AbstractPseudomonas aeruginosa is the predominant opportunistic bacterium that causes chronic respiratory infections in cystic fibrosis (CF) patients. This bacterium can develop a high level of resistance to multiple classes of antibiotics, and this is partially attributed to the ability of P. aeruginosa to grow as a biofilm. Such biofilm offers protection for bacterial cells from the host immune defences and antibiotics. In CF patients, tobramycin is one of the most commonly used antibiotics. However, because of the increasing level of P. aeruginosa resistance to tobramycin and other antibiotics, there is a significant challenge in the treatment and eradication of such infections. Therefore, this thesis investigates potential strategies to improve tobramycin efficacy in the eradication of P. aeruginosa biofilm. The main aim of this thesis is to evaluate the activity of tobramycin under a clinically relevant condition. Other specific aims are to investigate the influence of dry powder particle size on antibiofilm activity and to evaluate tobramycin combination therapy with non-antibiotic drugs.
The activity of tobramycin against planktonic and biofilms of P. aeruginosa was determined using artificial sputum medium (ASM), which is clinically more relevant than standard growth media. It was observed that the susceptibility of strains to tobramycin was reduced by four-fold when ASM was used as compared to the minimal MOPS medium. This highlights the importance of testing antibiotic susceptibility with a physiologically more relevant medium. Beside testing against planktonic cells, the results from biofilms using two different biofilm models indicated that eradication of P. aeruginosa biofilms was challenging to achieve with only 40-60% reduction in biofilm biomass or number of viable counts were observed in most strains, which suggest further research and improvement in tobramycin activity against P. aeruginosa is required.
Tobramycin dry powder inhaler (TOBI Podhaler®) is widely used in the treatment of P. aeruginosa lung infections in CF patients. Currently, the models that are used to test the activity of tobramycin are not representative of lung infections, and better models would provide a significant advantage as these could be used to improve the formulation of dry powder inhalers. For instance, one question that has not been addressed is whether antibiotic particle size influences the antibiofilm activity against P. aeruginosa. Therefore, in this thesis, the activity of tobramycin dry powder was investigated in terms of particle size. This was achieved using a Next Generation Impactor (NGI), which was used to fractionate tobramycin powder into different particle sizes. The results described here indicate that the antibiofilm activity of tobramycin dry powder inhaler can indeed be affected by the size of the powder particles. Against P. aeruginosa biofilms of LMG 27643 and LMG 27649 clinical isolates, smaller tobramycin particles (< 2.82 µm) showed better efficacy by ~ 20% as compared to larger tobramycin particles (< 11.7 µm) after 3 hours incubation time. No difference was seen after long incubation times, but these may not be clinically relevant as tobramycin is cleared from the lung in 2-3 hours. This work highlights that particle size of dry powder inhaler can affect antibacterial and antibiofilm activity.
Tobramycin activity in combination with non-antibiotic drugs was also determined, as a limited number of previous studies have investigated this issue against biofilms of P. aeruginosa. Moreover, most tobramycin combination studies employed standard growth media, whereas we analyzed this using a more clinically relevant ASM medium. This work was performed firstly by conducting a comprehensive literature review, where several non-antibiotic drugs from different classes that showed antibacterial activity against P. aeruginosa were found. These drugs include colistin, amitriptyline, chlorpromazine, niclosamide, furanone, quercetin, and diethylenetriaminepentaacetic acid. Checkerboard assays were used to evaluate tobramycin interactions, and Fractional Inhibitory Concentration (FIC) was used as an index to assess the type of interaction. The results from this work were mixed, with outcomes dependent on bacterial lifestyles (planktonic vs biofilm), type of strains (laboratory vs clinical isolates), and growth medium (ASM vs Mueller-Hinton Broth). For instance, using ASM medium resulted mostly in antagonistic interactions between tobramycin and non-antibiotic drugs against planktonic cells. Although a more clinically relevant ASM medium was used, the in vivo significance of our findings remains to be investigated and if it is confirmed, then this suggests careful attention should be considered when other drugs are prescribed simultaneously with tobramycin, particularly as most CF patients regularly use of many non-antibiotic drugs. Apart from antagonistic interactions, some of the non-antibiotic and antibiotic combinations did show synergistic interactions with tobramycin against biofilms of PAO1 but not against all clinical isolates. Such combinations were observed with amitriptyline, niclosamide, and colistin, which resulted in 80% reduction in biofilm biomass as compared to tobramycin alone. These combinations could be used as adjuvants with tobramycin for the treatment of P. aeruginosa infections, but further in vitro and in vivo experiments are warranted to ensure that they are effective.
|Date of Award||22 Jul 2020|
|Supervisor||Robert Price (Supervisor), Albert Bolhuis (Supervisor) & Matthew Jones (Supervisor)|