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

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 of bla_NDM-1 and plasmid copy number alteration under carbapenem exposure.

Three bla_NDM-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 bla_NDM-1. Horizontal transferability and stability of the plasmids encoding bla_NDM-1 were also determined. Changes in copy number of bla_NDM-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.

Under carbapenem stress over an interval of time there was a sharp variation in the transcriptional expression of bla_NDM-1 although it did not follow a specific pattern. All bla_NDM-1 carrying plasmids were transferable by conjugation. These plasmids were highly stable and complete loss was observed between 92^nd to 96^th serial passages when antibiotic pressure was withdrawn. High copy number of bla_NDM-1 was found for IncF type plasmids compared to the other replicon types.

This study suggests that the single dose of carbapenem pressure does not significantly influence the expression of bla_NDM-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.