Table 1.
MIC and MBC values of melimine and Mel4 against S. aureus.
Fig 1.
Growth of S. aureus in the presence and absence of LTA.
The antibacterial effect of melimine and Mel4 was significantly reduced by addition of LTA compared to peptides alone after 6 h of incubation. The LTA alone had higher effect than LTA with any peptides at 6 h. Mel4-LTA increased OD600 nm more than melimine-LTA at 8 h. Error bars represents the means (±SD) of three independent experiments performed in triplicate. * represent p≤0.001, ** p≤0.0001 and # p = 0.005.
Fig 2.
Cell membrane depolarization of S. aureus 38 as assessed with the release of membrane potential-sensitive dye DiSC3-(5) during treatment with peptides, measured spectroscopically at 622nm to 670nm excitation and emission wavelengths, and corresponding bacterial death as determined by plate counts. Each symbol represents data curve for increase in fluorescence (on the left y axis) and for reduction in bacterial count CFU/ml (on the right y axis). Data are presented as means (±SD) of three independent repeats in triplicate. DMSO (20%) was used as a positive control and buffer used as a negative control.
Fig 3.
Cell membrane permeabilization.
Cell membrane permeability of S. aureus 38 was assessed with as the increase in fluorescence intensity due to interaction of Sytox green dye with DNA (measured spectroscopically at 480nm excitation and 522nm emission wavelengths) at their MICs and MBCs of melimine and Mel4. Data are presented as means (±SD) of three independent repeats in triplicate. Triton-X 100 (1%) was used as a positive control and buffer only as a negative control.
Fig 4.
Cell membrane permeabilization of S. aureus 38 as determined with Syto-9 (membrane permeable) and Propidium Iodide (membrane impermeable stains) dyes using Flow cytometry.
Fig 5.
The leakage of cellular ATP (in percentage) from S. aureus 38 after treatment with MIC or MBC of each peptide shown at X axis. The corresponding change in the number of live cells (CFU/ml) estimated through viable count displayed at Y axis. Each symbol represents data curve for increase in fluorescence (on the left y axis) and for reduction in bacterial count CFU/ml (on the right y axis). Errors bars represent the means (±SD) of three independent experiments in triplicate compared with buffer-treated control.
Fig 6.
The release of UV absorbing materials (DNA/RNA) from S. aureus 38 determined spectroscopically at OD260nm.after incubation with MIC or MBC of melimine and Mel4. Data are presented as means (±SD) of three independent repeats in triplicate compared with buffer-treated control.
Fig 7.
Bacterial lysis of S. aureus 38 was determined spectroscopically as decrease in OD620nm after incubation with MIC and MBC of melimine and Mel4. Data are shown as means (±SD) of three independent repeats in triplicate compared with buffer-treated control. * represent p<0.001 and ** p<0.0001.
Fig 8.
Time kill kinetics of melimine and Mel4 against S. aureus 38. Aliquotes analyzed at different time intervals for viable count showed that peptides decrease the bacterial viability (log10 CFU/ml) with different rates compared to buffer treated (negative control).
Fig 9.
Detection of autolysins release.
(A) Release of autolysins was determined by measuring decrease in OD570nm of PGN suspensions following treatment with cell-free supernatants of melimine and Mel4 treated S. aureus 38 (S38). Cell-free supernatants from Mel4 treated S. aureus resulted in a greater degradation of PGN than those from melimine or buffer treated S. aureus. Data presented as means (±SD) of three independent repeats in triplicate. * represent p≤0.004 compared to melimine treated bacteria and ** p<0.001 compared to buffer control (B) Release of autolysins was also monitored by determining the zone of inhibition (arrow) of cell-free supernatants against Micrococcus lysodeikticus ATCC 4698 after (I) melimine and (II) Mel4 treatment. 1A = supernatant from melimine treated S. aureus 38, IIA supernatant from Mel4 treated S. aureus, IB and IIB zones produced by lysozyme (5 mg/ml), IC no zone produced when melimine was incubated with buffer alone, IIC Mel4 incubated with buffer alone, ID and IID S. aureus incubated without addition of melimine or Mel4.
Fig 10.
Hemolysis of horse erythrocytes.
Hemolysis was determined as increase in OD540nm after incubation with increasing concentration of the melimine and Mel4. Errors bars represents the means (±SD) of three independent experiments performed at three separate occasion in triplicate.
Fig 11.
Timeline of melimine or Mel4 interacting with S. aureus.
Both AMPs induced cell membrane depolarization after 30 seconds exposure to the staphylococci followed by release of ATP after 2 minutes. Cell membrane permeabilization occurred with melimine only after 10 minutes with near simultaneous release of DNA/RNA. Mel4 caused transient cell membrane after 30 minutes with no release of DNA/RNA. Mel4 showed autolysins induced killing after 5 hours. Complete bacterial lysis started after 6.5 hours of incubation with both the peptides.