Bactofencin A Displays a Delayed Killing Effect on a Clinical Strain of Staphylococcus aureus Which Is Greatly Accelerated in the Presence of Nisin

<b>Background/objectives:</b> Bacteriocins can be considered a novel source of natural alternatives to antibiotics or chemical food additives with the potential to fight against clinical and food pathogens. A number have already been commercialised as food preservatives, but they also have the potential to treat drug-resistant clinical pathogens and can play a role in immune modulation. To achieve their full potential, an understanding of their mode of action is required. <b>Methods:</b> Bactofencin A and nisin A were purified to homogeneity by reversed-phase HPLC and their effect on the mastitis pathogen <i>Staphylococcus aureus</i> DPC5246 was assessed by cell viability assays and flow cytometry. <b>Results:</b> We report that bactofencin A displays a delayed inhibitory effect against the mastitis pathogen, <i>Staphylococcus aureus</i> DPC5246, suggesting an unusual mode of action. This characteristic was clearly visible on BHI plate media, where formation of inhibition zones against the staphylococcal strain took 23 h compared to 6 h for the well-characterised nisin. This delayed killing and injury was also demonstrated using flow cytometry, where damage was evident 4 h after bacteriocin addition. Treatment with 2 μM bactofencin A resulted in approximately 20-fold higher numbers of injured and 50-fold higher numbers of dead cells when compared to untreated cells. Combining bactofencin A with the lantibiotic nisin A resulted in faster killing at lower bacteriocin concentrations. When combined in an equal ratio, the combination exhibited a 4-fold increase in inhibition compared to nisin A alone. These results demonstrate that the combination may be very effective in therapeutic applications against pathogenic staphylococci.