Adaptation to simulated microgravity in Streptococcus mutans

Abstract

Long-term space missions have shown an increased incidence of oral disease in astronauts’ and as a result, are one of the top conditions predicted to impact future missions. Here we set out to evaluate the adaptive response of Streptococcus mutans (etiological agent of dental caries) to simulated microgravity. This organism has been well studied on earth and treatment strategies are more predictable. Despite this, we are unsure how the bacterium will respond to the environmental stressors in space. We used experimental evolution for 100-days in high aspect ratio vessels followed by whole genome resequencing to evaluate this adaptive response. Our data shows that planktonic S. mutans did evolve variants in three genes (pknB, SMU_399 and SMU_1307c) that can be uniquely attributed to simulated microgravity populations. In addition, collection of data at multiple time points showed mutations in three additional genes (SMU_399, ptsH and rex) that were detected earlier in simulated microgravity populations than in the normal gravity controls, many of which are consistent with other studies. Comparison of virulence-related phenotypes between biological replicates from simulated microgravity and control orientation cultures generally showed few changes in antibiotic susceptibility, while acid tolerance and adhesion varied significantly between biological replicates and decreased as compared to the ancestral populations. Most importantly, our data shows the importance of a parallel normal gravity control, sequencing at multiple time points and the use of biological replicates for appropriate analysis of adaptation in simulated microgravity.

Co-adaptation of Streptococcus mutans to simulated microgravity and silver nitrate

“…High-aspect rotating vessels (HARVs) were purchased from Synthecon Inc. (Houston, TX) and used to culture S. mutans under sMG (Herranz, R. et al, 2013). Prior to inoculation, the HARVs were cleaned as described previously (Fernander et al, 2022). Using the S. mutans ancestral stock, 100 μL of an overnight culture was used to inoculate 100 mL of fresh BHI broth.…”

“…This was then split into 2 × 50 mL cultures, and 10 μg/mL silver nitrate was added to one 50 mL sample. The sub-culture in BHI alone was loaded into four HARVs and deemed sMG5-8 (to distinguish these four populations from our previous study that generated sMG1-4 (Fernander et al, 2022)). The BHI supplemented with AgNO 3 was split into four additional HARVs and deemed sMGAg1-4.…”

“…As the oral cavity will continuously encounter water from the PWD and, therefore, silver, if unfiltered, we explored the evolutionary consequences of silver nitrate exposure on the evolution of the oral human resident and pathogen S. mutans in simulated microgravity (sMG) as it may also alter treatment strategies. This is a follow-up study to our initial work evaluating the adaptive response of S. mutans to sMG alone (Fernander et al, 2022). Here, our objective is to better understand the consequences of using silver nitrate or silver-based compounds as the main antimicrobial mitigation strategy on future long-term space missions and provide data to support informed decisions regarding potential water treatment options and/or mitigation strategies.…”

Microgravity and evasion of plant innate immunity by human bacterial pathogens

“…Space vehicles represent evolutionarily novel ecosystems in which humans, the microbiome, and plants are exposed to a microgravity environment lacking in physical forces of weight, buoyancy, convection, and shear 14,15 . Numerous studies demonstrate that microgravity increases antibiotic resistance 9,16,17 , virulency [18][19][20][21] , and stress tolerance [22][23][24][25] in human pathogenic bacteria. Studies suggest microgravity increases plant susceptibility to fungal phytopathogens [26][27][28][29] , and yields differential expression of pathogen-related plant genes 30,31 .…”

“…Human bacterial pathogens display a wide range of altered behaviors associated with increased pathogenesis under spaceflight and modeled microgravity analogs (MMA). These include higher cell counts 15 , increased growth rate 23,32 , increased biofilm formation 16 , cell aggregation 20,32 , altered motility and chemotaxis 33 , increased expression of Type-III Secretion System (T3SS) related pathogenicity island genes 34,35 , altered virulency in animal hosts [18][19][20][21] , increased resistance to hydrogen peroxide 36 , increased macrophage survivability 20,21,23 , increased resistance to antibiotics 9,16,17 , increased tolerance to acidic conditions 21 , and reduced LD 50 in mice 20,21,23 . Salmonella Typhimurium cultured under MMA was more lethal compared to normal-gravity culture in mice with a 5.2-fold decrease in LD 50 and shorter average time to death 21 .…”