Studies on the effects of sub-minimal inhibitory concentrations of antibiotics on the virulence factors of biofilm bacteria
Pseudomonas aeruginosa is a notorious nosocomial opportunist. Planktonic forms of this pathogen have been traditionally studied for its pathogenicity. Such studies have shown that sub-minimal inhibitory concentrations (sub-MICs) of antibiotics are able to negatively modulate pathogenicity. However, more recent findings suggest a biofilm basis of infection. In this study, monospecies and binary biofilms of Pseudomonas aeruginosa ATCC 15692 (PAOl) and Escherichia coli ATCC 10000 were investigated for their pathogenic potential using resistance and virulence as key pathogenic determinants, in the presence of sub-MICs of selected antibiotics (Ampicillin, Nalidixic acid and Streptomycin). MICs of biofilms were observed to be at least 7-fold greater than those of the corresponding planktonic form of the same species (as judged from results obtained from MIC experiments). SDS-PAGE and 2D-PAGE analysis indicate alteration of outer membrane proteins (OMPs) within the envelope of the pathogen in sub-MIC antibiotic treated samples. The observed rearrangement of lipopolysaccharide (LPS; as observed in LPS gel experiments) may also contribute to the pathogens increased tolerance to antibiotics within the biofilm state. While LPS changes may possibly help the biofilm bacteria escape host immune system in vivo, more direct evidence of increases in virulence of the pathogen comes from investigation of its secreted proteases and cytotoxins (leucocidin). Virulence-specific azocasein and micro-culture tetrazolium (MTT) assays against both monospecies and binary biofilms of Pseudomonas aeruginosa indicate significant increases in virulence potential of proteases and cytotoxins, respectively. These results were further substantiated in phase contrast microscopy images showing advanced stages of oncosis in tissue cultured mouse spleen myeloma (Sp2) cells treated with leucocidin isolated from Ps. aeruginosa treated with sub-MIC of ampicillin (8 pg mL'1). The results reported in this thesis provide evidence of observed increases in virulence and pathogenicity in biofilm cells of Pseudomonas aeruginosa in the presence of sub-MICs of selected antibiotics, in vitro. Although these findings are those of in vitro experiments, they may have significant implications regarding the usage and therapeutic control of antibiotics in clinical situations.
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