Abstract
Despite following the recommended guidelines, mupirocin therapy may fail to decolonise colonised individuals and treatment relapses do occur, with reports of only 32 % of treated individuals remaining MRSA free one-year later. Cell tolerance leading to therapy failure may result from the ability of S. aureus to readily attach to mucin coated nasal epithelia (leading to attached cell populations), and a mupirocin induced stringent response (SR). This study aimed to evaluate the shortcomings of mupirocin therapy in vitro, by assessing surface attached cell susceptibility together with the impact of SR induction during decolonisation,with a view to proffering a fresh strategy for improving decolonisation therapy.To evaluate antibiotic tolerance and survival of surface adherent cells, surface attached clinical strains of S. aureus were exposed to mupirocin over a time-course of 1, to 24h. Three of these clinical isolates, were then evaluated for their ability to survive repeated exposure to mupirocin twice a day for five days (mimicking decolonisation therapy). Following redosing, the impact of a mupirocin-induced SR was investigated at the molecular level, which included assessment of gene modulation of key nasal colonisation factors. In parallel, and tomimic the in vivo environment, mupirocin exposure in a medium replicating an artificial nasal fluid (SNM3), was then assessed. Furthermore, the SR effect of in vivo levels of the Branched Chain Amino-Acids (BCAAs), present in nasal fluids, was also investigated in the SNM3. Finally, in an attempt to improve decolonisation therapy, biofilm associated cells we retreated with D-amino acids to initiate dispersal, cells were also exposed to a combination of antibiotics (mupirocin, rifampicin and D-tryptophan) to attempt to down-regulate the SR,and finally all three were combined to down-regulate colonisation factors, disperse surface attached cells and reinstate antibiotic susceptibility.
Surface attached cells, mimicking nasal colonisation, showed an ability to recover following exposure to mupirocin for 24h, but this was particularly apparent in strains which expressed low- level resistance to mupirocin (LL-MR), when compared to susceptible isolates (MS) cells.In addition, LL-MR cells also showed an enhanced ability to survive antibiotic treatment, overa five-day treatment regime. However, in both MS and LL-MR cells, mupirocin re-dosing induced an up-regulation of relA (indicative of induction of the SR), and a corresponding increase in the expression for clfB, isdA, fnbpA and sceD. Higher levels of the BCAAs in the SNM3, blocked the induction of the SR in SNM3. Finally, combination with D-tryptophan and rifampicin, lead to an inhibition in the induction of the SR and increased killing of both MS and LL-MR cells.
Decolonisation failure may be associated with the increased tolerance of surface attached MRSA in the nasal passages. The tolerance exhibited by LL-MR cells to repeated mupirocin exposure, might underlie the higher rate of relapse with these strains. Mupirocin induction of the SR, might be involved in the positive modulation of key nasal colonisation factors,aiding cell survival. Finally, the down-regulation of SR coupled with increased attached-cell killing, presents a fresh strategy for reducing cell tolerance to mupirocin induced stress, whilst increasing the susceptibility of attached cells in vivo.
| Date of Award | 2016 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Susan Lang (Supervisor) |