Multidrug Efflux Pumps in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that is increasingly a problem in the hospital setting. Some strains are resistant to all clinically useful antibiotics, and a large factor in this high level resistance is thought to be the overexpression of multidrug efflux pumps. However, at present only one RND type pump, SmeDEF, has been identified as a drug efflux pump in this species. In this work, a putative regulator of smeDEF expression, SmeT, is identified as being encoded adjacent to the smeDEF operon. Loss-of-function mutations in smeTlead to overexpression of smeDEF, and to decreased antibiotic susceptibility. Investigation of a collection of single step mutants, selected on different antibiotics and derived from clinical S. maltophilia isolates, identified some as overexpressing smeDEF in comparison to their parent isolates. Some of these spontaneous smeDEF overexpressing mutants had mutations in smeT which were associated with this smeDEF overexpression; in others, no change was found in either smeT or the smeDEF promoter region, indicating that transcriptional repression by smeT is not the only factor impacting on smeDEF expression. Evidence for the presence of other drugeffluxing pumps in S. maltophilia was also found, as many spontaneous multidrugresistant mutants were identified in which smeDEF overexpression did not occur. In addition, evidence for non-~-lactamase mediated ~-lactam resistance was found. A genome search of a group A isolate, K279a, identified many more genes which encode putative efflux pumps, and a preliminary screen for expression of these genes indicated that several are candidate drug efflux pumps. It is known that there is considerable variation amongst S. maltophilia isolates, and a correlation between ~-lactamase activity and induction and 16S rRNA sequence has previously been identified in a small collection of clinical isolates. In this study it was found that the smeT-smeD intergenic region also showed variation consistent with these groupings in the same collection of isolates. In order to investigate this further, collection of 50 clinical isolates from three continents was subjected to 16S rRNA and smeT-smeD intergenic region sequencing; it was found that 22 of the isolates fit into a highly homogeneous group, group I, that also includes the type strain and other frequently studied strains. However, another commonly used strain was found to belong to a much more diverse group. which contained 18 of the 50 clinical isolates. Two further groups were additionally identified. However, little, if any, correlation between gentypic grouping and phenotype was observed. This highlights the variability amongst Stenotrophomonas maltophilia clinical isolates, and the importance of using correctly controlled studies with well characterised parent isolates.EThOS - Electronic Theses Online ServiceGBUnited Kingdo