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Aims: To understand the bactericidal action of enterocin LD3 against Gramnegative bacteria. Methods and Results: Minimum inhibitory concentration (MIC) of enterocin LD3 against Micrococcus luteus MTCC 106 and Escherichia coli NCDC 135 was 80 and 112 lg ml À1 , and minimum bactericidal concentration (MBC) was 128 and 180 lg ml À1 , respectively. The efflux of potassium ion (K + ) was 14 and 13 ppm and electrical conductivity 10Á5 and 8Á3 mS cm À1 in cell-free supernatant of MIC-treated cells of M. luteus and E. coli respectively. The increased absorbance (OD 260/280 ) 0Á422/0Á260 and 0Á110/0Á075 in the bacteriocin-treated cells of M. luteus MTCC 106 and E. coli, NCDC 135, respectively, suggested the release of nucleic acids and proteins. The higher infrared absorbance at 1451Á82 and~1094Á30 cm À1 further suggested its interaction with cell membrane and nucleic acids of the target bacteria. The interaction of bacteriocin with nucleic acids was also confirmed using gel retardation assay. Transmission electron microscopy of the bacteriocin-treated cells revealed disruption of cell membrane and leakage of cytoplasmic contents. Conclusions: Enterocin LD3 demonstrates bactericidal activity against Gramnegative bacteria interacting with cell membrane and nucleic acids. Significance and Impact of the Study: The study discloses the possible mechanism of action of enterocin LD3 against Gram-negative bacteria which is a rare phenomenon.

Bacteriocins of Enterococcus hirae LD3 and Lactobacillus plantarum LD4 have been applied in milk for growth inhibition of Staphylococcus aureus. The enumeration of S. aureus cells in nutrient broth and milk was found log 10 9.7 and 10.2 CFU/ mL, respectively, whereas it was reduced with increasing concentration of bacteriocins suggesting loss of cell viability. The lethal concentration (LC 50 ) of enterocin LD3 and plantaricin LD4 against S. aureus was 160 and 220 µg/mL, respectively. Bacteriocin-treated cells were stained red with propidium iodide (PI) indicating dead cells further confirms bactericidal nature. The enterocin LD3-treated cells showed higher infrared absorbance at 1451.82 cm − 1 corresponding to phospholipids suggesting membrane-acting nature of the bacteriocin. However, plantaricin LD4-treated cells did not show such alterations suggesting different mode of action. Both bacteriocins caused disruption and shrinkage of target cells, and leakage of intracellular contents as observed in transmission electron microscope (TEM). The present study suggests killing of S. aureus in milk, therefore, enterocin LD3 and plantaricin LD4 may be applied in biopreservation of milk and related food products.

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.

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