An insight into selection specificity of quinolone resistance determinants within Enterobacteriaceae family.

OBJECTIVES: Quinolone antimicrobials are frequently misused due to self-medication and suboptimal dose administration, leading to the development of resistance as well as treatment failure. The present study aimed to characterise plasmid-mediated quinolone resistance (PMQR) determinants and their genetic selection in the presence of quinolone stress within members of the Enterobacteriaceae. METHODS: A total of 209 non-duplicate Enterobacteriaceae isolates were collected from hospital and community health centres over the period July 2013-June 2014. Molecular characterisation of phenotypically screened quinolone-resistant isolates was done by multiplex PCR. Plasmids bearing the qnr and aac(6')-Ib-cr genes were transformed into Escherichia coli DH5α and were selected on Muller-Hinton agar plates containing 0.25µg/mL and 0.5µg/mL ciprofloxacin, norfloxacin, ofloxacin, levofloxacin and moxifloxacin. Conjugation experiments were performed to determine whether the aac(6')-Ib-cr- and qnr-carrying plasmids were self-transferable. RESULTS: The transformation assay revealed that transformants carrying qnrA could be selected in media containing norfloxacin, ciprofloxacin and levofloxacin, whereas qnrB and aac(6')-Ib-cr were selected on media containing norfloxacin and ciprofloxacin. Transformed qnrD could be selected in media containing norfloxacin and ofloxacin, and qnrS was selected only in the presence of levofloxacin. CONCLUSIONS: The presence of qnr genes has been associated with an increase in quinolone minimum inhibitory concentrations (MICs) and therefore leads to treatment failure when quinolones are used as selective therapeutic drugs. Since PMQR determinants have a high prevalence, effective measures should be taken and surveillance should be performed in order to avoid treatment failures using this group of antimicrobials.

An unusual occurrence of plasmid-mediated blaOXA-23 carbapenemase in clinical isolates of Escherichia coli from India.

The blaOXA-23 group was considered as the first group of OXA-type ß-lactamases conferring carbapenem resistance and has been reported worldwide in Acinetobacter baumannii, however their presence in Escherichia coli is very rare and unique. This study describes an unusual occurrence of blaOXA-23 in 14 clinical isolates of E. coli obtained from intensive care unit patients admitted to a tertiary referral hospital in India. The blaOXA-23 gene was found located within a self-conjugative plasmid of IncFrepB and IncK incompatibility types and simultaneously carrying blaCTX-M-15, blaVEB-1, blaPER-1 and/or blaNDM-1. The copy number of blaOXA-23 within the IncK-type plasmid was inversely proportional to increasing concentrations of imipenem, whereas in the case of the IncFrepB-type the result was variable; and increased copy number of the IncK-type plasmid was observed with increasing concentrations of meropenem. Plasmids encoding blaOXA-23 could be successfully eliminated after single treatment and were found to be not highly stable, as complete loss of plasmids was observed within 5-10 days. This study emphasises that carbapenem stress invariably altered the copy number of two different Inc type plasmids encoding the blaOXA-23 resistance gene and also highlights a potential threat of clonal expansion of this class D carbapenemase through a heterologous host in this country, which is in second incidence globally.

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

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 of blaNDM-1 and plasmid copy number alteration under carbapenem exposure. METHODS: Three 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. RESULTS: Under 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. CONCLUSION: This 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.