Team:REC-CHENNAI/Characterization-Antimicrobial
ANTIMICROBIAL ACTIVITY
Minimum Inhibitory Concentration (MIC) of Latarcin is defined as the lowest concentration of the peptide that cause 100% inhibition of cell growth. In this assay, we determine the MIC of Latarcin-2a native (Wt) and Latarcin-2a F10K (Mt) in bacterial strains E.coli DH5α, Bacillus subtilis and yeast Saccharomyces cerevisiae.
Bacterial strains (Bacillus subtilis and Escherichia coli DH5α) were cultured in low salt LB broth, whereas yeast cells (Saccharomyces cerevisiae) were cultured in Nutrient Broth. Prior to MIC assay, growth kinetics for all the three strains was determined from which the mid-log phase was estimated as 6 hours from the addition of inoculum (Fig 1). Minimal Inhibitory Concentration (MIC) for native (Wt) and mutant (Mt) Latarcin-2a (Ltc-2a) was determined by adding the peptides to the media after 6 hours (to the mid log phase culture). The inhibition of cell growth was determined by measuring the absorbance at 630nm for every 30 minutes interval. The experiment was carried out in triplicate.
It has been observed that a higher concentration of the peptide inhibits the growth of the organisms immediately after the addition of the peptide whereas a lower concentration of the peptide takes comparatively more time to achieve 100% inhibition of cell growth.
Both Ltc-2a Wt and Ltc-2a Mt were found to be potential inhibitors of E. coli DH5α whereas B. subtilis showed some resistance to both the native and the mutant peptides. For S. cerevisiae, Ltc-2a Wt was a better inhibitor of growth when compared to Ltc-2a Mt (Fig 2).
Ltc-2a Mt efficiently inhibited the growth of E. coli but both Ltc-2a Wt and Ltc-2a Mt peptides showed lesser activity on B. subtilis (Fig 3).
The MIC values of Ltc-2a Wt and Ltc-2a Mt in the three microbial strains are given in Table 1.
TABLE 1: MIC of native and mutant latarcin-2a for E.coli, B.subtilis and S.cerevisiae.
FIGURE 1: Growth kinetics of E.coli, B.subtilis and S.cerevisiae.
The growth curve of the three strains were determined by measuring absorbance at 630nm for every 30 minutes interval. The plot represents the mean of the replicates (n=3) ± s.e.m. (For data clearance, the error bars are ignored).
A. Ltc-2a Wt B. Ltc-2a Mt
A. Ltc-2a Wt B. Ltc-2a Mt
A. Ltc-2a Wt B. Ltc-2a Mt
LEGEND
FIGURE 2: The growth inhibitory pattern of E. coli, B. subtilis and S. cerevisiae after the addition of Ltc-2a Wt (A) and Ltc-2a Mt (B) peptide. The plot represents the mean of the replicates (n=3) ± s.e.m. (For data clearance, the error bars are ignored)
Figure 3: Comparison of activity between Ltc-2a Wt and Ltc-2a Mt in E. coli, B. subtilis and S. cerevisiae.
To conclude, both Ltc-2a Wt and Ltc-2a Mt inhibited the growth of E. coli and S. cerevisiae but B. subtilis showed resistance towards both the peptides.
