Factors that enhance the ability of Pseudomonas aeruginosa to resist the action of antibiotics
P.aeruginosa is one of the most important pathogens in nosocomial infections and fails to respond to standard treatment, particularly in the case of patients subjected to prolong antibiotic treatment. To generate a more comprehensive understanding of the failure of antimicrobial treatment, focusing especially on the adaptive resistance could be the key area that the bacterium develops in this phenomenon. Most studies on antibiotic resistance in P.aeruginosa have focused upon genotypic studies. This study set out to develop an in vitro model to examine the effect of continual exposure of P.aeruginosa PA01 to the antibiotics studied. Experiments were initially conducted to consider the factors that having a significant influence on antibiotic susceptibility using a novel fluorescence based assay (OxoPlate® system). P.aeruginosa was subjected to the action of tobramycin, amikacin and colistin under various environmental factors. The results of the in vitro analysis showed that, from among the three antibiotics used, amikacin was the antibiotic where resistance was most readily developed. From these results, chemostat studies were designed to examine prolonged exposure of the antibiotic to planktonic cells. Chemostat cultures were exposed to amikacin at sub-inhibitory concentrations using Evans defined synthetic medium at different dilution rates (D) under glucose limitation. Both cultures grown at 0.025h-1 and 0.06h-1 developed the following characteristics i. low-level amikacin resistance, which exhibited an increase in the MIC 4-fold. ii. a clear development of phenotypic resistance and this resistance was not acquired as evidenced by the loss of resistance on culture into fresh medium lacking antibiotic. iii. adaptive resistance to amikacin conferred low-level resistance to other aminoglycosides such as tobramycin and antibiotics with different modes of action such as colistin. Low oxygen availability was seen in the cultures grown at 0.099 h-1 and 0.125 h-1, which lead to i. the appearance of the so called “persister” phenotype. These persisters are sub populations of cells that showed a reduction in bacterial cell size as evidenced from the flow cytometry output as well as being slow growing and resistant ii. extracellular polymeric fibrils were produced in the cells derived after 72h incubation time. In all cases, continual exposure resulted in phenotypically distinct mucoid and non- mucoid colony morphotypes, which were clearly observed on amikacin-free nutrient agar. Some of these selected morphotypes showed from the MIC and MBC data a high-level resistance to the antibiotic when left without antibiotics. The biological responses resulting from these studies offer valuable clues underlying unsuccessful treatment. Conducting experiments using robust systems renders this project extremely novel in the field of microbiology and this will contribute to the development of viable treatment options and ultimately the reduction of the emergence of antibiotic resistance.
QD Chemistry, QH301 Biology, RM Therapeutics. Pharmacology
Networked Digital Library of Theses and Dissertations