstract: BACKGROUND
  New Delhi metallo beta-lactamase is known to compromise carbapenem therapy and leading to treatment failure. However, their response to carbapenem stress is not clearly known. Here, we have investigated the transcriptional response ofblaNDM-1and plasmid copy number alteration under carbapenem exposure.
  
  
  METHODS
  ThreeblaNDM-1harboring plasmids representing three incompatibility types (IncFIC, IncA/C and IncK) were inoculated in LB broth with and without imipenem, meropenem and ertapenem. After each 1 h total RNA was isolated, immediately reverse transcribed into cDNA and quantitative real time PCR was used for transcriptional expression ofblaNDM-1. Horizontal transferability and stability of the plasmids encodingblaNDM-1were also determined. Changes in copy number ofblaNDM-1harboring plasmids under the exposure of different carbapenems were determined by real time PCR. Clonal relatedness among the isolates was determined by pulsed field gel electrophoresis.
  
  
  RESULTS
  Under carbapenem stress over an interval of time there was a sharp variation in the transcriptional expression ofblaNDM-1although it did not follow a specific pattern. AllblaNDM-1carrying plasmids were transferable by conjugation. These plasmids were highly stable and complete loss was observed between 92ndto 96thserial passages when antibiotic pressure was withdrawn. High copy number ofblaNDM-1was found for IncF type plasmids compared to the other replicon types.
  
  
  CONCLUSIONS
  This study suggests that the single dose of carbapenem pressure does not significantly influence the expression ofblaNDM-1and also focus on the stability of this gene as well as the change in copy number with respect to the incompatible type of plasmid harboring resistance determinant.
authors.affiliation: Department of Microbiology, Assam University, Silchar, India.
authors.forename: Deepjyoti
authors.initials: D
authors.lastname: Paul
authors.affiliation: Department of Microbiology, Assam University, Silchar, India.
authors.forename: Amitabha
authors.initials: A
authors.lastname: Bhattacharjee
authors.affiliation: Department of Statistics, Assam University, Silchar, India.
authors.forename: Dibyojyoti
authors.initials: D
authors.lastname: Bhattacharjee
authors.affiliation: Department of Microbiology, Silchar Medical College and Hospital, Silchar, India.
authors.forename: Debadatta
authors.initials: D
authors.lastname: Dhar
authors.affiliation: Department of Microbiology, Assam University, Silchar, India.
authors.forename: Anand Prakash
authors.initials: AP
authors.lastname: Maurya
authors.affiliation: Department of Microbiology, Silchar Medical College and Hospital, Silchar, India.
authors.forename: Atanu
authors.initials: A
authors.lastname: Chakravarty
keywords: Escherichia coli; Plasmid stability; Transcriptional expression; Transferability