Fluoride-sensitivity of growth and acid production of oral Actinomyces: comparison with oral Streptococcus

Authors


ABSTRACT

Actinomyces are predominant oral bacteria; however, their cariogenic potential in terms of acid production and fluoride sensitivity has not been elucidated in detail and compared with that of other caries-associated oral bacteria, such as Streptococcus. Therefore, this study aimed to elucidate and compare the acid production and growth of Actinomyces and Streptococcus in the presence of bicarbonate and fluoride to mimic conditions in the oral cavity. Acid production from glucose was measured by pH-stat at pH 5.5 and 7.0 under anaerobic conditions. Growth rate was assessed by optical density in anaerobic culture. Although Actinomyces produced acid at a lower rate than did Streptococcus, their acid production was more tolerant of fluoride (IDacid production 50 = 110–170 ppm at pH 7.0 and 10–13 ppm at pH 5.5) than that of Streptococcus (IDacid production 50 = 36–53 ppm at pH 7.0 and 6.3–6.5 ppm at pH 5.5). Bicarbonate increased acid production by Actinomyces with prominent succinate production and enhanced their fluoride tolerance (IDacid production 50 = 220–320 ppm at pH 7.0 and 33–52 ppm at pH 5.5). Bicarbonate had no effect on these variables in Streptococcus. In addition, although the growth rate of Actinomyces was lower than that of Streptococcus, Actinomyces growth was more tolerant of fluoride (IDgrowth 50 = 130–160 ppm) than was that of Streptococcus (IDgrowth 50 = 27–36 ppm). These results indicate that oral Actinomyces are more tolerant of fluoride than oral Streptococcus, and bicarbonate enhances the fluoride tolerance of oral Actinomyces. Because of the limited number of species tested here, further study is needed to generalize these findings to the genus level.

List of Abbreviations
A.

Actinomyces

ATP

adenosine-5′-triphosphate

EMP

Embden–Meyerhof–Parnas

G3PDH

glyceraldehyde-3-phosphate dehydrogenase

HF

hydrogen fluoride

IDacid production 50

50% inhibitory dose of fluoride on acid production

IDgrowth 50

50% inhibitory dose of fluoride on growth

LDH

lactate dehydrogenase

NAD

nicotinamide adenine dinucleotide (oxidized form)

NADH

nicotinamide adenine dinucleotide (reduced form)

OD

optical density

PEP

phosphoenolpyruvate

PEP–PTS

phosphoenolpyruvate-dependent phosphotransferase

PFL

pyruvate formate–lyase

PPB

potassium phosphate buffer

PPn

polyphosphate

S.

Streptococcus

Similarly to Streptococcus, Actinomyces are predominant oral bacteria [1] that are detected from dental plaque biofilm of healthy tooth surfaces and oral mucosa [2]. This bacterium is also frequently isolated from periodontitis lesions [3-6] and root surface caries [7-10], suggesting that it is associated with various oral diseases. In particular, its role in the causation of root surface caries has been a focus [7, 11]. The importance of this research has further been supported by recent comprehensive analyses of microbiota in root surface caries lesions [12-14].

The main cariogenic factor is acid production by carbohydrate metabolism. Actinomyces species can degrade carbohydrates and produce acids [15]; however, their metabolic pathways differ in part from those of Streptococcus species. In addition to glycolysis (EMP pathway), Actinomyces species have a pathway that utilizes part of the tricarboxylic acid cycle to combine PEP (a metabolic intermediate of the EMP pathway) with bicarbonate to produce succinate [16]. Bicarbonate also stimulates the growth [17, 18] and acid production [19] of Actinomyces. Because the concentration of bicarbonate is 4.6 mM in resting saliva, 30 mM in stimulated saliva [20], and 2.6 mM in plaque fluid of resting plaque (a type of dental plaque with no exogenous carbohydrate supply) [21], oral bacteria are always exposed to somewhere from 2–30 mM bicarbonate. Thus, although bicarbonate is one of the most significant environmental factors in the oral cavity, acid production and growth of the representative oral bacteria, Streptococcus and Actinomyces, have not been evaluated and compared in the presence of bicarbonate.

Fluoride is used to prevent caries worldwide. It is thought that the main preventive effect of topical application is to enhance remineralization and reduce demineralization of the tooth surface [22]. In addition, fluoride is known to inhibit acid production by and growth of various oral Streptococcus, such as Streptococcus mutans and Streptococcus sanguinis [23-27]. Fluoride is also known to inhibit acid production by and growth of Actinomyces species [28]. However, the sensitivity of acid production and growth ability to fluoride in Streptococcus and Actinomyces has not been directly compared under the same conditions.

In the oral cavity, bacteria are always surrounded and affected by several environmental factors, which should therefore be taken into consideration as much as possible when evaluating the physiological activity of bacteria in vitro. Therefore, this study aimed to compare the acid production and growth of representative genera of oral bacteria, Actinomyces and Streptococcus in the presence of bicarbonate and fluoride at acidic and neutral pHs to simulate important environmental factors in the oral cavity.

MATERIALS AND METHODS

Bacterial strains and growth conditions

Actinomyces naeslundii ATCC 12104 (formerly A. naeslundii genospecies 1 ATCC 12104), A. oris WVU 627 (formerly A. naeslundii genospecies 2 WVU 627), S. mutans NCTC 10449 and S. sanguinis ATCC 10556 were assessed in the present study [29]. These bacterial species were chosen because the Actinomyces strains are type strains of representative species of oral Actinomyces detected frequently in the oral cavity, particularly in supragingival plaque [29, 30], whereas S. mutans and S. sanguinis are type strains of representative species of well-known caries-associated bacteria and indigenous oral bacteria. These bacteria were maintained on blood agar plates. The purity of the cultures was checked by culturing on blood agar plates at 37°C for 2–3 days in an anaerobic glove box (N2, 80%; H2, 10%; CO2, 10%; NHC-type; Hirasawa Works, Tokyo, Japan). The bacteria were precultured in a complex medium containing 1.7% tryptone (Difco, Franklin Lakes, NJ, USA), 0.3% yeast extract, 0.5% NaCl, 0.1% NH4HCO3, 0.5% glucose and 50 mM PPB (pH 7.0) and incubated for 2 days (Actinomyces species) or overnight (Streptococcus species) at 37°C in the NHC-type glove box. The bacterial cultures were again transferred into the same medium (Actinomyces species; 0.5% inoculum size, Streptococcus species; 5% inoculum size) and grown under the same conditions. The bacterial cells were harvested by centrifugation (Actinomyces species; 21,000 g for 15 min at 4°C, Streptococcus species; 21,000 g for 7 min at 4°C) in the logarithmic growth phase after incubation for 19–21 hr (A. naeslundii), 13.5–15 hr (A. oris), 4.5–6 hr (S. mutans) and 3.5–6 hr (S. sanguinis). They were then washed three times (Actinomyces species) or twice (Streptococcus species) with 2 mM PPB (pH 7.0) containing 150 mM KCl and 5 mM MgCl2. The cells were suspended again in the same buffer (OD at 660 nm = 3.5). With the exception of centrifugation, washing and preservation of the cells were conducted under anaerobic conditions in an anaerobic glove box (N2, 90%; H2, 10%; NH-type; Hirasawa Works). Double-sealed centrifuge tubes were used to protect the cells from oxygen exposure during centrifugation.

Effects of fluoride on bacterial acid production

Reaction mixtures containing 2.7 mL cell suspensions and 4 mM NaCl or NaHCO3 were prepared, adjusted to pH 7.0 or 5.5 and pre-incubated for 3 min at 37°C. Potassium fluoride (0–225 ppmF) was added to the mixture, which was further pre-incubated for 4 min. Acid production was started by addition of 10 mM glucose. The rate of acid production was calculated by the titration volume of 50 mM KOH at pH 7.0 or 5.5 with a pH-stat (Actinomyces species: AUTO Titrator, model AUT-501, TOA Electronics, Tokyo, Japan; Streptococcus species: AUTO pH stat, model AUT-211S, TOA Electronics). The IDacid production 50 was obtained from fluoride concentration versus acid production curves. The experiments were carried out in NH-type boxes.

Measurement of acidic end-products

The acidic end-products, lactate, acetate, pyruvate, formate, and succinate were quantified, as described previously [31]. Before and 20 min after the addition of glucose, cell suspensions of Actinomyces species (0.45 mL) were sampled and immediately mixed with 0.05 mL of 6 N perchloric acid. The samples were removed from the anaerobic box, filtered through a membrane (pore size 0.20 µm; polypropylene; Toyo Roshi, Tokyo, Japan) and diluted tenfold with 0.2N HCl. The filtrates were analyzed with a carboxylic acid analyzer (Eyela S-3000, Tokyo Rika, Tokyo, Japan).

Effects of fluoride on bacterial growth

The bacterial species were grown under the same conditions for bacterial cell preparation, as described above, except for the presence of potassium fluoride (0–225 ppmF). The OD at 660 nm and pH of culture media were measured at 0, 6, 12 and 36 hr (Actinomyces species) or 0, 3, 6 and 24 hr (Streptococcus species) after inoculation. The growth rates were calculated from the growth curves at the logarithmic phase of growth, that is, at 6–12 hr for Actinomyces species and 0–3 hr for Streptococcus species. The growth rate was defined as ln2/doubling time. The IDgrowth 50 was calculated from the obtained growth rate.

The fluoride concentration of culture medium before and after bacterial growth was measured by a fluoride-combined electrode (Model 9609BNWP, Orion, Cambridge, MA, USA). This measurement was based on the method of Hallsworth et al. [32].

Statistical methods

The data are expressed as mean ± standard deviation. Acid production and growth were analyzed by the Student's one sample t-test and Tukey's test, respectively.

RESULTS

Effects of fluoride and bicarbonate on bacterial acid production

In the absence of fluoride and bicarbonate and at both pH 5.5 and 7.0, Actinomyces (solid lines) produced significantly less acid those did Streptococcus (broken lines, Fig. 1). The amounts of acid produced by Actinomyces were estimated as 12–33% and 9–13% of those produced by Streptococcus at pH 7.0 and pH 5.5, respectively. In the presence of fluoride, acid production by both bacteria decreased in a dose-dependent manner; however, Actinomyces was more tolerant to fluoride than Streptococcus (Fig. 1). For instance, at pH 7.0 and 90 ppmF, acid production by Streptococcus almost ceased, the IDacid production 50 being calculated as 36–53 ppmF, whereas acid production by Actinomyces was maintained and was even higher than that of Streptococcus, with IDacid production 50 of 110–170 ppmF. At pH 5.5, the tolerance of acid production to fluoride decreased to approximately 1/10. The IDacid production 50 of Streptococcus was 6.3–6.5 ppmF at pH 5.5, whereas that of Actinomyces was 10–13 ppmF at this pH.

Figure 1.

Effect of fluoride on acid production by Streptococcus and Actinomyces at pH 7.0 and 5.5. Mean values with standard deviations of three independent experiments are given. Within the same bacterial species and at the same concentrations of fluoride, there were significant differences in the amount of titrated KOH between with bicarbonate and without bicarbonate. *P < 0.05; **P < 0.01.

In the presence of bicarbonate, the amount of acid produced by Actinomyces increased (solid lines, Fig. 1). The IDacid production 50 of Actinomyces increased in the presence of bicarbonate to 220–320 and 17–22 ppmF at pH 7.0 and 5.5, respectively. However, the presence of bicarbonate rarely affected acid production by Streptococcus.

Effect of fluoride and bicarbonate on acidic end-products

In the absence of bicarbonate and at pH 7.0, 80% of the acidic end-products of A. oris was lactate, with small amounts of acetate and formate (Fig. 2). Addition of fluoride decreased the proportion of lactate to 70% at 225 ppmF. In the presence of bicarbonate, the proportion of lactate was about 70% with significant amounts of acetate, formate and succinate. However, the proportions of lactate, acetate, formate and succinate were almost constant, regardless of fluoride concentration.

Figure 2.

Effects of fluoride and bicarbonate on the profiles of acidic end-products from glucose by A. oris at pH 7.0. The data are the means of three independent experiments, except for 225 ppmF with bicarbonate (n = 2).

Effect of fluoride on bacterial growth

Fluoride inhibited growth of both Actinomyces and Streptococcus, the effect increasing in a dose-dependent manner (Fig. 3). However, Actinomyces had greater fluoride tolerance than did Streptococcus. For instance, the growth rates of Actinomyces and Streptococcus at 90 ppmF were estimated as 61–73% and 4–8% of those at 0 ppmF, respectively (Table 1). The IDgrowth 50 values of Actinomyces and Streptococcus were calculated as 130–160 and 27–36 ppmF, respectively. The presence of fluoride also decreased growth yield ([final OD] - [initial OD]) and increased final pH, depending on the growth inhibition (Table 1). In terms of these variables, Actinomyces was more tolerant to fluoride than Streptococcus. The concentration of fluoride in the culture media after bacterial growth was 95 ± 4% of that before bacterial growth, confirming that no consumption or adhesion of fluoride had occurred during growth.

Figure 3.

Effect of fluoride on growth of Streptococcus and Actinomyces. The mean values with standard deviations of three independent experiments are given. Within the same bacterial species, there were significant differences in the OD at 660 nm between with fluoride and without fluoride. *P < 0.05; **P < 0.01.

Table 1. Effect of fluoride on growth of Streptococcus and Actinomyces
Bacterial strainF (ppm)Growth rate (hr−1)Growth yield ([final OD] − [initial OD])Final pH
  • Data are presented as the mean and standard deviation of three independent experiments.
  • Within the same bacterial species, there were significant differences in growth rate between with and without fluoride according to the final OD at 660 nm and the pH.
  • **P <0.01;
  • *P <0.05.
Actinomyces naeslundii ATCC 1210400.11 ± 0.011.26 ± 0.626.30 ± 0.43
 900.08 ± 0.020.30 ± 0.266.70 ± 0.33
 2250.03 ± 0.02*0.01 ± 0.01*6.93 ± 0.17*
Actinomyces oris WVU 62700.28 ± 0.011.85 ± 0.145.33 ± 0.09
 900.17 ± 0.00**1.38 ± 0.08*6.20 ± 0.08**
 2250.06 ± 0.01**0.18 ± 0.02**6.83 ± 0.17**
Streptococcus mutans NCTC 1044900.50 ± 0.162.08 ± 0.065.12 ± 0.15
 450.19 ± 0.101.19 ± 0.33*6.18 ± 0.10**
 900.02 ± 0.02*0.03 ± 0.03**6.90 ± 0.16**
Streptococcus sanguinis ATCC 1055600.50 ± 0.112.40 ± 0.065.32 ± 0.02
 450.08 ± 0.02*0.93 ± 0.13**6.38 ± 0.13**
 900.04 ± 0.06*0.01 ± 0.01**7.00 ± 0.04**

DISCUSSION

In the absence of fluoride, Actinomyces produced significantly less acid than did Streptococcus (Fig. 1). Streptococcus has two sugar uptake mechanisms, the PEP–PTS and permease-mediated systems (reactions 1 and 2, Fig. 4) [33-35]. In contrast, Actinomyces is believed to incorporate sugars mainly by the permease-mediated system [16, 28]. In Actinomyces, incorporated glucose is further phosphorylated by hexokinase, which utilizes GTP or PPn as phosphoryl donors, in lieu of ATP, the latter typically being utilized by other bacteria such as Streptococcus. A previous report that the concentrations of intracellular GTP and PPn are lower than that of ATP in Actinomyces [36] suggests that glucose uptake by Actinomyces is less efficient than that of Streptococcus. This may explain why glycolysis and subsequent acid production are low in Actinomyces; however, further study is needed to clarify the differences in acid production rate among oral bacteria.

Figure 4.

Proposed mechanism of fluoride-derived inhibition of Actinomyces glucose metabolism. The numbers in circles indicate the following enzymes: 1, PEP (glucose phosphotransferase system); 2, hexokinase; 3, G3PDH; 4, enolase; 5, PEP carboxykinase; 6, PEP carboxylase; 7, malate dehydrogenase; 8, fumarate reductase; 9, lactate dehydrogenase; and 10, pyruvate formate-lyase. G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; G3P, glyceraldehyde 3-phosphate; 1,3 DPG, 1,3-diphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate.

Within the Actinomyces species tested, there was difference in acidogenicity; A. oris being more acidogenic than A. naesludnii (Fig. 1). This observation suggests that the former species might be more strongly caries-associated; however, so far, no reported oral microbiota analyses have supported that A. oris (previously A. naeslundii genospecies 2) is more strongly caries-associated than A. naeslundii [37, 38].

In the presence of fluoride, acid production by the bacteria studied was decreased by fluoride in a dose-dependent manner and Actinomyces was more tolerant of fluoride than was Streptococcus (Fig. 1). There are many published reports concerning the effects of fluoride on Streptococcus; these include inhibition of enolase (a glycolytic enzyme) [39-42] and the PEP–PTS pathway [42, 43] and enhancement of intracellular acidification and subsequent impairment of the entire glycolytic metabolisms [27, 42]. In contrast, the mechanism(s) by which fluoride inhibits Actinomyces is still unclear. Both enolases isolated and purified from Actinomyces and Streptococcus were inhibited by fluoride, the fluoride inhibition constant of Actinomyces enolase being only 1.2-fold higher than that of Streptococcus [44]. As stated above, Actinomyces' dependence on the permease-mediated system, that is, its lack of the fluoride-sensitive PEP–PTS pathway for sugar incorporation, might make it relatively insulated from fluoride inhibition. The fluoride sensitivity of both Actinomyces and Streptococcus was higher at acidic than at neutral pH (Fig. 1). It is believed that in Streptococcus some fluoride ions (F) convert to HF, which easily penetrates streptococcal cells. The HF dissociates to F in the cells, causing various inhibitory effects. Since the pKa of HF is 3.15, the acidic environment increases generation of HF and its penetration of cells; thus, the inhibitory effects of fluoride can be enhanced under acidic conditions [24, 27, 45-48]. This would also be the case for Actinomyces.

The presence of bicarbonate increased acid production by and decreased fluoride sensitivity of Actinomyces (Fig. 1). Because bicarbonate is in saliva (4.6–30 mM) [20] and plaque fluid (about 2.6 mM), oral bacteria are always exposed to it [21]. The concentration of bicarbonate in the present study (4 mM) was similar to that in dental plaque, supporting stimulation of Actinomyces acid production by bicarbonate in vivo in the oral cavity. G3PDH, a glycolytic enzyme, is a rate-limiting enzyme of glycolysis, the activity of which is regulated by the intracellular NADH/NAD ratio (Fig. 4). An increase in NADH (with an increase in NADH/NAD ratio) suppresses the activity of G3PDH, whereas a decrease in NADH (with an decrease in NADH/NAD ratio) promotes this activity [19]. In the absence of bicarbonate, re-oxidation of NADH to NAD is catalyzed only by LDH (reaction 9, Fig. 4), resulting in a high NADH/NAD ratio and subsequent low glycolytic activity through repression of G3PDH [19]. On the other hand, in the presence of bicarbonate, oxaloacetate is synthesized from PEP by assimilation of bicarbonate by PEP carboxykinase and PEP carboxylase (reactions 5 and 6, Fig. 4), and further reduced to succinate by malate dehydrogenase and fumarate reductase with efficient re-oxidation of NADH to NAD (reactions 7 and 8, Fig. 4). A low NADH/NAD ratio preserves the activity of G3PDH, resulting in efficient glycolysis and acid production [19]. The increase in fluoride tolerance of Actinomyces acid production in the presence of bicarbonate (Fig. 1) might be attributable to stimulation of glycolysis by bicarbonate, which compensates for the inhibitory effect of fluoride on glycolysis. In addition, a low NADH/NAD ratio decreases the dependence on LDH for NADH oxidation and some pyruvate can be catalyzed by PFL (reaction 10, Fig. 4), resulting in production of acetate and formate without NADH oxidation [19]. The increase in production of succinate, formate and acetate in the presence of bicarbonate (Fig. 2) is attributable to these biochemical mechanisms. In the absence of bicarbonate, fluoride decreased the proportion of lactic acid from 80% to 70% with increases in formate and acetate (Fig. 2), suggesting that the slowdown of glycolysis by fluoride causes a shift of pyruvate conversion enzyme from LDH to PFL, as previously reported for Streptococcus [49].

The growth of both Actinomyces and Streptococcus was decreased by fluoride in a dose-dependent manner, Actinomyces having greater fluoride tolerance than Streptococcus (Fig. 3 and Table 1). The similarity with fluoride inhibition of acid production (Fig. 1) suggests that the growth energy of Actinomyces and Streptococcus is mainly supplied by carbohydrate metabolism. However, growth of Actinomyces almost ceased in the presence of 225 ppmF, whereas acid production continued (Figs. 1,3 and Table 1), probably because fluoride suppresses not only glycolysis, but also other biochemical reactions involved in growth ability. Actinomyces species are known to synthesize glutamate and utilize it for growth [50], suggesting that fluoride can also inhibit amino acid metabolism such as glutamate synthesis.

In conclusion, acid production by and growth ability of Actinomyces were lower than those of Streptococcus; however, Actinomcyes was more tolerant to fluoride than Streptococcus. In addition, bicarbonate enhanced not only acid production by but also fluoride tolerance of Actinomyces. Most studies of fluoride inhibition of bacteria have used Streptococcus. The present study suggests that Actinomyces should also be considered in regard to use of fluoride for prevention of dental caries, especially root surface caries. However, because of the limited number of species tested, further study is needed to allow generalization to the genus level.

ACKNOWLEDGMENTS

This study was supported in part by Grants-in-Aid for Scientific Research B (No. 22390399) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

DISCLOSURE

None of the authors has any conflicts of interest associated with this study.

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