Evaluation of Antimicrobial Potential of Cold adapted Bacterial Isolates from Passu Glacier

Abstract

Microbial resistance to antibiotics has been a global concern nowadays and scientists are looking for new antimicrobial compounds from extreme environments like glaciers, snow, marine, deserts, hot springs etc. For this purpose, the study is designed to unearth the antimicrobial metabolites producing strains from unexplored Passu glacier and characterize its metabolites. Among the eleven isolates, four good antimicrobials producers HTP12, HTP13, HTP36, and LTP10 were selected based on results of agar diffusion assay against test strains. These isolates were identified by 16S rRNA sequencing and phylogenetic analysis. Effects of temperature, pH, incubation period, culture medium, carbon sources, nitrogen sources and salts on antimicrobial activity of four isolates were determined. The antimicrobial metabolites from LTP10 were extracted by liquid-liquid extraction technique and were dried. The stability of extract activity was evaluated at 25-65⁰C and pH 4-10. The crude extract was then evaluated by antibacterial, antifungal, antioxidant, cytotoxic, MIC and MBC assays and characterized by FTIR, GC-MS and LC-MS techniques. Molecular docking and in silico ADMET analysis of major compounds of extract was performed using online webservers CB-Dock, pkCSM, and swissADME. The isolates HTP12, HTP13, HTP36 and LTP10 were identified as Alcaligenes faecalis, Pseudochrobactrum saccharolyticum, Alcaligenes pakistanensis and Alcaligenes pakistanensis respectively. The optimum antibiotic production by isolates was found at 25⁰C and pH 7.0 after 96 hours of incubation in Luria-Bertani broth. Carbon sources, salts, and nitrogen sources have decreased antimicrobial activity of isolates except yeast extract which has positive effect on antimicrobial activity. Based on optimization study, best antibiotic producer LTP10 was selected for further evaluation. Ethyl acetate extract of LTP10 has shown best antimicrobial activity against clinical isolates. There was no significant decrease in activity of extract up to 45⁰C and pH 5.5-8.5. Antibiotic discs (penicillin, imipenem, cefepime) has significantly increased antimicrobial activity of LTP10 extract by synergistic effect. MIC and MBC values of extract against tested gram-positive and gram-negative strains was found in the range of 0.4-3.2 mg/ml. Brine shrimp lethality assay indicated non-cytotoxic nature while DPPH assay revealed antioxidant nature of LTP10 metabolites. FTIR analysis proved the presence of O-H, C-H, C=C, C-O, N-O, S=O, C-N and C-O functional groups denoting presence of important organic metabolites. GC-MS chromatogram of ethyl acetate and chloroform IX extracts of LTP10 validated the presence of about 14 important antimicrobial compounds. Thirteen metabolites with reported antimicrobial activity have been identified based on LC-MS analysis of ethyl acetate extract. Molecular docking and in silico bioactivity analysis of four major metabolites 7β-Hydroxy-3-oxochola-1,4-dien 24-oic acid, dianhydroaurasperone C, L-alpha-Amino-1H-pyrrole-1-hexanoic acid and L,L-Cyclo(leucylprolyl) has predicted that these compounds could be further considered for development into antibiotic drugs. These findings have revealed the significance of antimicrobial isolates from Passu glaciers. Due to immense increase in the antimicrobial resistance, investigation of such type of unexplored microbial habitat, is urgently required.