Strain improvement and characterization of antibiotic producing microorganisms from soil

Abstract

Since the discovery of Antibiotics in the 20th century, the idea of searching for antimicrobial compounds from natural sources came into existence. However natural products from microbial origin (especially soil microorganisms) have grasped a great devotion over the course of several decades. Recently, bacterial resistance have been observed against antibiotics of all classes, however it appears that the emergence of antimicrobial resistance is inevitable to almost every new drug.This necessitates carrying out studies that will generate new effective antibiotics. The present study is an attempt to identify, characterize and improve strains of bacteria for the ability to produce antibiotics. About 12 soil samples were screened for antibiotic-producing bacteria against 4 pathogenic microorganisms including Escherichia coli, Klebsiella pneumoniae Staphylococcus aureus and B. subtilis. After preliminary screening, active isolates with secondary metabolites showing activity were selected for secondary screening by agar well diffusion method to identify antibiotic potency. VITEK 2 system was used for rapid identification of the active isolates. The amplification of the 16s rRNA by PCR followed by sequencing and sequence analysis was used for the molecular identification of these strains. Optimization of chemical and physical culture conditions was carried out by manipulation of fermentation parameter such as pH, Temperature and incubation period. The results revealed 7 strains of antibiotic producing organisms. 4 bacterial strains demonstrated convincing growth inhibitory properties against pathogenic test organisms. Of these, were identified as Gram-positive cocci and 1 was identified as Gram-negative of the group of Rods and most of the isolates were active against the Gram positive than gram negative pathogens. Phylogenetic analysis of amplified 16S rRNA gene showed the isolates shared sequence identities of 99.65% with known Staphylococcus and Pseudomonas species. TSH2 and TSP3 clustered together with a sequence identity of 99.68% with Staphylococcus sciuri. Isolate TSP1 sequence had a sequence identity of 100% with Pseudomonas formosensis strain CC-CY503. The production of antimicrobial substances started on the 4th day and went on increasing till it reached a maximum peak on the 7th day. The optimum growth conditions were pH 7.5, temperature at 35°C, and incubation period in 7 days. In conclusion, the results of the present study indicate that soil contain great diversity of antibiotic producing organisms and the production of antimicrobial substances can be improved by manipulating the growth conditions.