School of Dentistry, Indiana University, Indianapolis, IN, USA
Department of Pathology and Laboratory Medicine, School of Medicine, Indiana University, Indianapolis, IN, USA
Correspondence: Richard L. Gregory, Indiana University School of Dentistry, 1121 W. Michigan Street, Indianapolis, IN 46202, USA. Tel.: +1 317 274 9949; fax: +1 317 278 1411; e-mail: email@example.com
Both Streptococcus mutans and Streptococcus sanguinis are normal bacterial inhabitants of dental plaque. Streptococcus mutans is the major agent causing dental caries. It has been well documented that nicotine affects the growth of S. mutans. This study investigated the effect of nicotine on mono- and dual-species growth of S. mutans and S. sanguinis. The results indicate that nicotine has no significant effect on S. sanguinis grown in either mono- or dual-species biofilms. However, nicotine significantly increased (P < 0.05) the growth of S. mutans in dual-species biofilm formation. In addition, the CFU level of S. sanguinis was higher than S. mutans without nicotine in the culture. With the addition of nicotine, the level of S. mutans biofilm was significantly enhanced as the nicotine concentration increased over the level of S. sanguinis in dual-species biofilm, and we also got the same result from the fluorescence in situ hybridization detecting the two bacteria grown in biofilm formation. The exopolysaccharide (EPS) of S. mutans has also been increased by the increasing nicotine concentration, while the EPS of S. sanguinis was decreased or inhibited by the affected nicotine. The data further confirm that nicotine is able to enhance the growth of S. mutans.
Streptococcus mutans and Streptococcus sanguinis are important members of the oral biofilm (Nyvad & Kilian, 1987; Marsh, 2003). Dental caries is one of the world's most major and prevalent diseases affecting 60–90% of school children and most of the adult population (Petersen et al., 2005). Streptococcus mutans is considered an important agent in the formation of human dental caries (Loesche, 1986). Streptococcus mutans and S. sanguinis inversely affect each other in the process of oral biofilm formation. We earlier reported that a low molecular weight compound from S. mutans is able to enhance the production of IgA1 protease activity by S. sanguinis (Gregory & Lloyd, 1993). Recent study has reported that S. sanguinis is able to inhibit S. mutans by producing H2O2 in initial biofilm formation, when there is enough oxygen for spxB expression (Zheng et al., 2011). An additional study reported that the existence of certain oral bacteria including S. sanguinis differentially influences biofilm and virulence gene expression of S. mutans (Wen et al., 2010).
Tobacco smoking has a documented effect on human health (Hamada & Slade, 1980), and an in vivo study demonstrated that long-term exposure to cigarette smoke and virus infections can act synergistically to increase the susceptibility of mice to secondary bacterial invasion (Mackenzie & Flower, 1979). Tobacco smoking has important effects on the oral cavity (Marcotte & Lavoie, 1998). Dental caries is considered a major disease in children and adults because of its high prevalence (Kaste et al., 1996; Winn et al., 1996). In recent years, more investigations find that smoking affects caries (Tomar & Winn, 1999; Aguilar-zinser et al., 2008; Tanaka et al., 2009; Baboni et al., 2010; Campus et al., 2011), and some reports looked at the effect of passive smoking by young children and their caries levels (Shenkin et al., 2004; Avşar et al., 2008). Nicotine is an alkaloid and a major component of cigarette smoke. Some studies investigated the influence that tobacco smoking has on both the components of the growing biofilm and the response from the host to the colonization (Vaananen et al., 1994), and additional studies investigated the risk of smoking on caries (Lindemeyer et al., 1981). Our previous study demonstrated that nicotine is able to increase the biofilm and metabolic activity of S. mutans in in vitro study (Huang et al., 2012). Although the close relationship between S. mutans and S. sanguinis has been well documented, no reports concerning on the effect of nicotine on the association between these two bacteria, and with regard to the biofilm formation and cariogenicity of S. mutans have been found. The present study was aimed at investigating the influence of nicotine on the competition between S. mutans and S. sanguinis in biofilm formation.
Materials and methods
Bacterial strains and growth conditions
Streptococcus mutans UA159 (ATCC 700610) and S. sanguinis ATCC 10556 were maintained in tryptic soy broth (TSB; Difco, Detroit, MI). For biofilm formation, both bacteria were grown in TSB with 1% (w/v) sucrose (TSBS) as the supplemental carbohydrate source. Biofilms were incubated in 5% CO2 (v/v) at 37 °C without agitation.
For planktonic growth curve assays of S. mutans and S. sanguinis, overnight bacterial cultures were added to TSB containing twofold serial dilutions of nicotine concentrations: 0–8 mg mL−1. Two hundred microlitre of the bacteria (final concentration 1 × 105 CFU mL−1) in TSB were put into selected wells of a sterile 96-well microtiter plate and incubated at 37 °C in air (keep out of uncontaminated) for 20 h. The turbidity was measured by optical density (OD) at 595 nm using a microplate reader (SpectraMax 190; Molecular Devices, Inc., Sunnyvale, CA) every 20 min. There were three replicates of each bacterium for each nicotine concentration.
Growth of dual-species biofilms
For the dual-species biofilm, a protocol described previously (Wen & Burne, 2002; Wen et al., 2010) was used with sterile glass slides as the substratum. Briefly, overnight bacterial cultures of S. mutans and S. sanguinis were passed by diluting the bacteria 1 : 50 with fresh TSB and incubating the cultures until mid-exponential phase (OD600 nm = 0.5). Each bacterial culture of 450 μL was added to 50-mL sterile polypropylene tubes (Fisher Scientific), which contained TSBS with different concentrations of nicotine (0, 0.25, 0.5, 1, and 2 mg mL−1). Ethanol-flamed sterile glass microscope slides (25 × 75 mm × 1 mm thick; Thermo Scientific) were placed into each tube aseptically to allow biofilm to grow on the slides at 37 °C. The slides were transferred daily to fresh tubes with TSBS containing nicotine. After 4 days, the biofilm was removed from slides using a disposable sterile cell scraper, and the biofilm cells were suspended in saline (0.9%, w/v). Then, biofilm cells were sonicated for 45 s to separate cells using an ultrasonifier (output control at 8, and Duty cycle of 70; Branson Sonifier 450; Fisher Scientific) for two separate cycles, with 2 min on ice between treatments. To determine the total number of viable bacterial cells (colony-forming units, CFU), the dispersed biofilm cells were diluted 1 : 1000 and plated on tryptic soy agar (TSA, total CFU of two bacteria) and mitis salivarius sucrose bacitracin agar (MSSB; for S. mutans) plates. The plates were incubated for 48 h at 37 °C in 5% CO2 (v/v), and the colonies counted using an automated colony counter. The numbers of S. mutans on MSSB were subtracted from the total number of bacteria (S. mutans and S. sanguinis) on TSA to provide the number of S. sanguinis. The data were reported as CFU mL−1.
Confocal laser scanning microscopy
Sterilized coverslips (1 cm in diameter) were placed in standard 24-well tissue culture plates, and bacteria (1 × 106 CFU mL−1) with different nicotine concentration cultures (0, 1, and 2 mg mL−1) were added to each well with the exopolysaccharide (EPS) dye Alexa Fluor® 647 (10 000 MW; Molecular Probes Inc.) to label formed EPS as previously described (Xiao et al., 2012; Zheng et al., 2013). The plate was incubated in 5% CO2 (v/v) at 37 °C for 24 h in the dark. Then, the planktonic cells were removed, and the coverslips were washed with sterile water and dried with a sterile filter paper, keeping in dark. The bacterial cells were labeled with SYTO® 9 nucleic acid stain (Banas et al., 2007; Molecular Probes Inc.) and incubated for 15 min. The coverslips were rinsed using deionized water to remove residual dye and dried with a sterile filter paper. The coverslips were placed on slides and sealed with nonfluorescence oil for laser scanning confocal microscopy (Leica TCS SP2; Leica Microsystems, Wetzlar, Germany) equipped with a 60× oil immersion objective lens. Briefly, for the nucleic acid stain and the EPS dye, 485 and 650 nm, respectively, was used as the absorption maxima wavelength and 498 nm as the emission maxima wavelength. Each sample was scanned at least three randomly selected positions. The three-dimensional reconstruction of the biofilms and the ratio of EPS to S. mutans were performed with Imaris 7.0.0 (Bitplane, Zürich, Switzerland).
Fluorescence in situ hybridization
Sterilized coverslips (1 cm in diameter) were placed in standard 24-well tissue culture plates. We inoculated 24-h dual-species biofilm of S. mutans and S. sanguinis in TSB media with 1% sucrose in different concentrations of nicotine (0, 0.5, 1, and 2 mg mL−1) using the 24-well tissue culture plate. Then, the formed biofilm was detected by fluorescence in situ hybridization (FISH). The oligonucleotide probe 5′-ACTCCAGACTTTCCTGAC-3′ labeled with FITC that identifies S. mutans (Paster et al., 1998) and oligonucleotide probe 5′-GCATACTATGGTTAAGCCACAGCC-3′ labeled with ROX that identifies S. sanguinis were designed in the current study with arb (Linux release arb 5.3) software. The probes are specific to the two bacterial 16S rRNA genes, respectively. We also inoculated dual-species biofilm of S. mutans and S. sanguinis at different growth time, such as 24, 48 and 72 h, with and without nicotine on the glass surfaces in 24-well plates, and the glass coverslips were transferred daily to fresh 24-well tissue culture plates with fresh TSBS containing the same concentrations of nicotine. Biofilm images were captured with a Leica DMIRE2 confocal laser scanning microscope (Leica) equipped with a 60× oil immersion objective lens. Then, the biofilm images were detected using FISH technology, the same as previously described (Zheng et al., 2013). Each sample was captured at least three randomly selected positions. And the ratio of S. mutans to S. sanguinis in dual-species biofilm treated with different nicotine concentrations (left) and different cultured time (right) was calculated with Image Pro Plus 6.0 (Media Cybernetics, Inc., Silver Spring, MD).
Each experiment was repeated at least three times. Differences between the nicotine-treated experimental and nontreated control groups were analyzed by spss software (version 16.0; Chicago, IL). Results are presented as means and standard deviations. One-way analysis of variance (one-way anova) and post hoc Tukey's multiple comparisons tests were performed to compare multiple means. Significance was set at P ≤ 0.05.
The growth curve of S. mutans indicates that mean growth rate was a little increased at logarithmic phase when the nicotine concentration was 0–0.5 mg mL−1 (Fig. 1). And at 0.25 and 0.5 nicotine concentration, the absorbance data were higher than the culture without nicotine at early stationary phase. The growth of S. mutans has been completely inhibited at 4 mg mL−1 of nicotine. While for S. sanguinis (Fig. 1), mean growth rate was not influenced by nicotine concentration at 0–2 mg mL−1. At 4 mg mL−1, the mean growth rate of S. sanguinis has been decreased, and at 8 mg mL−1 of nicotine, the growth of S. sanguinis was completely inhibited.
The dual-species experiments indicated that the CFU level of S. sanguinis was slightly decreased from 4.08 × 107 (without nicotine) to 3.04 × 107 (2 mg mL−1) as the nicotine concentration increased, although it was not statistically significant (Fig. 2). CFU of S. mutans in dual-species experiments was significantly increased (P < 0.05) from 1.91 × 107 (without nicotine) to 7.45 × 107 (2 mg mL−1; Fig. 2).
There was an obvious increase in S. mutans UA159 (green) biofilm mass treated with 1 and 2 mg mL−1 of nicotine compared to the biofilm without nicotine (Fig. 3a). Biofilm structure of S. mutans was much denser, and the bacterial density was increased in the presence of increasing nicotine. And nicotine also enhanced the production of EPS (red) of S. mutans. Each of S. mutans cell was also increased as increasing nicotine concentration (Fig. 3a Bar figure). In contrast, biofilm structure of S. sanguinis was much looser as the concentration of nicotine increased (Fig. 3b), and the EPS production of S. sanguinis biofilm was reduced with the additional nicotine.
At first 24-h biofilm formation of dual-species bacteria, as nicotine concentration increases, the degree of microbial coverage becomes thicker (Fig. 4a) and the ratio of S. mutans/S. sanguinis becomes increased, but lower than 1. The biomass of S. mutans increased by increasing nicotine but it was lower than that of S. sanguinis. At 1 mg mL−1 nicotine concentration (Fig. 4b), the first 24-h biofilm formation, microbial colonization was dominated by S. sanguinis. The degree of microbial coverage as well as the thickness of the biofilm is not very dense. For 48- and 72-h biofilm formation, the degree of microbial coverage became thicker and thicker. What's more, S. mutans grew much better, and the biomass was higher than S. sanguinis.
Previous study has shown the MIC (16 mg mL−1) and MBC (32 mg mL−1) of nicotine to S. mutans (Huang et al., 2012). In the present study, for the growth curve, 4 mg mL−1 nicotine completely inhibited the growth of S. mutans (Fig. 1), which is lower than previous study. The difference between them can be explained by the growth condition and detecting methods. Previous study detected the overnight culture of S. mutans incubated in 95% (v/v) air/5% CO2 (v/v) at 37 °C using a spectrophotometer. In the present study, we detected the growth curve of S. mutans incubated in the microplate reader, as the part of ‘'Planktonic growth' in Materials and methods’ statements. The final temperature of growth curve detected in the spectrophotometer was lower than 37 °C, because the room temperature is kept at 23 °C. And the environmental atmosphere for the growth of S. mutans could not be kept in a facultative anaerobic incubation. Whatever, we got the same result that the growth of S. mutans UA 159 was not significantly affected by nicotine. Planktonic growth curves also gave an indication of the effect on the log phase (mean generation time-MGT) of the bacteria. For S. mutans UA159, the MGT appears to be unchanged at nicotine levels between 0 and 1 mg mL−1, and level of 2 mg mL−1 slows the MGT marginally. It would have been useful therefore to have used 2–4 mg mL−1 for the dual- and mono-species biofilm experiments. The S. sanguinis planktonic experiments showed a slight change in MGT at a nicotine concentration of 4 mg mL−1, while 8 mg mL−1 was inhibitory. As 2 mg mL−1 had no effect in MGT, it was not surprising that the levels of S. sanguinis remained unchanged in the biofilm (mono- and dual-) and planktonic experiments where maximum nicotine levels were only 2 mg mL−1.
The physiological concentration of nicotine in the saliva of smoker ranges from 70 to 1560 μg mL−1 (Hoffmann & Adams, 1981). The levels of nicotine in saliva can be highly variable and depend upon the volume of saliva secreted, the duration of contact with tobacco, the amount of tobacco consumed, measuring time, and the methods used for measurement (Curvall et al., 1990; Schneider et al., 1997; Robson et al., 2010). Accordingly, it is difficult to know the concentration of nicotine in the dental plaque which is actually exposed.
In oral and dual-species biofilm, it is definitely existing bacterial cell–cell interactions, such as competition for binding sites and nutrients. It is well known that metabolites of one bacterium may influence the growth of another organism within the same biofilm. Streptococcus sanguinis is known to inhibit the growth of S. mutans by producing hydrogen peroxide (Kreth et al., 2005, 2008), although it has a limited impact on S. mutans growth when the bacteria were inoculated at the same time (Kreth et al., 2005), as they were in this study (Fig. 2). Streptococcus mutans did not grow as well as S. sanguinis did without nicotine in culture. With the addition of nicotine, the level of biofilm formed by S. mutans was significantly enhanced more than the level of S. sanguinis as the nicotine concentration increased. The amount of early colonizing S. sanguinis may produce sufficient H2O2 to inhibit the attachment of S. mutans to the glass surface. When nicotine was added to the culture, interestingly, the CFU level of S. mutans was increased above the level of S. sanguinis as the nicotine concentration (0.25–2 mg mL−1) was increased. Previous study showed that biofilm formation of S. mutans UA159 was significantly increased between 2 and 8 mg mL−1 nicotine concentration (Huang et al., 2012). The reason is with the competition of S. sanguinis to S. mutans, the activity of S. mutans may be highly stimulated and become more sensitive to nicotine. Nicotine has the ability to enhance the attachment of S. mutans to the glass surface and compete for binding sites with S. sanguinis. More produced EPS (Fig. 3a) demonstrated that nicotine enhanced the competitiveness of S. mutans to the detriment of S. sanguinis in biofilm formation. As EPS is mainly synthesized by glucosyltransferases (gtfs), our previous study also demonstrated that nicotine is able to upregulate gtf genes (Li et al., 2013). With the addition of nicotine to S. sanguinis biofilms, there was no EPS detected in CLSM images (Fig. 3b). We could speculate that probably the incorporation of Dextran, Alexa Fluor® 647; 10 000 MW to the cellular matrix of biofilm looks reduced or maybe nicotine effects the reduction in EPS. Some additional tests are needed to be carried out to demonstrate the reduction in the expression of components of the extracellular matrix or of EPS.
The S. mutans/S. sanguinis ratio (Fig. 2) also demonstrated that higher proportion of S. sanguinis is generally detected with lower levels of S. mutans at a 0 nicotine concentration, while higher levels of S. mutans were correlated with lower numbers of S. sanguinis at 0.25–2 mg mL−1 nicotine concentrations. It suggests that S. sanguinis should have an advantage over S. mutans for available nutrients when incubated in a mixed-species group without nicotine. The disadvantages of competition for nutrients may influence the capability of S. mutans to bind to glass surfaces, contributing to a decrease in biofilm formation when incubated together with S. sanguinis (Fig. 2). With the addition of nicotine, S. mutans was stimulated to produce more extracellular glucan to attach to the glass surfaces through increased EPS produced.
From the FISH result (Fig. 4), the competition between S. mutans and S. sanguinis with 1 mg mL−1 of nicotine in different incubation time was observed: After 24-h co-culture, the growth of S. sanguinis occupies the major position compared with S. mutans with or without nicotine in the culture. While after 48 h, there are more S. mutans growing on the glass slide than S. sanguinis, and both of the two bacteria form a much thicker biofilm on the glass slide than 24 h with and without nicotine. Interestingly, after growing for 72 h, both of the bacteria become less prevalent than at 48 h, and the biofilm is thinner. But the most important thing is that S. mutans is still dominant.
In conclusion, apparently nicotine benefits the growth of S. mutans in its competition with S. sanguinis in approximately half an order of magnitude when both microorganisms grow in dual-species biofilms. Nicotine was clearly able to increase the biofilm growth including EPS of S. mutans. While the exact mechanism still needs to be further investigated, the results obtained on the effect of nicotine between S. mutans and S. sanguinis may partially describe the prevalence and the close relationship of these two bacteria in cariogenic plaque of smokers.
This work was partially supported by NIH grants HL098960 and DE020614, the Indiana University Purdue University Indianapolis Tobacco Cessation and Biobehavioral Group (RLG), and the National Basic Research Program of China (‘973 Pilot Research Program’, 2011CB512108).