Gene expression analysis of the SdeAB multidrug efflux pump in antibiotic-resistant clinical isolates of Serratia marcescens.

Purpose: Many isolates of Serratia marcescens, a well-known opportunistic pathogen, can be multidrug resistant. Fluoroquinolones are among the most important groups of antibiotics used for treatment of these organisms. However, fluoroquinolone resistance among S. marcescens isolates is fast increasing. Drug extrusion through efflux pumps like SdeAB/ HasF is one of the major mechanisms of resistance to fluoroquinolones. This study was carried out to analyze, through gene expression analysis of sdeB, the relative contribution of this mechanism toward fluoroquinolone resistance in clinical isolates of Serratia. Materials and Methods: Total RNA from 45 clinical isolates of S. marcescens was isolated. Quantitative real-time RT PCR was performed on the extracted RNA to study the gene expression of sdeB and was normalized to the sdeB expression in the standard strain of S. marcescens. Results: Of the 45 isolates analyzed, sdeB expression was found to be elevated in 20 isolates (44%). Of these 20 isolates, eight (40%) were fully resistant to at least one of the fluoroquinolones studied. Conversely, of the 20 isolates that over-expressed sdeB, 12 (60%) were fully sensitive to all fluoroquinolones tested. Conclusions: Drug efflux pumps are an important means of fluoroquinolone resistance among clinically important species ofSerratia. The expression of these pumps can be up-regulated in the presence of antibiotics and have the potential for changing the phenotype from sensitive to resistant, thus contributing to therapeutic failures.

Simultaneous determination of intact lomefloxacin and ciprofloxacin in the presence of their acid degradation products

A reversed phase HPLC method was developed for the determination of lomefloxacin and its degradation product. In addition, two other methods have been developed for the determination of lomefloxacin hydrochloride [LF.HCl] and ciprofloxacin hydrochloride [CF.HCl] in presence of their acid induced degradation products. For the reversed phase HPLC method [determination of LF.HCl], the mobile phase used was a mixture of water: acetonitrile: triethylamine [80:20:0.6, v/v/v] adjusted to pH 3.0 with o-phosphoric acid. The flow rate was 1.5 ml/min. and the detection was carried out at 328 nm. The linearity range was found to be 0.5-6 micro g / 20 micro l for LF.HCl. The limits of detection and quantification [LOD and LOQ] were 0.22 micro g / 20 micro l and 0.74 micro g / 20 microl respectively. The second method was densitometric method for the determination of both LF.HCl and CF.HCl, the developing system used was a mixture of methanol and ammonia buffer [80:20, v/v]. Detection was carried out at 288 nm and 279 nm. for intact LF.HCl and CF.HCl respectively. The linearity ranges were found to be 1-6 micro g / 10 microl and 0.25-2.5 micro g / 10 microl for intact LF.HCl and CF.HCl respectively. LOD and LOQ were 0.1, 0.34 micro g/10 microl and 0.05, 0.18 micro g /10 microl for both drugs, respectively. The third method was derivative spectrophotometric method for the determination of [LF.HCl] and [CF.HCL]. The linearity ranges were found to be 2-8 micro g/ml and 5-12 micro g/ml for LF.HCl and CF.HCl respectively. LOD and LOQ were 0.39 micro g, 1.29 micro g/ml and 1.03, 3.45 micro g/ml for LF.HCl and CF.HCl respectively. Separation and identification of the acid degradation products of lomefloxacin hydrochloride and ciprofloxacin hydrochloride were carried out. The three described methods proved to be sensitive, precise and applicable to both dosage forms and laboratory prepared mixtures of the intact drugs and their acid degradation products