Antimicrobial Activity of a Bacteriocin Produced by Enterococcus faecalis KT11 against Some Pathogens and Antibiotic-Resistant Bacteria

Abstract

In this study, the antimicrobial activity of a bacteriocin produced by Enterococcus faecalis KT11, isolated from traditional Kargı Tulum cheese, was determined, and bacteriocin KT11 was partially characterized. The results showed that bacteriocin KT11 was antagonistically effective against various Gram-positive and Gram-negative test bacteria, including vancomycin- and/or methicillin-resistant bacteria. The activity of bacteriocin KT11 was completely abolished after treatment with proteolytic enzymes (proteinase K, α-chymotrypsin, protease and trypsin), which demonstrates the proteinaceous nature of this bacteriocin. Additionally, bacteriocin KT11 remained stable at pH values ranging from 2 to 11 and after autoclaving at 121℃ for 30 min. In addition, the activity of bacteriocin KT11 was stable after treatment with several surfactants (EDTA, SDS, Triton X-100, Tween 80 and urea) and organic solvents (chloroform, propanol, methanol, ethyl alcohol, acetone, hexane and ethyl ether). Cell-free supernatant of E. faecalis KT11 was subjected to ammonium sulfate precipitation and then desalted by using a 3.5-kDa cut-off dialysis membrane. The bacteriocin activity was determined to be 711 AU/mL in the dialysate. After tricine-SDS-PAGE analysis, one peptide band, which had a molecular weight of ~3.5 kDa, exhibited antimicrobial activity. Because the bacteriocin KT11, isolated from E. faecalis KT11, exhibits a broad antimicrobial spectrum, heat stability and stability over a wide pH range, this bacteriocin can be used as a potential bio-preservative in foods. Additionally, bacteriocin KT11 alone or in combination with conventional antibiotics may provide a therapeutic option for the treatment of multidrug-resistant clinical pathogens after further in vivo studies.

Keywords: Enterococcus faecalis; Kargı; Tulum cheese; antimicrobial peptides; bacteriocin.

Fig. 1. Effect of heat treatments on bacteriocin KT11 stability.

Heat resistance of bacteriocin KT11 was determined by heating the bacteriocin KT11 samples to 60℃, 80℃, 100℃, and 121℃. Untreated bacteriocin KT11 samples were used as a control. Residual antimicrobial activity was determined by AWDA and S. aureus ATCC 25923 was used as indicator bacteria. Then percentage of activity was calculated (% activity=inhibition zone of the treated sample/inhibition zone of the control sample×100).

Fig. 2. Stability of bacteriocin KT11 at different pH levels.

The pH of the bacteriocin KT11 samples was adjusted and the samples were maintained at 4℃ for 0, 6, 12, 18 or 24 h. Untreated bacteriocin KT11 was used as a control. Their residual antimicrobial activities were determined by AWDA and S. aureus ATCC 25923 was used as indicator bacteria.

Fig. 3. Effect of organic solvents on bacteriocin stability.

The effect of various organic solvents on bacteriocin KT11 activity was determined by adding chloroform, propanol, methanol, ethyl alcohol, acetone, hexane or ethyl ether to the bacteriocin KT11 samples. Untreated bacteriocin KT11 was used as a control. Their residual antimicrobial activities were determined by AWDA and S. aureus ATCC 25923 was used as indicator bacteria.

Fig. 4. Production of bacteriocin KT11 during growth of E. faecalis KT11 in MRS broth and activity of bacteriocin KT11.

Growth of E. faecalis KT11 was determined spectrofotometrically (OD 600 nm). While line in the graph indicates the optic density (OD) of E. faecalis KT11 culture, bars indicate activity (AU/mL) of bacteriocin KT11. Antimicrobial activity was determined by AWDA for each sampling time, S. aureus ATCC 25923 was used as indicator bacteria.

Fig. 5. Antimicrobial activity of lyophilized bacteriocin KT11 stored at different temperatures.

Lyophilized bacteriocin KT11 samples were stored at 4℃, –20℃, and –80℃ for 3 months. During storage, at timepoints of 1, 15, 30, 60, and 90 d, samples were taken, resuspended in sterile deionized water and their residual antimicrobial activities were determined by AWDA, S. aureus ATCC 25923 was used as indicator bacteria.

Fig. 6. Tricine-SDS-PAGE analysis and direct antimicrobial activity assay of the partially purified bacteriocin KT11 (Lane M; marker, Lane 1; partially purified bacteriocin KT11, Lane 2; gel overlaid with soft nutrient agar seeded with S. aureus ATCC 25923, 10⁶ CFU/mL).