Abstract
Bovine mastitis (BM) is a serious disease burdening the global dairy industry. Staphylococcus aureus, a leading BM pathogen, poses challenges due to its various virulence mechanisms and ability to develop resistance towards conventional antibiotics. Biofilm and persister cell formation complicate S. aureus-induced BM. Biofilms are communities of bacteria encased in a protective matrix of extracellular polymeric substances. Persister cells are metabolically inactive, phenotypic variants of bacteria that display increased tolerance to antibacterial treatments. These factors promote resilience towards antibiotics and contribute to treatment failure and chronic infections in BM. My Honours project explored a novel strategy involving antibiotics combined with innovative peptoids. Peptoids are peptide analogues that mimic the structural elements and functionality of natural peptides. Unlike antibiotics, peptoids function through nonspecific mechanisms and bacteria are therefore less likely to develop resistance towards them. Additionally, they are able to target biofilms and persister cells.
My project assessed the antibacterial and antibiofilm activities of novel TM series peptoids, their synergistic potential with BM-relevant antibiotics and their ability to eradicate persister cells in vitro against S. aureus. Broth microdilution assays in standard laboratory media were used to determine minimum inhibitory concentrations (MICs). Antibiofilm activity was determined under the same experimental conditions. Synergy checkerboard assays established fractional inhibitory concentration indices (FICI) between lead peptoids and antibiotics. Time-kill curve experiments over 24 hours established the concentration-dependent antibacterial effects of gentamicin (1.89-18.9 μg/ml) and TM14 (3.13-31.3 μg/ml) on S. aureus, both independently and in combination. These were specifically used to target persister cells.
Six antibiotics and five peptoids displayed potent antibacterial activity with MIC values ranging from 0.63-12.5 μg/ml. Gentamicin exhibited the lowest MIC of 0.63 μg/ml. TM1 and TM14 with MICs of 3.13 μg/ml, demonstrated antibiofilm activity at concentrations ranging from 0.78-50 μg/ml, inhibiting ~50% and 30% of biofilm formation respectively. TM14 displayed synergy with ciprofloxacin and erythromycin indicated by FICIs of 0.38 and 0.5 respectively. Time-kill assays showed that combination treatments of gentamicin (at 6.3 μg/ml and 18.9 μg/ml) with TM14 (15.65 μg/ml) eliminated almost all cells by 24 hours, effectively targeting persister cells. To conclude, TM14 displayed potent antibacterial and antibiofilm activity, synergy with two conventional antibiotics and eliminated persister cells in the presence of gentamicin. Biofilms and persister cells impose huge challenges in treating S. aureus-induced BM. I have investigated a novel molecule that overcomes these issues where antibiotic therapy has failed and may have the potential to target S. aureus in BM.