Greenfield, Hayley (2018) Finding new potential antimicrobials in natural environments : do bacterial isolates from ancient and modern woodland, and farmland show the ability to produce inhibitory compounds? (MSc(R) thesis), Kingston University, .
Abstract
Research and development into the production of novel antimicrobials is greatly needed due to the current global threat that antimicrobial resistance is presenting. In the present study, modern and ancient forestlands, Friston Forest, (Sussex, UK), and New Forest, (Hampshire, UK), and Dawes Farm, (Warnham, West Sussex, UK) were sampled in the hope of isolating organisms with inhibitory activity and/or similar morphology to that of the Streptomyces spp. for further assessment of antagonistic activity against selected test bacteria. Perpendicular screening, and the newly developed starvation method, were used to characterise the inhibitory ability of organisms isolated against sensitive test strains of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Proteus mirabilis. Organisms displaying inhibitory activity towards sensitive organisms were further tested using clinically isolated test organisms from the same genus. Perpendicular screening confirmed 11 (15.9%), 5 (14.7%), and 13 (30.2%) of the total organisms isolated from Friston Forest, New Forest and Dawes Farm respectively, with inhibitory activity towards one or more of the test organisms used, with eight of these displaying further inhibition of at least one of the clinical test organisms used in this method. The starvation method, used for further antibacterial screening, involving the incubation of the potential antimicrobial producing organisms in minimal media for long incubation periods, confirmed 21 (30.4%) 12 (35.3%), and 9 (20.9%) isolates from Friston Forest, New Forest, and Dawes Farm vets with inhibitory ability. All organisms isolated were tested using Gram staining, with those displaying resemblance to the Streptomyces spp. selected for further determinative identification using the polymerase chain reaction, with primers specific to the 16S rRNA gene of the Streptomyces genus. Nineteen isolates were determined as belonging to this genus, with 42.1% of the confirmed Streptomyces spp. displaying inhibitory activities. The same soil samples were used for the cultivation of potentially pathogenic bacteria, with biochemical testing used to determine the identity of each of the 222 organisms isolated, to a genus level. Across the three locations three isolates were identified as belonging to Salmonella spp., 21 as Staphylococcus aureus, nine Pseudomonas spp., two Staphylococcus epidermidis, four Klebsiella spp. and four Escherichia coli. The screening carried out in this study confirmed the ability for the isolation of pathogenic organisms as well as soil organisms with antibiotic producing capability, from the previously untested farm and forestland sampled.
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