|Keywords (Extracted from title, table of contents and abstract of thesis)
bacteriocins, staphylococci, antagonistic substances, bacterial interactions, grampositive organisms, staphylococcus spp, staphylococcal isolates, staphylococcus aureus, gramnegative bacteria, streptococcus pyogenes
Production of antagonistic substances is highly important factor in microbial ecology. Among the many different substances known to play a role in bacterial interactions, bacteriocins are the most specific and efficient antagonists. Bacteriocins, the bacterial peptides and protein antibiotics are ribosomally made or derived from gene-encoded precursor peptides and towards which the producing strain possesses a specific self-protection mechanism i.e. immunity. Such bacteriocins have been found in all bacterial species in which they have been looked for, it has been assumed they will be found to occur in all bacteria, provided enough indicator strains are tested and growth conditions resemble those found in vivo, i.e. not in pure culture, not on rich media and not at optimum growth conditions. In a classical sense, although bacteriocin activity is considered to be species specific, bacteriocins produced by Grampositive organisms have proved to have a greater spectra of activity and thus possibly have a broader application.
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. The inhibitory substances produced by these isolates were in agreement with the criteria that would allow us to define them as bacteriocins i.e. a narrow spectrum of activity centered around its species, an essential protein moiety, a bactericidal mode of action, attachment to cell receptors on susceptible cells, and plasmid-encoded function.
Initial characterization (both physical and chemical) was taken into account for six staphylococcal isolates i.e. ABl50, ABl77, AB188, AB20l, AB202 and AB308. Staphylococcus aureus AB20 1 was selected for detailed study. The bacteriocin-like inhibitory substance from Staphylococcus aureus AB201 was designated as Bac201. It was inhibitory to both Grampositive and Gramnegative bacteria. Among Gramnegative bacteria it was active against Neisseria meningitidis and Acinetobacter calcoaceticus (both being Gramnegative cocci). Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. The bacteriocin could not be induced or eluted. The bacteriocin was partially purified by ammonium sulfate precipitation. Proteolytic enzymes rapidly inactivated the antagonistic activity of the partially purified material, whereas glycolytic and lipolytic enzymes had no effect. It remained stable in the presence of organic solvents. It could be stored at -20°C without loss of activity, is stable at 60°C and 80°C for 30 min, l00°C for 20 min wide autoclaving temperature (121 °C for 15min), and exhibited activity within a wide range of pH (2.5-10). AB20 had an estimated Mr of 41kDa, as indicated by activity detection after SDS-PAGE. Temperature-mediated (44°C) plasmid curing studies suggested linkage of bacteriocin production to a 4.8-MDa plasmid. The AB201 was bactericidal rather than bacteriolytic. AB20 1 was purified to homogeneity by ammonium sulfate precipitation, gel PLC on BioSil-SEC-125, and by reverse phase HPLC on Vydac C4. The purification of AB201 resulted in 461-fold increase in specific activity. The purified AB201 migrated as single band on SOS-PAGE. The molecular weight was estimated to be 41kDa on SOS-PAGE. Purified AB201 was sensitive to proteolytic enzymes, resistant to heat and organic solvents, and active over a wide range of pH. The purified AB201 was glycine rich Protein. The amino acid composition showed predominance of glycine (39.2%), proline(12.9) and alanine (7.8%) residues. The electron microscopic studies revealed numerous more or less circular bodies averaging from 12-30nm in diameter. Purified AB20 1 binds to the cell surface of Staphylococcus aureus AB211, its resistant mutant and several other sensitive and resistant Grampositive bacteria, and to Neisseria meningitidis and Acinetobacter calcoaceticus. The bactericidal effect on sensitive cells was not produced by either cell lysis or apparent loss of membrane permeability. The antagonistic activity of Bac201 was increased when combined with Bac308 from Staphylococcus aureus AB308, however, no notable increase was observed when combined with Sam53, a bacteriocin from Streptococcus pyogenes Sam53.