Isolation of novel antibiotic producing microorganisms from invertebrates

Because of the increased incidence of antibiotic resistance there is an increasing need to isolate new microorganisms producing new antibiotics. Because conventional sources such as soil have been exhausted, attention has been turned to unexplored sources. The aims of this research were to investigate terrestrial and aquatic invertebrates as a source of antibiotic producing microorganisms, to isolate the organisms and screen them for activity against Gram-positive and Gram-negative bacteria. Those with activity would be further characterised. Forty-seven antibiotic producers were isolated from 35 samples of different terrestrial and aquatic invertebrates using media designed to isolate streptomycetes. Identification by PCR and sequencing of 16S and 18S rRNA showed that the majority were in fact fungi, mainly Penicillium spp. and only 5 were Streptomyces spp. Three strains isolated from Helix aspersa (ES1), Lumbriculus variegate (La1a) and Baetis larvae (M1 11) that had strong activity against Gram-positive and Gram-negative bacteria including Methicillin-resistant Staphylococcus aureus (MRSA) were chosen for further study. PCR and sequencing of rRNA and Internal Transcribed Spacer (ITS) regions confirmed that the strains were closely related and were probably strains of P. chrysogenum. The organisms were grown in liquid media and on agar and the active components were extracted with ethyl acetate and fractionated by HPLC. As facilities for large scale liquid cultures were not available large scale preparation was performed from agar medium and the active fractions. HPLC showed that the extracts were complex and contained more than one active component. The purified fractions were chemically characterised by NMR, FTIR and MS. The results showed that the active components were novel flavones and isoflavones. Such compounds have extracted from plants and only previously been shown to be produced by endophytic fungi. The minimal inhibitory concentrations (MIC) against test bacteria and inhibitory concentration IC50 (IC50 is the concentration of a compound at which 50 % of human cancer cell tissue cultures are killed) showed potential for use in chemotherapy as all purified fractions except Es1 F3/3 and Es1 F6 were found to have IC50 values>100 μg/ml, High IC50 and low MIC (0.39 to 50 μg/ml) may indicate fractions suitable for testing for treatment of human infections. The results showed that the invertebrates were indeed a source of antibiotic producing organisms. Whether the organisms were acquired by the invertebrates from the environment or whether they were in a symbiotic association needs further study