Transcriptional analysis of blaNDM-1 and copy number alteration under carbapenem stress

Abstract

BackgroundNew 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 of blaNDM-1 and plasmid copy number alteration under carbapenem exposure.MethodsThree blaNDM-1 harboring 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 of blaNDM-1. Horizontal transferability and stability of the plasmids encoding blaNDM-1 were also determined. Changes in copy number of blaNDM-1 harboring 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.ResultsUnder carbapenem stress over an interval of time there was a sharp variation in the transcriptional expression of blaNDM-1 although it did not follow a specific pattern. All blaNDM-1 carrying plasmids were transferable by conjugation. These plasmids were highly stable and complete loss was observed between 92nd to 96th serial passages when antibiotic pressure was withdrawn. High copy number of blaNDM-1 was found for IncF type plasmids compared to the other replicon types.ConclusionThis study suggests that the single dose of carbapenem pressure does not significantly influence the expression of blaNDM-1 and 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.