| Popis: |
It is estimated that there are over 700,000 deaths annually worldwide due to multi-drug resistant (MDR) bacterial infections and by 2050 this is predicted to rise to 10 million. One of these MDR organisms is Acinetobacter baumannii, an opportunistic pathogen implicated in pneumonia, meningitis and septicaemia. It is especially difficult to treat due to its ability to become rapidly resistant to a wide range of antimicrobials and persist due to its ability to form biofilms. Biofilms are sessile communities of bacterial cells, attached to surfaces and embedded within a protective polysaccharide matrix, making them 10-1000 times more resistant than their planktonic form. Antimicrobial peptides (AMPs) are a diverse group of polypeptides that are present in nearly all living things as part of the innate immune system. They are being extensively researched as potential alternatives to classical antibiotics. The objective of this research project was to compare the bactericidal and biofilm activity of two synthetic nature inspired antimicrobial peptides, bicarinalin and BP100, with colistin sulphate. Colistin sulphate is an antimicrobial peptide, currently used as a last resort treatment of A. baumannii infections. The bacteriostatic and bactericidal effects of colistin sulphate, bicarinalin and BP100 on A. baumannii 19606 were assessed using microbiological assays and the consequences of peptide action was visualised on single cells of A. baumannii using atomic force microscopy (AFM). The changes in the roughness and area of the cell surface at increasing peptide concentrations were determined to quantify the effect of the three different peptides. The effect of the peptides on biofilm formation was investigated by determining the minimum biofilm inhibition concentration (MBIC) and on preformed biofilms using the biofilm removal assay. Peptide action on the biofilms was visualised by scanning III electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The nucleic acid stain SYTO9 was used with CLSM to visualise and quantify biofilm bio-volume reduction at increasing concentrations of peptide. Bactericidal results showed concentrations of 0.5 μg/ml for colistin sulphate and 4 μg/ml for both bicarinalin and BP100. Biofilm removal assay results highlighted that at higher concentrations, both bicarinalin and BP100 had significantly greater biofilm removal potential than colistin sulphate. At peptide concentrations of 1024 μg/ml, biofilm reduction was only circa 50% for colistin sulphate but 65% and 83% for the AMPs bicarinalin and BP100 respectively. At 2048 μg/ml, the highest concentration analysed, BP100 eradicated 95% of the biofilm, nearly twice that of colistin. AFM illustrated dramatic changes in cell size and membrane conformity when treated with peptides of concentrations at and above the MBC. SEM images showed the decrease in biofilm presence and cellular changes as peptide concentration increased. CLSM images and bio-volume quantification, with increasing peptide concentrations, further reinforced the evidence that bicarinalin and BP100 were more effective than colistin against A. baumannii biofilms at high concentrations. At a concentration of 1000 μg/ml, bicarinalin and BP100 removed approximately 50-55% of the biofilms, twice as much as colistin, a trend consistent with the biofilm removal assay results. The results demonstrated the potential for AMPs as therapeutic alternatives to the currently used treatment against A. baumannii infections. Compared to colistin sulphate, bicarinalin and BP100 had generally better removal potential against A. baumannii biofilms. |
