An investigation into some antibiotics produced by Pseudomonas antimicrobica
thesisposted on 19.09.2012, 13:45 by Ernest Attafuah
Two strains (NCm 9897 and 9898; strains A and B respectively) of a Pseudomonas· species have been shown to display antifungal and antibacterial activity on solid media. Biochemical tests indicate that the organisms may be two distinct strains of a new species. Cell morphology was studied using scanning electron microscopy. Chemically defined media, established for the organisms, indicate non-fastidous characteristics. Four liquid media, able to elicit antibiotic production from Strain A have been developed: a chemically defined medium (antibacterial), a chemically defined medium and a complex medium (antifungal) and a chemically defined medium (antibacterial and antifungal). Nitrogen and magnesium limitation significantly increased yields. Magnesium content in a medium (without a magnesium salt component) and in whole cell samples grown in the said medium were assessed using atomic absorption spectroscopy and elemental analysis respectively. Optimization experiments for antibacterial and antifungal activity, assessed by a disc diffusion assay, increased yields, in 250 ml conical flasks by a factor of X9 and 109% respectively. A 6 litre laboratory-scale fermentor was used for larger batch cultivations . Procedures for extraction of the active compounds from the biological matrices were . developed leading to the isolation of one antibacterial compound, ABl (yellow crystalline) and three antifungal compounds, AFl, AF2 and AF3 (pale yellow and amorphous). Structure determination of ABl, involving mass spectrometry, IR/UV spectroscopy, lH-NMR and x-ray diffraction, indicated it to be 1.6 dimethyl pyrimido[5,4-e ]-1,2,4- triazine-5,7(IH,6H)-dione (Xanthothricin; Toxoflavin), a toxic metabolite previously detected in foods contaminated with Ps. cocovenenans. Selective media, developed ' for Strain A and Strain B, did not support growth of Ps. cocovenenans. Preliminary structural analysis suggests that AFl may possess a mono-substituted ring system with CHz chain and a terminal hydroxyl group; that AF2 may belong to the polyene group of antifungal antibiotics and that AF3 may be an aliphatic ketone with hydroxyl group . Agar diffusion, minimum inhibitory concentration, assays for the compounds, indicate activity to be in the ~gml range for sensitive microorganisms. Antibiotic challenge against test microorganisms suggest bacteriostatic activity for ABl, fungistatic activity for AFl and AF3 and fungicidal activity for AF2.