| Serratia marcescens is a prominent opportunistic pathogen in clinical settings, responsible for serious infections in immunocompromised individuals. This organism has been shown to be resistant to several classes of antibiotics, making the treatment of infections very difficult. Recently, S. marcescens has been shown to be resistant to fluoroquinolone, a relatively new group of antibiotics. The major mechanism for fluoroquinolone resistance in various Gram-negative pathogens is the active efflux of the antibiotic molecule mediated by inner membrane efflux pumps belonging to the Resistance-Nodulation-Celt Division (RND) family. RND pumps work in conjunction with a periplasmic protein and an outer membrane protein in Gram-negative organisms. In order to design effective chemotherapy to cure S. marcescens infections, identification and characterization of efflux pump(s) in this organism is essential. This thesis establishes active efflux as a resistance mechanism in S. marcescens, and presents molecular characterization of two different efflux pumps in this organism. Clinical isolates of S. marcescens were examined, and results established active efflux as a mechanism of resistance in S. marcescens. Two RND pump-encoding genes were identified by using the PCR targeting conserved motifs found within RND pump-encoding genes. To clone the complete genes of these two putative RND complexes, a S. marcescens genomic DNA library was constructed and screened with DNA probes synthesized from the PCR products. Both operons were isolated and sequenced. Sequence analysis revealed that both operons contained RND complex-encoding genes. The operons were named sdeAB and sdeCDE (sde , S&barbelow;erratia d&barbelow;rug e&barbelow;fflux). Functional analysis of the sdeAB and sdeCDE gene products revealed that SdeAB is in fact a multidrug efflux pump with a wide range of substrates. The sdeAB locus was also found to be over-expressed in laboratory-derived fluoroduinolone resistant mutants, suggesting that it is a major pump in S. marcescens. Substrate specificity could not be ascertained for SdeCDE, as it was not found to efflux any of the compounds tested. A gene encoding the outer-membrane component was also identified, cloned, and characterized. Functional analysis of the outer-membrane component revealed that it is involved in energy-dependent efflux of antimicrobial agents, and could therefore be acting in conjunction with the active efflux pump(s) in S. marcescens. A MarA homolog was also identified upstream of the sdeAB locus and the gene was named sdeR. SdeR may be involved in the over-expression of sdeAB in multidrug resistant isolates of S. marcescens . A putative binding site for SdeR upstream of sdeAB was identified. |
