Evaluation of some pseudomonas species isolated from Hogsback forest reserve for the production of antibacterial compounds

Pseudomonas species are Gram-negative bacteria most abundant in soil and water bodies, with the capacity to thrive in varied environments. They are largely associated with resistant pathogenic bacteria linked to human and plant diseases. Species such as Pseudomonas aeruginosa have been particularly targeted as case studies due to the extremity to which they pose a threat to human health. With more focus directed at using these species for biocontrol and bioremediation purposes, their role in bioactive compound production may be equally important. As the crisis on antimicrobial resistance still persists, the need for effective antimicrobial compounds is ever more urgent and solutions may possibly still be dormant in bacterial species whose potential has not been fully investigated. On a bid to source out potential antimicrobial compound producers, soil samples were collected from Hogback forest reserve in the province of the Eastern Cape, South Africa. For bacterial screening, M1 and R2A agar were used and the cultures grown at 37˚C for a period of seven days. After the presumed Pseudomonas species were identified, antimicrobial production was determined by submerged fermentation method using nutrient broth as media of choice. Active isolates were further studied to determine the optimum conditions which best facilitate for antimicrobial compound production, with parameters such as temperature (25˚C – 40˚C) and pH (4 – 9) considered. The role plasmids play in antimicrobial compound production was also investigated. Each isolate was grown in fermentation media containing Sodium dodecyl sulphate and Ethedium Bromide, at varying concentrations, to facilitate for plasmid curing. With each sample, distinct colonies were identified with varying pigmentations most dominant being a cream colour. The identity of the isolated strains was achieved through sequencing of 16S rDNA. Phylogenetic analysis showed that isolate A16 had 80 percent homology with Pseudomonas plecoglossicida strain P4 and share a close ancestor with isolates Y52 and Y81, also isolate Y89 showed a 90 percent homology with Pseudomonas sp. Co-11a. With the exception of isolate A16, the isolates which were active against Gram-negative bacteria lost activity as the screening processes continued. When looking at temperature variations, isolates Y81 and A16 were highly active with maximum activity observed at 35˚C while Y89 performed best at 25˚C and Y52 showed constant activity across all studied temperatures. The plasmids in all isolates were found to be 48.5 kb in size with the exception of isolate Y89 which was 20 kb. The plasmids were cured at concentrations of (1 mg/ml; 5 mg/ml; 7 mg/ml; 10 mg/ml; 11 mg/ml) SDS and (125 μg/ml; 6.5 μg/ml; 5μg/ml) EtBr. The curing process also showed changes in both the antimicrobial activity of the isolates as well as their physical characteristics. The isolates are the first reported Pseudomonas species from Hogsback forest reserve with the ability to produce antimicrobial compounds which are active against Gram-positive and Gram-negative bacteria. These mesophilic bacteria also show that plasmids do not pay any role in the production of antimicrobial compounds and that the biosynthesis genes are highly likely to be chromosomal borne meaning that the production cannot be linked to horizontal transfer of genes. Therefore, these isolated Pseudomonas species provide a potential reservoir of antimicrobial compounds which may play an important role in the antimicrobial resistance phenomenon