Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91

Staphylococcus aureus strain CH-91, isolated from a broiler chicken with atopic dermatitis, has a highly proteolytic phenotype that is correlated with the disease. We describe the isolation and biochemical and molecular characterization of the AI-type lantibiotic BacCH91 from S. aureus CH-91 culture medium. The bacteriocin was purified using a three-stage procedure comprising precipitation with ammonium sulfate, extraction with organic solvents, and reversed-phase HPLC. The BacCH91 peptide is thermostable and highly resistant to cleavage by both prokaryotic and eukaryotic peptidases. The MIC for the Gram-positive bacteria ranged from 2.5 nM for Microococcus luteus through 1.3–6.0 μM for staphylococcal strains up to more than 100 μM for Lactococcus lactis. BacCH91 was ineffective against the Gram-negative strains tested at the maximal concentration (100 μM). The amino acid sequence of BacCH91 is similar to that of epidermin and gallidermin. The encoding gene (bacCH91) occurred in two allelic variants distinguishable in the restriction fragment length polymorphism assay. Variant I, identified in S. aureus CH-91, dominated in S. aureus strains of poultry origin, although strains with variant II were also identified in this group. S. aureus strains of human origin were characterized exclusively by variant II

Biological and physicochemical characterization of a bacteriocin produced by Staphylococcus aureus CH91

Bacteriocins are ribosomally–synthesized peptides or proteins produced by different groups of bacteria to inhibit the growth of similar or closely related bacterial strains. Bacteriocins are categorized in several ways, including their structure, source of origin and mechanism of action.The aim of this work was physicochemical and biological characterization of the bacteriocin produced by Staphylococcus aureus CH91. The isolation procedure comprised the following steps: ammonium sulphate precipitation, extraction with organic solvents and separation on reverse-phase high performance liquid chromatography column.Determination of the minimal inhibition concentration assay showed that purified bacteriocin is the most active toward the strain Sarcina lutea ATCC 9341. Bacteriocin was also assessed for its sensitivity to various hydrolytic enzymes, pH and heat treatment.Bakteriocyny są syntetyzowanymi na rybosomach peptydami lub białkami produkowanymi przez liczne grupy bakterii w celu hamowania wzrostu innych blisko spokrewnionych szczepów. Bakteriocyny zostały podzielone według różnych kryteriów, biorąc pod uwagę ich strukturę, gatunek produkujący czy mechanizm działania. Celem niniejszej pracy była biologiczna i fizykochemiczna charakterystyka bakteriocyny produkowanej przez szczep Staphylococcus aureus CH91. Procedura izolacji bakteriocyny składała się z następujących etapów: wysalanie siarczanem(VI) amonu, ekstrakcja z użyciem rozpuszczalników organicznych oraz rozdział z użyciem wysokosprawnej chromatografii cieczowej w układzie odwróconych faz.Biologiczna charakterystyka badanej bakteriocyny obejmowała oznaczenia dawki MIC (ang. minimal inhibition concentration). Testy te wykazały, że szczepem najbardziej wrażliwym na działanie bakteroriocynę jest Sarcina lutea ATCC 9341. Badano także wrażliwość bakteriocyny na rożne enzymy hydrolityczne, pH oraz temperaturę