2015Horvath-PappEPhD.pdf (3.98 MB)
Analysis of the fitness cost of integrons in clinical isolates of acinetobacter baumannii
thesisposted on 2015-05-12, 08:54 authored by Eva Horvath-Papp
Antibiotic resistance determinants carried on mobile genetic elements such as integrons are widespread in Acinetobacter baumannii. A total of 76 clinical isolates of A. baumannii were screened for class 1, 2 and 3 integron structures using PCR-based assays. Further chromosome walking and Southern blot-based analyses revealed a significant proportion of incomplete integrons, primarily due to IS element-mediated deletions or truncations at the 5’ end of these integrons. Based on these multiple detection methods, a total of 72 integron structures distributed among 58 isolates were identified. Remarkably, 29 (40.3 %) were either incomplete, or had a truncated integrase gene. These data also revealed that using established PCR-based detection methods alone would yield high rates of false-negative and –positive results. The high frequencies of intI1 and/or 5’ end integron truncations suggest that IntI1 amelioration confers a significant selective advantage. This hypothesis was investigated in E. coli and A. baumannii backgrounds using an IPTG-inducible IntI1-expressing plasmid. Significant fitness costs were observed through growth curve analysis when IntI1 was overexpressed in recA-deficient E. coli lab strains, and also in an A. baumannii strain. The latter was predicted to have a ~1.5-fold higher number of non-canonical IntI1 recombination sites in a typical E. coli strain. The archetypal intI1 repressor, LexA, is not present in the A. baumannii genome, so the role of the closest homologue, ∆UmuDAb, was investigated through knockout studies. ΔumuDAb mutants were created through allelic exchange, and although the mutants showed a significant upregulation of intI1 expression, when the mutants were complemented with a umuDAb-overexpressing plasmid, intI1 expression was also increased, albeit to a lesser extent. This study demonstrates a complex DNA-damage response in A. baumannii, and will aid further investigation for a full understanding of the development and maintenance of antibiotic resistance in this important nosocomial pathogen.
Date of award2015-05-01
Author affiliationDepartment of Infection, Immunity and Inflammation
Awarding institutionUniversity of Leicester