Geomicrobiological Investigation and Environmental Implications of Bacterial Isolates of Kashmir Caves, Pakistan

Abstract

Much of the current microbial research has focused on biotechnological applications of bacterial strains isolated from soil and water. There exists limited data on potential of bacteria isolated from extreme environments like caves. Our understanding regarding extreme habitats of this earth are still limited and there is a need to explore them for better understanding of their role in natural regulation of earth’s resources. Geological formations are closely associated with microbiological processes. Microorganisms of all types play an important role in formation and evolution of various geological features of the earth. In the current study, an attempt was made to illustrate geochemical and microbiological properties Bat guano from a karstic limestone cave in Khyber Pathunkhwa Province of Pakistan. Analysis was carried out for 3 layers of bat guano pile (top, middle and bottom). Geochemical profiling for various minerals and heavy metals was carried out using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Nutrient composition including total organic carbon, total nitrogen, phosphorus and potassium was evaluated using C-N analyzer. Culturable bacterial diversity was established by culturing bacterial species from each layer on Nutrient Agar plates. DNA extraction was done for sequencing of 16S ribosomal RNA gene analysis. In this study, we report antimicrobial compounds producing bacterial strain isolated from Bats guano of a limestone cave. Screening of bacterial strains was carried out from top, middle and bottom layer of the guano sample. Out of 20 isolated strains, 3 showed antimicrobial activity against all tested ATTC strain of common bacterial (both gram positive and gram negative) and fungal species. 16s RNA gene sequencing of the strain showing maximum activity revealed the isolated strain as Bacillus subtilis. The current study was aimed to investigate occurrence of multidrug resistance in bacterial species inside Kashmir cave of province Khyber Pakhtunkhwa, Pakistan. A total of 14 bacterial strains were isolated from 3 different layers of bat guano samples of the cave and screened for their potential to resist 6 commonly used antibiotic classes namely, aminoglycosides, beta lactams, carbapenems, fluroquinolones, macrolides, and glycopeptides. The occurrence of antibiotic resistance genes namely, bla-CTXM, bla-NDM, Gyr A and vanA gene was screened through viii polymerase chain reaction by amplifying the DNA. This study focused on evaluation of biodegradation potential of Alcaligenes faecalis bacterial strain, isolated from cave bat guano samples. The guano sample was collected from deep end of the cave and screening for LDPE bacteria was carried out from three layers of the bat guano. GTL 3 strain was selected for the experimentation based on maximum growth shown during the screening process. Biodegradation was carried out in MSM medium amended separately with glucose, Tween 80 and CaCO3. Assessment of biodegradation process was done on the basis of growth of the bacterial strain (OD 620nm), weight loss percentage of LDPE pieces, mechanical characterization including young’s modulus, tensile strength, tear strength and elongation at break at every 10th of the experiment till 90th day. Lipase production of GTL 3 strain was also evaluated at every 10th day for 90 days using NPP as substrate. Chemical changes and change in functional groups were checked using Fourier Transform Infra Red spectroscopy at the end of the experiment. Physical changes were observed using Scanning Electron Microscopy at the end of the experiment. ICP-MS showed presence of various minerals including heavy metals in different layer of bat guano. The mineral concentration was high in fresh guano samples (top layer) and decreased as we move towards lower layer (old deposits). Carbon-Nitrogen Analysis (CN Analyzer) of Guano showed top layers to be rich in organic carbon, total nitrogen, potassium and phosprus content that decreased in amount we moved from top layer to the bottom layer. Phylogenetic analysis of Bat guano revealed 64.28% bacteria belonged to phylum firmicutes, 35.70% to proteobacteria, and 7.10% to phylum bacteroides. Optimum production of antimicrobial compounds took place in TSB medium after 72h of incubation at 30˚C. FTIR of crude extract showed chemical similarity with bacitracin. GCMS analysis of the crude extract revealed a range of antimicrobial and anticancer compounds. Almost all the isolates showed resistance to more than 1 of the tested antibiotic classes. Firmicutes demonstrated high resistance against carbapenems, while Proteobacteria and Bacteroides were more resistant against the β lactam and aminoglycoside class of drugs. 78.5% of the strains were positive for bla-CTXM gene, 64.2% for bla-NDM, 42.8% for Gyr A and 35.70% for vanA gene. The results of the study revealed an initial decrease in in OD that stabilized with increasing time of incubation. Maximum growth (OD 1.9) was observed in culture with added CaCO3 in it. Weight loss % increased continuously throughout the experiment with maximum loss of 6.9% was observed by the end of the experiment in CaCO3 medium. Maximum decrease in Young’s modulus was from 385.423Mpa to 316.964Mpa, tensile strength ix from 15.6Mpa to 2.62Mpa, tear strength from 126.486 N/mm to 65.581 N/mm and elongation at break from 17.98mm to 1.98mm in CaCO3 amended medium by end of experiment. Lipase production increased in the initial days of the experiment, with a maximum production of 18.4 U/mL at day 30 and decreased continuously form hereafter. FTIR showed peak formation at 2345cm-1, 3493cm-1, 726cm-1,1145cm-1, 1875cm-1, 2250cm-1, and 3560cm-1 suggests oxidative breakdown taking with the polyethylene polymer. SEM images revealed clear signs of physical damage, erosion, abrasion of LDPE pieces. Bacterial adherence and biofilm formation by GTL 3 on LDPE surface was also observed in SEM images. This study is a first comprehensive research on biodiversity assessment and environmental implications of bacterial isolates of bat guano samples of Kashmir cave, Pakistan. It can be concluded form this research that the guano samples contained various essential and non essential elements and diverse bacterial flora. The isolates were capable of producing a variety of bioactive compounds, resist multiple commonly used drugs (MDR) and efficiently biodegrade polyethylene polymer. The future work should focus on metagenomic studies to document unculturable biodiversity of bat guano. Metabolomics studies should be conducted to decipher hidden metabolic range of these organisms. Studies on biogeochemical cycles should be carried to determine niche of bacteria isolates of bat guano in cave ecosystem.