Potential of cave bacteria in drug discovery: Investigation of sphingopyxis terrae and other Bacteria from a BC cave

The discovery of new and more effective antibiotics continues to be a priority given the frequency of the emerging multi-drug resistant pathogenic microorganisms. Thus, scientists are searching for new antibiotics from microorganisms selected from extreme habitats such as very old caves. Various cave bacteria species were isolated and could be sources of new antibiotics. The objective of our work is to isolate cave bacteria from a volcanic cave in Wells Gray Provincial Park in BC and to test if they produce metabolites with antimicrobial activity against some microorganisms including multi- drug resistant pathogens. This study used 16 cave strains previously isolated and screened against a panel of microorganisms including drug resistant pathogens. Upon retesting, 4 out of 16 cave bacterial isolates, RA001, RA003, RA004, and PM58B-RA, demonstrated antimicrobial activity against Mycobacterium smegmatis, Micrococcus luteus, Acinetobacter baumannii and MDR-Staphylococcus aureus. To study the conditions for best growth and antimicrobial production, these four bacteria were cultured in different fermentation media (namely Hickey-Tresner, R2A, V-8 juice and ISP-2) and incubated at 12 and 25°C for 14 days. During the course of fermentation, the percentage of packed cell volume (%PCV), antimicrobial activity and pH were observed and recorded daily. It was found that each of the bacteria demonstrated antimicrobial activity against different microorganisms at various times of fermentation and temperature. Overall, R2A broth medium and the lower temperature of 12°C appear to be best for antimicrobial production by the cave bacteria used in this study. Isolation and purification of the antimicrobial compounds produced by these isolates is under investigation using the best growth conditions determined in this study. We identified these bacteria using chemotaxonomic studies; 16S rRNA sequencing and Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF), all isolates were identified to the species level. PM58B was found to be Bacillus licheniformis, RA001, and RA004 were identified as Arthrobacter agilis that may be of different variants. While RA003 was identified as Sphingopyxis terrae. Active compounds from RA003 fermentation broth were further studied by extraction and purification. In conclusion, cave bacteria are promising sources of potential novel antimicrobial compounds. Isolation, optimization of screening, growth media and conditions of cave bacteria may be useful in the discovery of new antimicrobial drugs. Additionally, the knowledge obtained from this study with respect to cave bacteria and their roles in cave formation and degradation will add to existing information on cave conservation.