RESEARCH LETTER
Effects of simulated microgravity on Streptococcus mutans physiology and biofilm structure Xingqun Cheng, Xin Xu, Jing Chen, Xuedong Zhou, Lei Cheng, Mingyun Li, Jiyao Li, Renke Wang, Wenxiang Jia & Yu-Qing Li State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
Correspondence: Yu-Qing Li, State Key Laboratory of Oral Diseases, Sichuan University, No. 14, Section 3, Renmin Road South, Chengdu 610041, China. Tel.: +86 28 85501232; fax: +86 28 85582167; e-mail: [email protected] Received 3 June 2014; revised 5 August 2014; accepted 5 August 2014. Final version published online 28 August 2014. DOI: 10.1111/1574-6968.12573
MICROBIOLOGY LETTERS
Editor: Robert Burne Keywords acid tolerance; astronauts; biofilm architecture; dental caries; interspecies competition; mutacin production.
Abstract Long-term spaceflights will eventually become an inevitable occurrence. Previous studies have indicated that oral infectious diseases, including dental caries, were more prevalent in astronauts due to the effect of microgravity. However, the impact of the space environment, especially the microgravity environment, on the virulence factors of Streptococcus mutans, a major caries-associated bacterium, is yet to be explored. In the present study, we investigated the impact of simulated microgravity on the physiology and biofilm structure of S. mutans. We also explored the dual-species interaction between S. mutans and Streptococcus sanguinis under a simulated microgravity condition. Results indicated that the simulated microgravity condition can enhance the acid tolerance ability, modify the biofilm architecture and extracellular polysaccharide distribution of S. mutans, and increase the proportion of S. mutans within a dual-species biofilm, probably through the regulation of various gene expressions. We hypothesize that the enhanced competitiveness of S. mutans under simulated microgravity may cause a multispecies micro-ecological imbalance, which would result in the initiation of dental caries. Our current findings are consistent with previous studies, which revealed a higher astronaut-associated incidence of caries. Further research is required to explore the detailed mechanisms.
Introduction With the development of manned space technology, long-term spaceflights have been discussed and planned, such as prolonged stays at the International Space Station and missions to Mars (18 months at least). Previous studies have indicated that spaceflight dental emergencies would increase during these extended duration missions (Rai & Kaur, 2011). Moreover, although in-flight dental emergencies have been historically rare, oral diseases such as dental caries and periodontal disease were found to be more prevalent following exposure to a simulated microgravity condition (a condition of near-weightlessness, 1 9 10 6 g), as compared with normal, 1 g, earth environments (Rai et al., 2011). Dental caries has been recognized as one of the most common oral infectious diseases in humans. The interactions of bacteria, host, dietary and time can all eventually ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
lead to tooth decay. Although the energy needs, diet and water intake during flight are similar to that on Earth (Lane et al., 2013), a previous short-term study revealed that astronauts might become more susceptible to dental caries due to a decrease in the salivary flow, to bone resorption, and to the formation of dental calculus (Rai & Kaur, 2011). However, the relationship between bacterial physiological changes and the caries status of astronauts remains unclear. Streptococcus mutans, the primary etiologic agent of dental caries (Loesche, 1986), can adhere firmly to the tooth surface and form stable biofilm by producing extracellular polysaccharides (EPS; Koo et al., 2009). It is also able to produce acid efficiently by metabolizing carbohydrates and is highly aciduric. The comprehensive actions of these unique features of S. mutans result in prolonged periods of dental plaque acidification, demineralization of the tooth enamel, and ultimately the initiation of dental FEMS Microbiol Lett 359 (2014) 94–101
Characteristics of S. mutans biofilm under microgravity
caries (Banas, 2004). Meanwhile, the synergism or antagonism within the oral microbial community is important for bacterial equilibrium and disease development. The acid-producing bacteria S. mutans within the biofilm ecosystem has the ability to produce acid efficiently by metabolizing carbohydrates, actively inhibiting the growth of other ‘beneficial’ commensal colonizers such as Streptococcus sanguinis (Takahashi & Nyvad, 2011). In contrast, cytotoxic hydrogen peroxide (H2O2)-producing bacteria, such as S. sanguinis, can antagonize mutacin-generating S. mutans (Kreth et al., 2008). Changes in any key environmental factors will then trigger a shift from the balance state of the microflora to a disease-associated species composition (Marsh, 1994). However, the effect of space microgravity environment on the bacterial virulence and microbial equilibrium within the oral biofilm remains to be explored. Several studies have demonstrated that the behavior and virulence determinants of microorganisms can be affected by an exposure to microgravity (Nickerson et al., 2004; Rosenzweig et al., 2010), resulting in a potential impact on human diseases. In microgravity environments, microbial pathogens such as Pseudomonas aeruginosa (Crabbe et al., 2011) and Salmonella Typhimurium (Wilson et al., 2007) demonstrate an enhanced virulence and increased biofilm formation (Crabbe et al., 2008). Since the effects of both the microgravity condition on the virulence factors of S. mutans and the ecological balance of the oral ecosystems remain unknown, we were interested in conducting a study to characterize S. mutans under a simulated microgravity condition. Therefore, in the present study, we explored the effects of simulated microgravity on the physiology and biofilm structure of the main caries pathogen, S. mutans. The dual-species interaction between S. mutans and S. sanguinis under simulated microgravity conditions was also investigated.
Materials and methods Superconducting magnet
To investigate the effects of microgravity on bacteria, we used a ground-based experimental platform for gravitational biology simulated by a large gradient superconducting magnet (JMTA-16T50MF; Japan Superconductor Technology Inc., Tokyo, Japan) (Fig. 1a) (Qian et al., 2013). The superconducting magnet generated different magnetic force fields corresponding to different apparent body force levels. Decreased gravity environments were achieved by changing the location of the cell sample within the magnet.
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Bacterial strains and growth media
Streptococcus mutans UA159 and S. sanguinis ATCC10556 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All bacteria were routinely anaerobically grown at 37 °C (90% N2, 5% CO2, 5% H2) in a brain-heart infusion broth (BHI; Difco, Sparks, MD). Bacteria were seeded in 35-mm tissue culture plates sealed with parafilm. To distinguish gravitational and magnetic field effects, three groups were designed in this study (lg*, 1 g* and 1 g; ‘g’ means gravity and ‘*’ means gravity environment generated by the magnet). The plates of lg* and 1 g* groups were put into special positions on the superconducting magnet (Fig. 1a). The control sample, 1 g, was put in the incubation chamber outside the superconducting magnet. Cultures of S. mutans were sampled at hourly intervals for 10 h. These samples were then plated to determine the viable counts (as colonyforming units, CFU, per mL of culture) over the incubation period for plotting growth curves. Glycolytic pH drop assay
The effect of simulated microgravity on S. mutans glycolysis was measured similar to Xu et al. (2011) with slight modifications. Briefly, bacteria cultured under the environment of lg*, 1 g* and 1 g were harvested at midlogarithmic phase by centrifugation (4000 g, 20 °C, 10 min), washed with 0.5 mM potassium phosphate buffer containing 37.5 mM KCl and 1.25 mM MgCl2 (pH 6.5), and re-suspended (OD600 nm = 0.5) in the same solution. Glucose was added to give a final concentration of 1% (w/v). The decrease in pH as a result of glycolytic activity in the S. mutans cells was monitored over a period of 120 min (Corning pH meter 240; Corning Inc., NY). Acid killing and hydrogen peroxide challenge
The ability of S. mutans cells to withstand acid challenge or oxidative stress was determined using a method modified from a previous study (Wen et al., 2005). Briefly, bacteria used for acid killing and H2O2 challenge assays were first grown under the environment of lg*, 1 g* or 1 g. Cells were then harvested immediately at mid-exponential phase (OD600 nm = 0.5), washed with an equal volume 0.1 M glycine buffer (pH 7.0) and subjected to acid or oxidative stress. For acid-killing assays, S. mutans cells were incubated in 5 mL 0.1 M glycine buffer (pH 2.8) for 30 and 60 min. For oxidative stress tolerance assays, S. mutans cells were re-suspended in 5 mL 0.1 M glycine buffer (pH 7.0). Hydrogen peroxide (H2O2; Fisher Scientific, Fair Lawn, NJ) was then added to the cell
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Fig. 1. Effects of simulated microgravity on cell growth, acid production, and stress tolerance of Streptococcus mutans. (a) Superconducting magnet (JMTA-16T50MF). The superconducting magnet could generate different magnetic force fields, corresponding to different body force levels (lg* and 1 g*). The height of the superconducting magnet is 195 cm, with a diameter of 50 mm, and a 450-mm cylindrical cavity. To develop the biological experiment, equipment matched with the superconducting magnet was designed, including a temperature control system, an object stage, a gas control system, and an observing system. (b) Growth curves of S. mutans under different gravity conditions. (c) Effect of simulated microgravity on acid production of S. mutans UA159. Glycolytic acid production was determined by monitoring the pH decrease in sucrose solution (1%, w/v) over a period of 120 min. Results are the average of three independent experiments and are presented as mean SD. (d) Acid-killing assay. Acid tolerance was determined by measuring the survival rate of S. mutans cells at pH 2.8 at different time points (0, 30, 60 min). (e) H2O2 challenge assay. H2O2 sensitivity was determined by measuring the survival rate of S. mutans cells at an H2O2 concentration of 0.2% at different time points (0, 15, 30 min).
suspension to give a final concentration of 0.2% (v/v). After incubation for 30 and 60 min, catalase (5 mg mL 1; Sigma) was added to inactivate H2O2. The control group was incubated in 0.1 M glycine buffer (pH 7.0) for the same period of time. Serial dilutions were performed in 0.1 M glycine buffer (pH 7.0) and the survival rate was determined by plating the samples in triplicate on BHI plates. Biofilm analysis and structural imaging
The bacterial cells and EPS of the 24-h S. mutans biofilm were labeled with SYTO 9 (Molecular Probes, Invitrogen Corp., Carlsbad, CA) and Alexa Fluor 647-labeled dextran conjugate (Molecular Probes), respectively, as previously described (Xiao et al., 2012). Biofilm images were captured with a Leica DMIRE2 confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with a 609 oil immersion objective lens. The image collection gates were set to 495–515 nm for SYTO 9 and 655–690 nm for Alexa Fluor 647. Each biofilm was scanned at five randomly selected positions, and then the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
confocal image series were generated by optical sectioning each of these positions. Three-dimensional reconstruction of the biofilms and the quantification of EPS/bacteria biomass were performed with IMARIS 7.0.0 (Bitplane, Zurich, Switzerland). Dual-species interaction within the biofilm
Cultures of S. mutans and S. sanguinis were inoculated on saliva-coated glass coverslips in fresh BHI supplemented with 1% sucrose in a 24-well cell culture plate under different gravity conditions. Biofilms established at 24 h were fixed in 4% paraformaldehyde (PFA), bacteria were labeled with oligonucleotide probes (Supporting Information, Table S1) and investigated by speciesspecific fluorescence in situ hybridization (FISH) as described previously (Zheng et al., 2013). Micrographs were sampled from at least five randomly selected positions of each sample. The ratio of S. mutans to S. sanguinis was calculated based on the coverage area of each bacterium, analyzed by IMAGE PRO PLUS 6.0 (Media Cybernetics, Inc., Silver Spring, MD). FEMS Microbiol Lett 359 (2014) 94–101
Characteristics of S. mutans biofilm under microgravity
Quantitative real-time PCR (qRT-PCR)
For quantitative real-time PCR, gene-specific primers were designed (Supporting Information, Table S2). Total bacterial RNA isolation, purification, cDNA reverse transcription, and PCR reactions were performed as previously described (Li et al., 2013). Amplification specificity was assessed using melting curve analysis. Different gene expressions were normalized to the levels of 16S rRNA gene transcripts. The data were analyzed by Bio-Rad CFX MANAGER software (version 2.0) according to the 2 ΔΔ CT method. Statistical analysis
All experiments were performed in triplicate and reproduced at least three separate times. Data were computerized and analyzed using SPSS 18.0 software. One-way ANOVA and Student’s t-test were used to compare the data of two groups (lg* or 1 g* with 1 g). Data were considered significantly different if the two-tailed P-value was < 0.05.
Results Effects of simulated microgravity on cell growth, acid production, and stress tolerance of S. mutans
The superconducting magnet (Fig. 1a) was used to simulate microgravity conditions and to explore the effects of a simulated microgravity condition (lg*) on the growth properties of S. mutans. As shown in Fig. 1b and c, no significant differences with respect to the S. mutans growth kinetics or acid production were observed under the lg* condition, as compared with those under a normal gravity condition (1 g* and 1 g). To rule out the possibility that the cells may re-adapt quickly to gravity, we also measured both the initial and end pH of the cultures under different gravity conditions; no significant differences among the three groups were found (lg*, 1 g* and 1 g) (data not shown). However, as shown in the acid-killing assay (Fig. 1d), the survival rate of the lg* group was significantly increased (3.5-fold) after short-term (30 min) acid treatment (pH 2.8), indicating that the acid resistance ability, which is considered a major cariogenic virulence factor of S. mutans, was enhanced under a simulated microgravity condition. Meanwhile, as demonstrated by the H2O2 sensitivity assay (Fig. 1e), there were no significant differences with respect to the survival rate of S. mutans under different gravity conditions. We also examined the cell morphology and membrane surface characteristics of S. mutans by scanning electron microscopy (SEM) assay. However, as FEMS Microbiol Lett 359 (2014) 94–101
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demonstrated in Supporting Information, Fig. S1, there were no significant differences with respect to the cell morphology and membrane surface characteristics of S. mutans under different gravity conditions. Streptococcus mutans showed abnormal biofilm structure and EPS distribution under simulated microgravity conditions
The biofilm structure and EPS production ability of S. mutans under different gravity conditions were determined by confocal laser scanning microscopy (Fig. 2a). There was no significant difference with respect to the total amount of bacteria and EPS under different gravity conditions (Fig. 2c). However, as shown in Fig. 2a, the biofilm that formed under lg* was thinner and denser than those formed under 1 g* and 1 g. Moreover, we found that the architecture and composition of the S. mutans biofilm formed under lg* was abnormal. The distribution of bacteria and EPS of the lg* group and those of the 1 g* and 1 g groups were significantly different at designated heights (Fig. 2b,H1,H2). The EPS of the lg* group was distributed from top to bottom. By contrast, almost all of the EPS under 1 g* and 1 g was distributed at the bottom (Fig. 2b). The EPS/S. mutans ratio of the lg* group at intermediate heights (3–8 lm) was significantly higher than that of the 1 g* and 1 g groups at relative heights (4–14 lm) (Fig. 2d). However, the EPS/S. mutans ratio of the lg* group at the bottom (1– 2 lm) was significantly lower than that of the 1 g* and 1 g groups at relative heights (1–3 lm). Streptococcus mutans showed a superior competitive advantage over S. sanguinis under a simulated microgravity condition
A unique characteristic of multispecies biofilms is the constant interspecies interaction, either synergistic or antagonistic, among its microbial residents. The dynamic antagonistic interactions are very important for maintaining the balance of microbial ecology and oral health. Therefore, in addition to exploring the biofilm formation of a single bacteria (S. mutans only), we used an S. mutans and S. sanguinis dual-species biofilm model (Zheng et al., 2013) to construct a micro-ecological condition under simulated gravity. FISH was used to visualize the spatio-temporal interactions within the dual-species biofilm community. As shown in Fig. 3a, S. mutans of lg* group showed a superior competitive advantage over S. sanguinis. This result was further confirmed by a CFU counting assay, which revealed that the S. mutans/S. sanguinis ratio of the lg* group was significantly increased (Fig. 3b). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Fig. 2. Effects of simulated microgravity on biofilm architecture and EPS distribution of Streptococcus mutans. (a) Double-labeling of 24-h S. mutans biofilms. Green, bacteria (SYTO 9); red, EPS (Alexa Fluor 647). Images were taken at 609 magnification. The 3-D reconstruction of biofilms and the quantification of EPS/bacteria distribution were both performed with IMARIS 7.0.0. The distributions of EPS and bacteria at different heights (H1: 2 lm and H2: 5 lm) are shown in (b). (c) Quantification of bacteria/EPS biomass was performed with IMARIS 7.0.0. Results are the average of five randomly selected positions of each sample and are presented as mean SD. (d) The ratio of EPS to bacteria at different heights was quantified with IMARIS 7.0.0. Results are the average of five randomly selected positions of each sample and are presented as mean SD.
Expression of bacteriocin-, ATR-, and EPSrelated genes of S. mutans under a simulated microgravity condition
Previous studies have shown that the interspecies interaction between S. mutans and S. sanguinis possibly was mediated through the production of inhibitory compounds, such as bacteriocins and hydrogen peroxide. In this study, we found no differences in the S. sanguinis growth and H2O2 production under different gravity conditions (data not shown). Therefore, we speculated that the ecological imbalance of the S. mutans/S. sanguinis biofilm was probably due to the competitive advantage of S. mutans. Interestingly, as demonstrated by the qRTPCR assay (Fig. 3c), S. mutans genes related to mutacin production (nlmA and nlmC, encoding non-antibiotic ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
bacteriocins mutacin IV, and mutacin V) were significantly up-regulated under a lg* condition by 4.6- and 2.0-fold (P < 0.05), respectively. To validate whether ATR was induced under a simulated microgravity condition, we investigated the expression of ATR-related genes, such as atpA, atpE and ffh (Fig. 4). Although the expression of atpA/E showed no significant difference between three groups, the expression level of ffh, which encodes a bacterial signal recognition particle that is linked to the ATR (Gutierrez et al., 1999), was increased by 1.68-fold (P < 0.05). We also explored how the simulated microgravity condition influences the expression profiles of EPS-related genes, including genes encoding glucosyltransferase (gtfB, gtfC and gtfD), dextranase (dexA), fructosyltransferase (ftf) and fructan hydrolase (fruA). Of these, only the FEMS Microbiol Lett 359 (2014) 94–101
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Fig. 3. Bacterial competition within dual-species biofilms and the expression change of bacteriocin-related genes under a simulated microgravity condition. (a) Streptococcus mutans (green) and Streptococcus sanguinis (red) were labeled with species-specific FISH probes (Zheng et al., 2013). Images were taken at 609 magnification, and representative images are shown. (b) Ratio of S. mutans to S. sanguinis under different gravity conditions in dual-species biofilms. The ratio was calculated as CFU of S. mutans in different gravity conditions/CFU of S. sanguinis in the same gravity condition as S. mutans. (c) qRT-PCR results of bacteriocin genes (nlmA and nlmC) under simulated microgravity condition. Results are the average of three independent experiments and are presented as mean SD. Asterisks indicate significant differences (P < 0.05) compared with 1 g.
Fig. 4. Effects of simulated microgravity on the expression of ATR and EPS synthesis related genes of Streptococcus mutans. qRT-PCR assays were carried out as described in Materials and methods. All target genes were amplified using specific primers. Different gene expressions were normalized to the levels of 16S rRNA gene transcripts, and the folds of expression change were calculated. Representative data are shown. Asterisks indicate significant differences (P < 0.05) compared with 1 g.
expression of dexA was significantly enhanced (1.66-fold, P < 0.05), whereas the expression of gtfC was reduced (0.72-fold, P < 0.05).
Discussion A limitation in the professional dental care available during long space flights would cause difficulties in maintaining oral hygiene and treating tooth decay, resulting in an increased prevalence of dental caries. Previous studies have shown a higher caries incidence in astronauts during FEMS Microbiol Lett 359 (2014) 94–101
prolonged space missions. Caries is a chronic infectious disease that depends on a number of factors, including bacteria, host, diet and time. In the present study, we explored the effects of simulated microgravity on the phenotypic characteristic and interspecies interactions of S. mutans, the main pathogen of dental caries, through a ground-based experimental platform (JMTA-16T50MF) that has been widely used in research and medical applications (Qian et al., 2013; Yu & Shang, 2014). We found that S. mutans under the lg condition could withstand a longer time of acid stress (30 min) (Fig. 1d), indicating that the acid tolerance ability, one of the major cariogenic factors associated with S. mutans, was enhanced under the lg condition. S. mutans has evolved two distinct mechanisms to withstand acid shock – constitutive mechanisms and acid tolerance response (ATR) (Matsui & Cvitkovitch, 2010). In this study, we found that the expression of ffh, which encodes a bacterial signal recognition particle involved in resistance to acid stress (Gutierrez et al., 1999), was significantly up-regulated by 1.68-fold, indicating that the increased resistance of S. mutans to decreased pH (2.8) under the microgravity condition might depend on the activation of ATR. Interestingly, we also noticed that, in contrast to S. mutans of the 1 g group, S. mutans under the microgravity condition formed a denser biofilm (Fig. 2a). Cell density has been shown to modulate the ability of S. mutans to adapt to acid challenge. Moreover, biofilm cells at low cell density exhibited a decreased tolerance to a killing pH of 3.0 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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when compared with cells extracted at high cell density (Li et al., 2001). Our results also indicated that the simulated microgravity was able to alter the EPS distribution of S. mutans (Fig. 2). EPS is mainly responsible for cellular aggregation and surface attachment and plays an important role in dental plaque formation (Koo et al., 2009). A previous study has shown that the structure of EPS can be modified by dextranase (encoded by dexA) (Hayacibara et al., 2004). The presence of dextranase altered the proportions of soluble to insoluble glucan, but had a modest effect on the total amount of glucan formed. In this study, our data revealed that the expression of dexA was significantly enhanced by 1.66-fold, whereas the expression of gtfC was reduced by 0.72-fold. We hypothesize that these changes might influence the EPS composition and thus further modulate the three-dimensional (3D) structure of the S. mutans biofilm. Streptococcus mutans also showed a superior competitive advantage over S. sanguinis under the lg condition (Fig. 3a and b), even though its H2O2 resistance ability was not enhanced (Fig. 1e). As identified by qRT-PCR, genes relating to bacteriocin production (nlmA and nlmC) were significantly up-regulated. Production of mutacin is one of the mechanisms employed by S. mutans to maintain a competitive advantage in the multi-species oral biofilm (Hillman, 2002; Hossain & Biswas, 2011). Previous studies have demonstrated that the transcription of the mutacin gene and the production of mutacin greatly increased with cell density (Merritt et al., 2005; Kreth et al., 2006). Furthermore, the competence regulatory system ComDE (Kreth et al., 2007), as well as many other regulators (Qi et al., 2004; Tsang et al., 2006; Nguyen et al., 2009; Senadheera et al., 2012), are directly or indirectly involved in the regulation of mutacin production. Thereby we hypothesize that, under the lg condition, S. mutans likely enhances its competitiveness through an up-regulation of mutacin production. However, the detailed mechanism of these experimental phenomena warrants further investigation. We believe that it is also necessary to investigate the global changes of S. mutans gene expression levels during lg treatment, as well as to identify the specific regulatory pathways responses to lg signal. In conclusion, we found that the simulated microgravity condition could enhance the acid tolerance ability, modify the biofilm structure of S. mutans, and increase the proportion of S. mutans within a dual-species biofilm, likely through the regulation of gene expression. The enhanced competitiveness of S. mutans under simulated microgravity may cause a multispecies micro-ecological imbalance, which would ultimately result in the initiation of dental caries. Our current findings are consistent with ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
previous studies that have shown an increased incidence of caries in astronauts. Further research is required to explore the detailed mechanisms involved in this phenomenon.
Acknowledgements This work was supported by the National Natural Science Foundation of China (31200985, 81170959 and 81200782), the Scientific Research Foundation for Young Investigators, Sichuan University, China (2012SCU11019), International Science and Technology Cooperation Program of China (2011DFA30940), and State Key Laboratory of Oral Diseases. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
Authors’ contribution X.C. and X.X. contributing equally as first authors.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Scanning electron microscopy assays of Streptococcus mutans cell morphology under the simulated microgravity condition. The experiment was carried out as described in the Supporting Information, Materials and Methods. Representative images are shown. The images were taken at 20 0009 magnification. Scale bars: 5 lm. Table S1. Probes used for FISH assays in this study. Table S2. Primers used for qRT-PCR assays in this study. Data S1. Materials and methods.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Effects of simulated microgravity on Streptococcus mutans physiology and biofilm structure Xingqun Cheng, Xin Xu, Jing Chen, Xuedong Zhou, Lei Cheng, Mingyun Li, Jiyao Li, Renke Wang, Wenxiang Jia & Yu-Qing Li State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
Correspondence: Yu-Qing Li, State Key Laboratory of Oral Diseases, Sichuan University, No. 14, Section 3, Renmin Road South, Chengdu 610041, China. Tel.: +86 28 85501232; fax: +86 28 85582167; e-mail: [email protected] Received 3 June 2014; revised 5 August 2014; accepted 5 August 2014. Final version published online 28 August 2014. DOI: 10.1111/1574-6968.12573
MICROBIOLOGY LETTERS
Editor: Robert Burne Keywords acid tolerance; astronauts; biofilm architecture; dental caries; interspecies competition; mutacin production.
Abstract Long-term spaceflights will eventually become an inevitable occurrence. Previous studies have indicated that oral infectious diseases, including dental caries, were more prevalent in astronauts due to the effect of microgravity. However, the impact of the space environment, especially the microgravity environment, on the virulence factors of Streptococcus mutans, a major caries-associated bacterium, is yet to be explored. In the present study, we investigated the impact of simulated microgravity on the physiology and biofilm structure of S. mutans. We also explored the dual-species interaction between S. mutans and Streptococcus sanguinis under a simulated microgravity condition. Results indicated that the simulated microgravity condition can enhance the acid tolerance ability, modify the biofilm architecture and extracellular polysaccharide distribution of S. mutans, and increase the proportion of S. mutans within a dual-species biofilm, probably through the regulation of various gene expressions. We hypothesize that the enhanced competitiveness of S. mutans under simulated microgravity may cause a multispecies micro-ecological imbalance, which would result in the initiation of dental caries. Our current findings are consistent with previous studies, which revealed a higher astronaut-associated incidence of caries. Further research is required to explore the detailed mechanisms.
Introduction With the development of manned space technology, long-term spaceflights have been discussed and planned, such as prolonged stays at the International Space Station and missions to Mars (18 months at least). Previous studies have indicated that spaceflight dental emergencies would increase during these extended duration missions (Rai & Kaur, 2011). Moreover, although in-flight dental emergencies have been historically rare, oral diseases such as dental caries and periodontal disease were found to be more prevalent following exposure to a simulated microgravity condition (a condition of near-weightlessness, 1 9 10 6 g), as compared with normal, 1 g, earth environments (Rai et al., 2011). Dental caries has been recognized as one of the most common oral infectious diseases in humans. The interactions of bacteria, host, dietary and time can all eventually ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
lead to tooth decay. Although the energy needs, diet and water intake during flight are similar to that on Earth (Lane et al., 2013), a previous short-term study revealed that astronauts might become more susceptible to dental caries due to a decrease in the salivary flow, to bone resorption, and to the formation of dental calculus (Rai & Kaur, 2011). However, the relationship between bacterial physiological changes and the caries status of astronauts remains unclear. Streptococcus mutans, the primary etiologic agent of dental caries (Loesche, 1986), can adhere firmly to the tooth surface and form stable biofilm by producing extracellular polysaccharides (EPS; Koo et al., 2009). It is also able to produce acid efficiently by metabolizing carbohydrates and is highly aciduric. The comprehensive actions of these unique features of S. mutans result in prolonged periods of dental plaque acidification, demineralization of the tooth enamel, and ultimately the initiation of dental FEMS Microbiol Lett 359 (2014) 94–101
Characteristics of S. mutans biofilm under microgravity
caries (Banas, 2004). Meanwhile, the synergism or antagonism within the oral microbial community is important for bacterial equilibrium and disease development. The acid-producing bacteria S. mutans within the biofilm ecosystem has the ability to produce acid efficiently by metabolizing carbohydrates, actively inhibiting the growth of other ‘beneficial’ commensal colonizers such as Streptococcus sanguinis (Takahashi & Nyvad, 2011). In contrast, cytotoxic hydrogen peroxide (H2O2)-producing bacteria, such as S. sanguinis, can antagonize mutacin-generating S. mutans (Kreth et al., 2008). Changes in any key environmental factors will then trigger a shift from the balance state of the microflora to a disease-associated species composition (Marsh, 1994). However, the effect of space microgravity environment on the bacterial virulence and microbial equilibrium within the oral biofilm remains to be explored. Several studies have demonstrated that the behavior and virulence determinants of microorganisms can be affected by an exposure to microgravity (Nickerson et al., 2004; Rosenzweig et al., 2010), resulting in a potential impact on human diseases. In microgravity environments, microbial pathogens such as Pseudomonas aeruginosa (Crabbe et al., 2011) and Salmonella Typhimurium (Wilson et al., 2007) demonstrate an enhanced virulence and increased biofilm formation (Crabbe et al., 2008). Since the effects of both the microgravity condition on the virulence factors of S. mutans and the ecological balance of the oral ecosystems remain unknown, we were interested in conducting a study to characterize S. mutans under a simulated microgravity condition. Therefore, in the present study, we explored the effects of simulated microgravity on the physiology and biofilm structure of the main caries pathogen, S. mutans. The dual-species interaction between S. mutans and S. sanguinis under simulated microgravity conditions was also investigated.
Materials and methods Superconducting magnet
To investigate the effects of microgravity on bacteria, we used a ground-based experimental platform for gravitational biology simulated by a large gradient superconducting magnet (JMTA-16T50MF; Japan Superconductor Technology Inc., Tokyo, Japan) (Fig. 1a) (Qian et al., 2013). The superconducting magnet generated different magnetic force fields corresponding to different apparent body force levels. Decreased gravity environments were achieved by changing the location of the cell sample within the magnet.
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Bacterial strains and growth media
Streptococcus mutans UA159 and S. sanguinis ATCC10556 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All bacteria were routinely anaerobically grown at 37 °C (90% N2, 5% CO2, 5% H2) in a brain-heart infusion broth (BHI; Difco, Sparks, MD). Bacteria were seeded in 35-mm tissue culture plates sealed with parafilm. To distinguish gravitational and magnetic field effects, three groups were designed in this study (lg*, 1 g* and 1 g; ‘g’ means gravity and ‘*’ means gravity environment generated by the magnet). The plates of lg* and 1 g* groups were put into special positions on the superconducting magnet (Fig. 1a). The control sample, 1 g, was put in the incubation chamber outside the superconducting magnet. Cultures of S. mutans were sampled at hourly intervals for 10 h. These samples were then plated to determine the viable counts (as colonyforming units, CFU, per mL of culture) over the incubation period for plotting growth curves. Glycolytic pH drop assay
The effect of simulated microgravity on S. mutans glycolysis was measured similar to Xu et al. (2011) with slight modifications. Briefly, bacteria cultured under the environment of lg*, 1 g* and 1 g were harvested at midlogarithmic phase by centrifugation (4000 g, 20 °C, 10 min), washed with 0.5 mM potassium phosphate buffer containing 37.5 mM KCl and 1.25 mM MgCl2 (pH 6.5), and re-suspended (OD600 nm = 0.5) in the same solution. Glucose was added to give a final concentration of 1% (w/v). The decrease in pH as a result of glycolytic activity in the S. mutans cells was monitored over a period of 120 min (Corning pH meter 240; Corning Inc., NY). Acid killing and hydrogen peroxide challenge
The ability of S. mutans cells to withstand acid challenge or oxidative stress was determined using a method modified from a previous study (Wen et al., 2005). Briefly, bacteria used for acid killing and H2O2 challenge assays were first grown under the environment of lg*, 1 g* or 1 g. Cells were then harvested immediately at mid-exponential phase (OD600 nm = 0.5), washed with an equal volume 0.1 M glycine buffer (pH 7.0) and subjected to acid or oxidative stress. For acid-killing assays, S. mutans cells were incubated in 5 mL 0.1 M glycine buffer (pH 2.8) for 30 and 60 min. For oxidative stress tolerance assays, S. mutans cells were re-suspended in 5 mL 0.1 M glycine buffer (pH 7.0). Hydrogen peroxide (H2O2; Fisher Scientific, Fair Lawn, NJ) was then added to the cell
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Fig. 1. Effects of simulated microgravity on cell growth, acid production, and stress tolerance of Streptococcus mutans. (a) Superconducting magnet (JMTA-16T50MF). The superconducting magnet could generate different magnetic force fields, corresponding to different body force levels (lg* and 1 g*). The height of the superconducting magnet is 195 cm, with a diameter of 50 mm, and a 450-mm cylindrical cavity. To develop the biological experiment, equipment matched with the superconducting magnet was designed, including a temperature control system, an object stage, a gas control system, and an observing system. (b) Growth curves of S. mutans under different gravity conditions. (c) Effect of simulated microgravity on acid production of S. mutans UA159. Glycolytic acid production was determined by monitoring the pH decrease in sucrose solution (1%, w/v) over a period of 120 min. Results are the average of three independent experiments and are presented as mean SD. (d) Acid-killing assay. Acid tolerance was determined by measuring the survival rate of S. mutans cells at pH 2.8 at different time points (0, 30, 60 min). (e) H2O2 challenge assay. H2O2 sensitivity was determined by measuring the survival rate of S. mutans cells at an H2O2 concentration of 0.2% at different time points (0, 15, 30 min).
suspension to give a final concentration of 0.2% (v/v). After incubation for 30 and 60 min, catalase (5 mg mL 1; Sigma) was added to inactivate H2O2. The control group was incubated in 0.1 M glycine buffer (pH 7.0) for the same period of time. Serial dilutions were performed in 0.1 M glycine buffer (pH 7.0) and the survival rate was determined by plating the samples in triplicate on BHI plates. Biofilm analysis and structural imaging
The bacterial cells and EPS of the 24-h S. mutans biofilm were labeled with SYTO 9 (Molecular Probes, Invitrogen Corp., Carlsbad, CA) and Alexa Fluor 647-labeled dextran conjugate (Molecular Probes), respectively, as previously described (Xiao et al., 2012). Biofilm images were captured with a Leica DMIRE2 confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with a 609 oil immersion objective lens. The image collection gates were set to 495–515 nm for SYTO 9 and 655–690 nm for Alexa Fluor 647. Each biofilm was scanned at five randomly selected positions, and then the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
confocal image series were generated by optical sectioning each of these positions. Three-dimensional reconstruction of the biofilms and the quantification of EPS/bacteria biomass were performed with IMARIS 7.0.0 (Bitplane, Zurich, Switzerland). Dual-species interaction within the biofilm
Cultures of S. mutans and S. sanguinis were inoculated on saliva-coated glass coverslips in fresh BHI supplemented with 1% sucrose in a 24-well cell culture plate under different gravity conditions. Biofilms established at 24 h were fixed in 4% paraformaldehyde (PFA), bacteria were labeled with oligonucleotide probes (Supporting Information, Table S1) and investigated by speciesspecific fluorescence in situ hybridization (FISH) as described previously (Zheng et al., 2013). Micrographs were sampled from at least five randomly selected positions of each sample. The ratio of S. mutans to S. sanguinis was calculated based on the coverage area of each bacterium, analyzed by IMAGE PRO PLUS 6.0 (Media Cybernetics, Inc., Silver Spring, MD). FEMS Microbiol Lett 359 (2014) 94–101
Characteristics of S. mutans biofilm under microgravity
Quantitative real-time PCR (qRT-PCR)
For quantitative real-time PCR, gene-specific primers were designed (Supporting Information, Table S2). Total bacterial RNA isolation, purification, cDNA reverse transcription, and PCR reactions were performed as previously described (Li et al., 2013). Amplification specificity was assessed using melting curve analysis. Different gene expressions were normalized to the levels of 16S rRNA gene transcripts. The data were analyzed by Bio-Rad CFX MANAGER software (version 2.0) according to the 2 ΔΔ CT method. Statistical analysis
All experiments were performed in triplicate and reproduced at least three separate times. Data were computerized and analyzed using SPSS 18.0 software. One-way ANOVA and Student’s t-test were used to compare the data of two groups (lg* or 1 g* with 1 g). Data were considered significantly different if the two-tailed P-value was < 0.05.
Results Effects of simulated microgravity on cell growth, acid production, and stress tolerance of S. mutans
The superconducting magnet (Fig. 1a) was used to simulate microgravity conditions and to explore the effects of a simulated microgravity condition (lg*) on the growth properties of S. mutans. As shown in Fig. 1b and c, no significant differences with respect to the S. mutans growth kinetics or acid production were observed under the lg* condition, as compared with those under a normal gravity condition (1 g* and 1 g). To rule out the possibility that the cells may re-adapt quickly to gravity, we also measured both the initial and end pH of the cultures under different gravity conditions; no significant differences among the three groups were found (lg*, 1 g* and 1 g) (data not shown). However, as shown in the acid-killing assay (Fig. 1d), the survival rate of the lg* group was significantly increased (3.5-fold) after short-term (30 min) acid treatment (pH 2.8), indicating that the acid resistance ability, which is considered a major cariogenic virulence factor of S. mutans, was enhanced under a simulated microgravity condition. Meanwhile, as demonstrated by the H2O2 sensitivity assay (Fig. 1e), there were no significant differences with respect to the survival rate of S. mutans under different gravity conditions. We also examined the cell morphology and membrane surface characteristics of S. mutans by scanning electron microscopy (SEM) assay. However, as FEMS Microbiol Lett 359 (2014) 94–101
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demonstrated in Supporting Information, Fig. S1, there were no significant differences with respect to the cell morphology and membrane surface characteristics of S. mutans under different gravity conditions. Streptococcus mutans showed abnormal biofilm structure and EPS distribution under simulated microgravity conditions
The biofilm structure and EPS production ability of S. mutans under different gravity conditions were determined by confocal laser scanning microscopy (Fig. 2a). There was no significant difference with respect to the total amount of bacteria and EPS under different gravity conditions (Fig. 2c). However, as shown in Fig. 2a, the biofilm that formed under lg* was thinner and denser than those formed under 1 g* and 1 g. Moreover, we found that the architecture and composition of the S. mutans biofilm formed under lg* was abnormal. The distribution of bacteria and EPS of the lg* group and those of the 1 g* and 1 g groups were significantly different at designated heights (Fig. 2b,H1,H2). The EPS of the lg* group was distributed from top to bottom. By contrast, almost all of the EPS under 1 g* and 1 g was distributed at the bottom (Fig. 2b). The EPS/S. mutans ratio of the lg* group at intermediate heights (3–8 lm) was significantly higher than that of the 1 g* and 1 g groups at relative heights (4–14 lm) (Fig. 2d). However, the EPS/S. mutans ratio of the lg* group at the bottom (1– 2 lm) was significantly lower than that of the 1 g* and 1 g groups at relative heights (1–3 lm). Streptococcus mutans showed a superior competitive advantage over S. sanguinis under a simulated microgravity condition
A unique characteristic of multispecies biofilms is the constant interspecies interaction, either synergistic or antagonistic, among its microbial residents. The dynamic antagonistic interactions are very important for maintaining the balance of microbial ecology and oral health. Therefore, in addition to exploring the biofilm formation of a single bacteria (S. mutans only), we used an S. mutans and S. sanguinis dual-species biofilm model (Zheng et al., 2013) to construct a micro-ecological condition under simulated gravity. FISH was used to visualize the spatio-temporal interactions within the dual-species biofilm community. As shown in Fig. 3a, S. mutans of lg* group showed a superior competitive advantage over S. sanguinis. This result was further confirmed by a CFU counting assay, which revealed that the S. mutans/S. sanguinis ratio of the lg* group was significantly increased (Fig. 3b). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Fig. 2. Effects of simulated microgravity on biofilm architecture and EPS distribution of Streptococcus mutans. (a) Double-labeling of 24-h S. mutans biofilms. Green, bacteria (SYTO 9); red, EPS (Alexa Fluor 647). Images were taken at 609 magnification. The 3-D reconstruction of biofilms and the quantification of EPS/bacteria distribution were both performed with IMARIS 7.0.0. The distributions of EPS and bacteria at different heights (H1: 2 lm and H2: 5 lm) are shown in (b). (c) Quantification of bacteria/EPS biomass was performed with IMARIS 7.0.0. Results are the average of five randomly selected positions of each sample and are presented as mean SD. (d) The ratio of EPS to bacteria at different heights was quantified with IMARIS 7.0.0. Results are the average of five randomly selected positions of each sample and are presented as mean SD.
Expression of bacteriocin-, ATR-, and EPSrelated genes of S. mutans under a simulated microgravity condition
Previous studies have shown that the interspecies interaction between S. mutans and S. sanguinis possibly was mediated through the production of inhibitory compounds, such as bacteriocins and hydrogen peroxide. In this study, we found no differences in the S. sanguinis growth and H2O2 production under different gravity conditions (data not shown). Therefore, we speculated that the ecological imbalance of the S. mutans/S. sanguinis biofilm was probably due to the competitive advantage of S. mutans. Interestingly, as demonstrated by the qRTPCR assay (Fig. 3c), S. mutans genes related to mutacin production (nlmA and nlmC, encoding non-antibiotic ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
bacteriocins mutacin IV, and mutacin V) were significantly up-regulated under a lg* condition by 4.6- and 2.0-fold (P < 0.05), respectively. To validate whether ATR was induced under a simulated microgravity condition, we investigated the expression of ATR-related genes, such as atpA, atpE and ffh (Fig. 4). Although the expression of atpA/E showed no significant difference between three groups, the expression level of ffh, which encodes a bacterial signal recognition particle that is linked to the ATR (Gutierrez et al., 1999), was increased by 1.68-fold (P < 0.05). We also explored how the simulated microgravity condition influences the expression profiles of EPS-related genes, including genes encoding glucosyltransferase (gtfB, gtfC and gtfD), dextranase (dexA), fructosyltransferase (ftf) and fructan hydrolase (fruA). Of these, only the FEMS Microbiol Lett 359 (2014) 94–101
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Fig. 3. Bacterial competition within dual-species biofilms and the expression change of bacteriocin-related genes under a simulated microgravity condition. (a) Streptococcus mutans (green) and Streptococcus sanguinis (red) were labeled with species-specific FISH probes (Zheng et al., 2013). Images were taken at 609 magnification, and representative images are shown. (b) Ratio of S. mutans to S. sanguinis under different gravity conditions in dual-species biofilms. The ratio was calculated as CFU of S. mutans in different gravity conditions/CFU of S. sanguinis in the same gravity condition as S. mutans. (c) qRT-PCR results of bacteriocin genes (nlmA and nlmC) under simulated microgravity condition. Results are the average of three independent experiments and are presented as mean SD. Asterisks indicate significant differences (P < 0.05) compared with 1 g.
Fig. 4. Effects of simulated microgravity on the expression of ATR and EPS synthesis related genes of Streptococcus mutans. qRT-PCR assays were carried out as described in Materials and methods. All target genes were amplified using specific primers. Different gene expressions were normalized to the levels of 16S rRNA gene transcripts, and the folds of expression change were calculated. Representative data are shown. Asterisks indicate significant differences (P < 0.05) compared with 1 g.
expression of dexA was significantly enhanced (1.66-fold, P < 0.05), whereas the expression of gtfC was reduced (0.72-fold, P < 0.05).
Discussion A limitation in the professional dental care available during long space flights would cause difficulties in maintaining oral hygiene and treating tooth decay, resulting in an increased prevalence of dental caries. Previous studies have shown a higher caries incidence in astronauts during FEMS Microbiol Lett 359 (2014) 94–101
prolonged space missions. Caries is a chronic infectious disease that depends on a number of factors, including bacteria, host, diet and time. In the present study, we explored the effects of simulated microgravity on the phenotypic characteristic and interspecies interactions of S. mutans, the main pathogen of dental caries, through a ground-based experimental platform (JMTA-16T50MF) that has been widely used in research and medical applications (Qian et al., 2013; Yu & Shang, 2014). We found that S. mutans under the lg condition could withstand a longer time of acid stress (30 min) (Fig. 1d), indicating that the acid tolerance ability, one of the major cariogenic factors associated with S. mutans, was enhanced under the lg condition. S. mutans has evolved two distinct mechanisms to withstand acid shock – constitutive mechanisms and acid tolerance response (ATR) (Matsui & Cvitkovitch, 2010). In this study, we found that the expression of ffh, which encodes a bacterial signal recognition particle involved in resistance to acid stress (Gutierrez et al., 1999), was significantly up-regulated by 1.68-fold, indicating that the increased resistance of S. mutans to decreased pH (2.8) under the microgravity condition might depend on the activation of ATR. Interestingly, we also noticed that, in contrast to S. mutans of the 1 g group, S. mutans under the microgravity condition formed a denser biofilm (Fig. 2a). Cell density has been shown to modulate the ability of S. mutans to adapt to acid challenge. Moreover, biofilm cells at low cell density exhibited a decreased tolerance to a killing pH of 3.0 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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when compared with cells extracted at high cell density (Li et al., 2001). Our results also indicated that the simulated microgravity was able to alter the EPS distribution of S. mutans (Fig. 2). EPS is mainly responsible for cellular aggregation and surface attachment and plays an important role in dental plaque formation (Koo et al., 2009). A previous study has shown that the structure of EPS can be modified by dextranase (encoded by dexA) (Hayacibara et al., 2004). The presence of dextranase altered the proportions of soluble to insoluble glucan, but had a modest effect on the total amount of glucan formed. In this study, our data revealed that the expression of dexA was significantly enhanced by 1.66-fold, whereas the expression of gtfC was reduced by 0.72-fold. We hypothesize that these changes might influence the EPS composition and thus further modulate the three-dimensional (3D) structure of the S. mutans biofilm. Streptococcus mutans also showed a superior competitive advantage over S. sanguinis under the lg condition (Fig. 3a and b), even though its H2O2 resistance ability was not enhanced (Fig. 1e). As identified by qRT-PCR, genes relating to bacteriocin production (nlmA and nlmC) were significantly up-regulated. Production of mutacin is one of the mechanisms employed by S. mutans to maintain a competitive advantage in the multi-species oral biofilm (Hillman, 2002; Hossain & Biswas, 2011). Previous studies have demonstrated that the transcription of the mutacin gene and the production of mutacin greatly increased with cell density (Merritt et al., 2005; Kreth et al., 2006). Furthermore, the competence regulatory system ComDE (Kreth et al., 2007), as well as many other regulators (Qi et al., 2004; Tsang et al., 2006; Nguyen et al., 2009; Senadheera et al., 2012), are directly or indirectly involved in the regulation of mutacin production. Thereby we hypothesize that, under the lg condition, S. mutans likely enhances its competitiveness through an up-regulation of mutacin production. However, the detailed mechanism of these experimental phenomena warrants further investigation. We believe that it is also necessary to investigate the global changes of S. mutans gene expression levels during lg treatment, as well as to identify the specific regulatory pathways responses to lg signal. In conclusion, we found that the simulated microgravity condition could enhance the acid tolerance ability, modify the biofilm structure of S. mutans, and increase the proportion of S. mutans within a dual-species biofilm, likely through the regulation of gene expression. The enhanced competitiveness of S. mutans under simulated microgravity may cause a multispecies micro-ecological imbalance, which would ultimately result in the initiation of dental caries. Our current findings are consistent with ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
previous studies that have shown an increased incidence of caries in astronauts. Further research is required to explore the detailed mechanisms involved in this phenomenon.
Acknowledgements This work was supported by the National Natural Science Foundation of China (31200985, 81170959 and 81200782), the Scientific Research Foundation for Young Investigators, Sichuan University, China (2012SCU11019), International Science and Technology Cooperation Program of China (2011DFA30940), and State Key Laboratory of Oral Diseases. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
Authors’ contribution X.C. and X.X. contributing equally as first authors.
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Characteristics of S. mutans biofilm under microgravity
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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Scanning electron microscopy assays of Streptococcus mutans cell morphology under the simulated microgravity condition. The experiment was carried out as described in the Supporting Information, Materials and Methods. Representative images are shown. The images were taken at 20 0009 magnification. Scale bars: 5 lm. Table S1. Probes used for FISH assays in this study. Table S2. Primers used for qRT-PCR assays in this study. Data S1. Materials and methods.
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