Evaluating the long-term adaptive response of Streptococcus mutans to microgravity

Strategies for maintaining life on extended missions in space are becoming a priority for NASA and other agencies as the desire to explore and colonize planets like Mars increases. Life on the International Space Station (ISS) presents many microbial challenges to the humans that inhabit it. Genomic and phenotypic traits of Streptococcus, an organism that causes dental infections, is well studied on Earth but little knowledge exists on the organism’s microbial changes on extended space missions. In this study, we evaluate the long-term (100-day) adaptive response of S. mutans to normal gravity (NG)(x4) and microgravity (MG)(x4) with the use of 8 High Aspect Rotating Vessel (HARV) which simulates the MG environment. In addition, we analyze the genetic changes that arise in response to MG by DNASeq as well as perform several assays including minimum inhibitory concentration (MIC), competition, and biofilm. Hypothesis: S. mutans cultured in a MG environment will develop genomic and phenotypic traits that will aid in virulence and resistance to antimicrobials than S. mutans cultured in a NG environment. Methods: HARVs inoculated with S. mutans and 10 ml of Brain Heart Infusion (BHI) media. HARVs are sub-cultured every 24-36hr with 10 ml of BHI. 5 ml of samples from each sub-culture are used in detecting possible contamination via microscopy, nanodrop, and plating. Noncontaminated samples are stored in glycerol stocks that call for 100 ug of sample to 400 ug of glycerol. The remaining of sample is centrifuged to collect cells. Glycerol stocks and centrifuged cells are stored at -80 Celsius. Stocks are used in case of future contamination, and cells are used for DNA seq. Results/Conclusions: The evaluation of S. mutans signified that growth rate is not a factor although the growth formation of biofilms is significant to survival. NG biofilms formed along the base opposed to MG biofilms formed sporadically throughout media, attaching to wall inside of HARVs. Not many bacteria resemble S. mutans; we were able to detect the presence of a contaminant specifically, staphylococcus. Plated S. mutans appear white, small and circular whereas staphylococcus appear yellow and grow at an increased rate. Under the microscope, S. mutans are tiny, circular and form in chains and staphylococcus are circular but, clump together to form in a larger colony. Future Direction: To evaluate the long-term (100- day) adaptive response of S. mutans to microgravity (MG) and the co-adaptation to microgravity and silver (MGAg) to better understand the consequences of using silver to filter potable water on the host microbiome. Significance: The ability to filter, recycle, and reuse water for extended missions is vital to sustaining life on the ISS. Silver is used as the primary biocide in the portable water dispenser (PWD) at a standard level of 400ppb although, the Environmental Protection Agency states no more than 100 ppb of silver to be considered non-toxic to human consumption. These studies will allow us to evaluate the rate and genetic mechanisms in which S. mutans may evolve resistance to levels of silver presently used as a biocide for potable water on the ISS. The acquired genetic data will also help in the potential development of treatments and/or control strategies to combat the dental issues that may arise in space due to this pathogen