Effects of chlorophyll-derived efflux pump inhibitor pheophorbide a and pyropheophorbide a on erythromycin resistance of Staphylococcus aureus, Enterococcus faecalis, Salmonella Typhimurium and Escherichia coli

The purpose of this study was to validate the hypothesis that pheophorbide a and pyropheophorbide a reduce erythromycin resistance of reference strains of facultative anaerobic bacteria with multidrug or macrolide efflux pumps, as indicative of their effect on bacteria indigenous to anaerobic swine waste (feces and stored manure). Minimal inhibitory concentrations of erythromycin (MICs; µg erythromycin ml^-1) were determined for erythromycin-naïve and serial passage-selected mutant Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Salmonella enterica serovar Typhimurium ATCC 14028 and porcine Escherichia coli P286.10.99.C3 and P475.10.99 using CLSI-standard broth macrodilution assays with or without pheophorbide a or pyropheophorbide a (0.5 and 50 µg ml^-1) under static aerobic and anaerobic (100 % CO2; Coy Anaerobic chamber) conditions for up to seven days. The results showed that the greatest effects were obtained using pyropheophorbide a against S. aureus: MICs for naïve cultures were low (max. 1) and reduced twofold by 0.5 and 50 µg pyropheophorbide a ml^-1 under both aerobic and anaerobic conditions. Mutant cultures exhibited a 4096-fold increase of basal MICs, whereby 50 µg pyropheophorbide a ml^-1 caused an up to threefold reduction under anaerobic conditions. Effects on the other strains were not noticed (E. faecalis, E. coli P286.10.99.C3) or significant only on mutant cultures under anaerobic conditions (twofold MIC reduction for E. coli P475.10.99 and S. Typhimurium by 50 µg pyropheophorbide a ml^-1). In conclusion, this is the first study indicating that chlorophyll-derived pyropheophorbide a can reduce antibiotic resistance of bacteria in anaerobic habitats, such as food chain-relevant swine waste.