An Antimicrobial Metabolite from Bacillus sp.: Significant Activity Against Pathogenic Bacteria Including Multidrug-Resistant Clinical Strains

Abstract

In this study, the cell free modified tryptone soya broth (pH 7.4 ± 0.2) of Bacillus subtilis URID 12.1 showed significant antimicrobial activity against multidrug-resistant strains of Staphylococcus aureus, S. epidermidis, Streptococcus pyogenes and Enterococcus faecalis. The partially purified antimicrobial molecule was found to be resistant to extremes of pH and temperatures and also to higher concentrations of trypsin and proteinase K. The antimicrobial molecule was purified by a three-step method that included reversed-phase high performance liquid chromatography (RP-HPLC). The minimum inhibitory concentration (MIC) values were determined for 14 species of bacteria using a microbroth dilution technique. The HPLC-purified fraction showed the MICs ranging from 0.5 to 16 μg/ml for methicillin and vancomycin-resistant Staphylococcus aureus (MVRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) strains. The molecular mass of the antimicrobial compound was determined to be 842.37 Da. The same antimicrobial fraction showed negligible haemolytic activity against human red blood cells even at a concentration as high as 100 μg/ml. Because of its significant antimicrobial activity at low MIC values coupled with its non-haemolytic property, it may prove to be a novel antimicrobial lead molecule.

Keywords: Bacillus sp.; Staphylococcus aureus; minimum inhibitory concentration; multidrug resistant-bacteria; non-haemolytic property; promising antimicrobial agent.

Figure 1

Phylogenetic tree showing the similarity of URID 12.1 among other Bacillus species. Pseudomonas mendocina was used as an outgroup.

Figure 2

Growth and production kinetics of URID 12.1; the antimicrobial compound production is expressed as AU/mL.

Figure 3

Effect of pH (3.0, 6.0, 8.0, and 12.0) and temperatures (100 and 121°C) on the antimicrobial activity. (A,B) Effect on antimicrobial activity after boiling for 30 min at different pH values tested against methicillin resistant S. epidermidis ATCC 12228 (MRSE) and S. aureus MTCC 737, respectively. (C,D) Effect on antimicrobial activity after autoclaving at different pH values against MRSE and S. aureus MTCC 737, respectively.

Figure 4

(A) SDS-PAGE with molecular marker (lane M) and sample (lane S). (B) Antimicrobial activity shown as zone of inhibition after SDS-PAGE and zymogram. (C) Antimicrobial activity shown by Native PAGE and zymogram.

Figure 5

(A) Bioautography assay showing zone of inhibition, against S. aureus MTCC 737. (B) Bioautography assay (using the TTC dye) showing antimicrobial activity against MVRSA 4 showing an Rf value 0.80.

Figure 6

(A) Reversed Phase HPLC chromatogram wherein the antimicrobial compound had a retention time of 30.5 min (encircled peak). (B) MALDI-TOF analysis showing the molecular mass as 842.37 Da.

Figure 7

Haemolytic activity of antimicrobial compound. One-hundred microgram per milliliter of the HPLC-purified antimicrobial fraction showed merely 4.69% hemolysis to cause any adverse haemolytic effects.