Purification, Characterization and Applied Studies of Protein Antibiotics from Indigenous Staphylococci

Abstract

Production of antagonistic substances is highly important factor in microbial ecology. Among many different substances known to play role in bacterial interactions, bacteriocins are the most specific and efficient antagonists. Bacteriocins or bacteriocin like inhibitory substances produced by staphylococcus spp were studied in more than 350 clinical staphylococcal isolates. 7% of the isolates exhibited bacteriocin like antagonistic activity. Initial characterization (both physical and chemical) was taken into account for six staphylococcal isolates i.e., AB 150, AB 177, AB 188, AB201, AB202 and AB308. Staphylococcus aureus AB201 was selected for detailed study. The bacteriocin like inhibitory substance produced by Staphylococcus aureus AB201 was designated as staphylococci Bac201. It was inhibitory to both Gram-positive and Gram-negative bacteria. Among Gram-positive bacteria it was active against Neisseria meningitides and Acinetobacter calcaemic (both being Gram-negative cocci). It was partially purified to 80% saturation by ammonium sulphate precipitation, gel filtration and reversed phase high performance liquid chromatography (Vydac C4). The native Bac201 was sized at approximately 170-KDa as determined by GF HPLC. The purification of Bac201 resulted in 466-fold increase in specific activity and recovery of 0.94% of total antibacterial activity. The proteolytic enzymes rapidly inactivated the antagonistic activity of the partially purified material, whereas glycolytic and lipolytic enzymes had no effects. It remained stable in the presence of mild organic solvents. It could be stored at -20°C without loss of activity, stable at 60°C and 80°C for 30 min, 100°C for 20 minutes and autoclaving temperature (121 °C for 15 minutes), and exhibited activity within a wide range of pH (2.5-10). The amino acid composition revealed a general resemblance with other reported high molecular mass bacteriocins and predominance of glycine (39%), proline (13%) and alanine (8%) residue. Further results showed that Bac201 has bactericidal effects on sensitive cells which is not produced by either cell lysis or apparent loss of membrane permeability. The MIC of staphylococci Bac201 against all the dermatophytes except E. floccosum was in the range of 20µg/ml. Mycelial plugs taken from the zones of inhibition were found to revive their growth after re-incubation into fresh media. These results suggest that the staphylococci Bac201 was "fungistatic" in its inhibitory action. Staphylococci Bac201 was found to be effective against 15 out of 30 strains and the range of MIC was found to be 0.5 to 0.03 l mg/ml. The antigenicity profile studies of staphylococci Bac201 revealed that staphylococci Bac201 is antigenically (immunogenically) poor (with reference to rabbit immune system) and the antibody titre was also not found to be much significant as so precipitation was observed in Ouchterlony technique and ring precipitation test. Staphylococci Bac201 was tested for its mutagenic potential in the Ames Salmonella screening system and in Pseudomonas aeruginosa PA0286 system (met, trp). The studies revealed that staphylococci Bac201 has no tendency for reversion mutations on his (auxo trophy) and (prototrophy) marker in Salmonella typhimurium tester strains TA98 and TA 100 compared with their spontaneous reversions. Staphylococci Bac201, therefore, found not to possess genetic activity. The pharmacological studies of Staphylococci Bac201 does not show any toxic/side effects on experimental animals indicating that this product is safe and could be used as chemotherapeutic agent.