Characterisation of Antibiotic Resistance in Clostridium difficile

Abstract
Clostridium difficile is a Gram-positive, obligate anaerobe and an opportunistic pathogen that causes antibiotic associated diarrhoea. The incidence of C. difficile infection (CDI) increased dramatically in the early years of this century, an epidemic caused by the previously rare ribotype 027. In addition to causing large hospital outbreaks this lineage was also associated with seemingly more severe disease. Ribotype 027 strains have been reported to produce more spores and more toxin, perhaps going someway to explaining the efficient transmission and poor clinical outcome.
We sought to understand the peptidoglycan biosynthetic pathways active in both vegetative cells and during sporulation, in order to identify proteins playing a role in resistance to cell wall targeting antibiotics. A total of 11 genes predicted to encode penicillin-binding proteins (PBPs) were identified in the genome of R20291, the UK prototypic ribotype 027 strain. Two putative PBPs were taken forward for further study: one class B PBP, SpoVD, required for both sporulation and cephalosporin resistance, and one class C PBP, Cwp20, that contributes to cephalosporin resistance.
A ΔspoVD mutant showed two strong phenotypes: a sporulation defect and cephalosporin sensitivity. In addition, an interaction between SpoVD and SpoVE that appears to be crucial in both sporulation and cephalosporin resistance was demonstrated. A Δcwp20 mutant had a clear defect in cephalosporin resistance. Disruption of cwp20 in a strain that completely lacked the S-layer provided further evidence for a role cephalosporin resistance. Cwp20 was determined to be a class A β-lactamase.
The third part of this thesis is devoted to identification of genes that are responsible for ceftazidime, cefoxitin and ciprofloxacin resistance. A total of 6,000 transposon mutants were screened for resistance to each antibiotic. Three genes with defects in resistance to these antibiotics were chosen for detailed analysis. SpoVE, a membrane protein and putative lipid II flippase, was found to be involved in both cefoxitin and ceftazidime resistance. A CD0398 mutant was found to have a defect under ceftazidime selection. Complementation restored ceftazidime resistance and CD0399 was identified as a class A β-lactamase. A CD0622 mutant was found to have a defect under ciprofloxacin selection and CD0622 was demonstrated to act as an efflux pump.