The efficacy of antimicrobials is an important aspect during their applications in food and therapeutics. In this study, combination of two bacteriocins, enterocin LD3 and plantaricin LD4, was studied against two pathogenic bacteria, Staphylococcus aureus subsp. aureus ATCC25923 and Salmonella enterica subsp. enterica serovar Typhimurium ATCC13311 for increasing their potency and bactericidal activity. The minimal inhibitory concentrations (MICs) of enterocin LD3 and plantaricin LD4 against Staph. aureus subsp. aureus ATCC25923 were 180 and 220 μg/mL, whereas in combination, reduced to 115 μg/mL, respectively. The MICs of enterocin LD3 and plantaricin LD4 against Salm. enterica subsp. enterica serovar Typhimurium ATCC13311 were 240 and 320 μg/mL, respectively, whereas in combination, these were found to be 130 μg/mL, respectively. The fractional inhibitory concentration (FIC) indices calculated as 0.50 against Staph. aureus subsp. aureus ATCC25923 and 0.43 against Salm. enterica subsp. enterica serovar Typhimurium ATCC13311 were found to be ≤ 0.5 indicating the synergy. The isobologram showed MIC of combined bacteriocins falls below the plotted straight line further signifies synergy. The growth response of Staph. aureus subsp. aureus ATCC25923 and Salm. enterica subsp. enterica serovar Typhimurium ATCC13311 was significantly reduced in the presence of combined bacteriocins in comparison with their individual effects. The number of dead cells was higher as a result of combined effect as compared with their independent effect evidenced by fluorescent microscopy. Transmission electron microscopy (TEM) revealed the higher disruption of cell membrane in the combined bacteriocin-treated cells as compared with alone effects. The FTIR spectra of enterocin LD3-treated cells showed alteration at ~ 1,451.82 and ~ 1,094.30/cm corresponding to nucleic acids and phospholipids suggesting its interaction with cell membrane and nucleic acids. In contrast, plantaricin LD4-treated cells did not show such alterations suggesting plantaricin LD4 may kill target cells using other mechanism. Our data suggest that different mode of action of both bacteriocins results in division of labour and may be responsible for their synergistic activity against target cells. Similarly, the synergistic effect of bacteriocins was also observed against other pathogenic bacteria such as Proteus mirabilis ATCC43071, Pseudomonas aeruginosa ATCC27853 and Escherichia coli ATCC25922. These bacteriocins, therefore, act synergistically against target pathogens and may be applied in appropriate combinations for food safety and medical applications.
Keywords: Enterocin LD3; Fluorescence microscopy; Plantaricin LD4; Salmonella Typhimurium; Staphylococcus aureus; Synergistic effect; Transmission electron microscope.