1
Inhibition of different strains of Streptococcus mutans at different concentrations of Fluoride and Chlorhexidine
André Lund & Christian Tutumlu Tutor: Nicklas Strömberg
Abstract 207 Text 3907
2 ABSTRACT
Background: The most common species associated with dental caries is Streptococcus mutans. Different Streptococcus mutans adhesion biotypes, SpaP A/B/C and Cnm/Cbm, with ability to predict individual caries development have recently been identified.
Aim: The aim of the study was to investigate if there was a difference in growth ability of the Streptococcus mutans adhesion biotypes and their relative sensitivity to biocides such as fluoride and chlorhexidine in vitro. We also aim to compare the potency of biocides in vitro to those concentrations used in the clinic.
Methods: Culturing of the Streptococcus mutans biotypes in a planktonic solution with and without fluoride and chlorhexidine. Used concentrations: 3.5-4500 parts per million fluoride and 15-500 parts per million chlorhexidine. Optical density was used to measure growth under the different conditions.
3 BACKGROUND
The most common species of bacteria and one of the earliest colonizers, from birth, that can be found in the oral cavity is Streptococcus spp. (Jewetz et al. 2007; Fejerskov et al. 2015). Although knowing that Streptococcus mutans is the main causative microorganism associated with dental caries (Sundas S, Rao A. 2011) numerous studies have shown a distinct but weak relationship between mutans streptococci and caries (Fejerskov et al. 2015).
With dental caries being a common disease worldwide, it has over the years decreased in occurrence and a shift in caries prevalence has surfaced. This has been most common in industrialized countries with low caries prevalence. In Sweden, 10-15% of the population with high caries burden do not respond effectively to traditional standard prevention and dental caries is poorly explained by the traditionally life related factors like oral hygiene, sugar consumption etc. (Källestål 2005). In regard to these high-risk patients, better predictive and preventive tools are needed.
In the treatment and prevention of dental caries fluoride has a good effect (Emilson C.G. 1997; Fejerskov et al. 2015). And with fluoride containing toothpaste a good caries prophylactic method arise with daily usage in conjunction with toothbrushing (SBU 2002), but with the arise of caries-active and high-risk individuals fluoride varnish were developed with the intention to prolong the contact time between fluoride and dental enamel (Bonetti D., Clarkson J.E. 2016). The evidence of fluoride varnish, as a caries inhibitory agent, has been judged to be fair with a caries increment reduction of 25-45% (Bader J.D. et al., 2001; Bonetti D., Clarkson J.E. 2016; Källestål C. 2005).
Fluoride can be stored in plaque for some time before released gradually, post-brushing F concentrations in saliva and plaque has shown to have a marked effect on both experimental caries lesions and caries clinically (Nordström A., Birkhed D., 2013).
4 Over the years it has been shown that S. mutans is a weak predictor of individual caries development. Recently, it was shown that S. mutans biotypes with sucrose-independent adhesion to the host salivary agglutinin (DMBT1), i.e. SpaP (Surface presentation of antigens Protein) A, B or C, and to collagen, i.e. Cnm, Cbm, were better predictors of individual caries development (Esberg et al., 2017). The SpaP B and Cnm biotypes appeared to be of higher virulence and coincided with increased 5-year caries increment. Moreover, their binding to DMBT1 and saliva correlated with higher individual caries scores, with SpaP B being more acid-tolerance. These adhesion properties of the S. mutans biotypes, allows them to colonize naked and cavitated tooth surface and promote plaque growth, which in turn enhance the cariogenic effect (Nobbs et al. 2009).
These findings provide a path for individualized oral care in terms of risk assessment and prevention, with more focus on the high caries risk patients (Esberg et al., 2017). With fluoride having a effectiveness in the control of dental caries with its capability of remineralization, make the tooth more resistant to demineralization, counteract dental biofilm formation and metabolization, but also with its antibacterial and bacteriostatic effect, it is an interesting biocide to further investigate (Fejerskov et al. 2015; Van Loveren C., 2001; Loesche WJ et al., 1975). The effect on bacterial metabolism by fluoride can act directly as an inhibitor of the enzyme glycolytic enolase, by direct action on inhibition of heme-based peroxidases with binding of fluoride to heme, and by formation of metal-fluoride complexes which inhibit the proton-translocation F-ATPases (Marquis R.E., 1990). Although, it is not clear to what extent antimicrobial activity contribute to caries prevention (Van Loveren C., 2001). With this information are the recommended concentrations of fluoride in our prophylactic dental products, which is recommended in the medical guidelines of “Region Västerbotten” and the “Swedish agency for health technology assessment and assessment of social services” (SBU) the year 2002, sufficient to establish an antibacterial and/or an anticaries effect? We know that for the oral bacteria to resist the toxic effect of fluoride, they are able to develop fluoride-resistance through either temporary fluoride-resistance with phenotypic adaptation or more long-lasting resistance with genotypic changes (Liao Y. et al., 2017).
5 sensitive. CHX may also inhibit the enzyme glucosyltransferase, which is essential for bacterial accumulation on tooth surfaces, and the enzyme phosphoenolpyruvate phosphotransferase which is essential for sucrose metabolism (Fejerskov et al. 2015).
The aims of the study i) were to investigate differences in growth between S. mutans adhesion biotypes, ii) examine if there is a difference in sensitivity to biocides regarding growth inhibition or tolerance in vitro between the different biotypes of S. mutans; SpaP A, -B, -C, Cnm positive and negative and Cbm.
METHODS
S. mutans adhesion biotypes used
The biotypes of S. mutans comprise of Cnm positive, Cnm negative, Cbm positive, SpaP A, -B and -C. The isolates of S. mutans biotypes, used in this study, were stored in Brain Heart Infusion (BHI) containing 17% glycerol and frozen in -80°C.
Growth of bacterial strains
The 6 different biotypes of S. mutans were cultured on Blood agar plates (BAPs) for 20h (37°C) in two consecutive repetition to wake the frozen bacteria to a more active and metabolized form. Each biotype was then harvested with sterile swap and suspended in separate test tubes in a solution media, Jordan’s Streptococci Broth, prepared according to the recipe of C. G. Emilson (Gold et al. 1973; Emilson & Bratthall 1976; Jordan et al. 1987; Wan 2002; Hildebrandt & Bretz 2006).
Measurement and dilution of strain-solutions with Fluoride and CHX
6 concentration of biocide, NaF or CHX. A negative control (only Jordan broth) was also present.
Regarding NaF, the sterile fluoride solution used in this experiment was made by mixturing NaF powder with autoclaved H2O. The solution was then filtered through a sterile filter (SARSTEDT Filtropur S 0.45) before mixed with the Jordan broth. In purpose to minimize the dilute factor when mixing the nutrient Jordan broth with the fluoride solution we mixed the fluoride solution with a 2:1 concentration of Jordan broth, which gives us a fluoride-Jordan solution with a 1:1 concentration of Jordan broth which coincides with the Jordan-concentration used for the bacterial solutions. We then mixed the fluoride-Jordan solution with the bacterial solution in the ratio 1:1. By doing this we avoided nutritional deficiency. After biocide addition OD measurements were taken every 30 min, in 550nm wavelength, under 3h until 6 measurements were taken.
Regarding the experimental test with CHX, only two S.mutans biotypes were subject of testing, Cnm+ and SpaP B. The manner of conduction was in similarity with the one for fluoride. But in preparing the CHX-Jordan solution, CHX (Corsodyl 2mg/ml) was mixed directly with a 1:1 Jordan broth, achieving the same broth concentration as the bacterial solutions.
Every diluting and testing process were conducted constantly in a heating chamber (34-37°C) for achieving optimal growth and with clinical nylon gloves and mouthguard for minimal contamination risk. This experimental conduction was executed for three repetitions, in the purpose of achieving accurate results and to minimize the risk of error and blunder factors. Biocide concentrations
In order to establish fluoride concentration-intervals relevant to our experimental cause, several experimental tests in a gradient of different concentrations were conducted, with basis from dental-product concentrations.
7 Regarding the biocide CHX, an experimental test with the concentrations of 500, 250, 125, 62, 31 and 15 ppm CHX were conducted in one repetition.
Ethical considerations
In this study the testing and experimental conduction only uses the bacterial isolates derived from patients, further cultured in the laboratory without handling any personal information. These bacterial isolates cannot be traced to any of the sampled children and thus does not trespass in any personal integrity. With regard to our laboratory type of study we assess, according to the ethical recommendations of the Helsinki Convention, that an ethical trial is not needed. We assess there’s a usefulness in search for a better understanding and new approaches in treating patients with high caries susceptibility due to colonization by S. mutans biotypes with higher virulence.
Statistical analysis
The statistical analysis was done by visualizing the growth of S. mutans biotypes in their natural growth and in presence of biocides in different concentrations. When a distinct difference in growth was observed between any of the biotypes (both with and without biocide) a more thorough analysis was made by implementing confidence intervals (95%) to visualize and further clarify the difference. This was done by calculating mean values for SpaP B and SpaP C for all three measurement (see Table 2 suppl.)
RESULTS
Growth of the adhesion biotypes
When observing the natural growth of each biotype without biocide present, similar growth patterns were observed for all three separate experiments (Fig 1a). The growth tended to be slower for the SpaP C adhesion biotype and somewhat higher for SpaP B (Fig 1b). Comparison of mean values between SpaP B and SpaP C for all occasions of measure can be seen in suppl. Table 2 with integrated confidence intervals (95%). The separation in growth between these two curves are apparent and most prominent at time 4h and 5,5h where the confidence intervals separates (Fig 1b). The difference in OD between the mean values for SpaP B and SpaP C are 0.674 at 4h and 0.883 at 5.5h (suppl. Table 2).
Dose-response and minimum inhibition concentration for F-
8 three experimental repetitions on high fluoride concentrations. The lower concentrations of fluoride (112-3.5ppm F-) showed a more variability in growth inhibition between different mutans biotypes (Fig 3 and 4). A rather consistent pattern of growth-inhibition was observed between the different S. mutans biotypes with a gradual degree of inhibition dependent on the concentration of fluoride. Fig 3 shows the six lowest concentrations of fluoride that were tested only on two S. mutans biotypes, Cnm- and Cnm+. The results were similar and showed that the three lowest concentrations (14, 7 and 3.5ppm F-) had a minimal to no inhibition in growth while the concentrations 112ppm, 56ppm and 28ppm had the largest variation in growth-inhibition of the biotypes, from minimal to high growth-inhibition. The growth growth-inhibition seen was dose dependent, with higher inhibition when higher fluoride concentration was added. Fig 4 show one representative diagram of each biotype that were tested with the fluoride concentrations 112ppm, 56ppm and 28ppm, which were conducted in three repetitions. The inhibition pattern shown regarding fluoride was consistent and with no difference among all mutans biotypes. Over 225ppm fluoride concentration caused a total inhibition of growth, 112ppm resulted in approximately 50% inhibition of the growth, and lower concentration of fluoride had a lower degree of inhibition on the different S. mutans biotypes (Fig 3 and 4). No difference in response/sensitivity to fluoride and in growth inhibition was observed between the six different biotypes (Fig 2, 3 and 4).
Dose-response and minimum inhibition concentration for CHX
In the tests with chlorhexidine on the more cariogenic S. mutans strains Cnm+ and SpaP B an inhibition of bacterial growth was observed for all concentrations of chlorhexidine, but a different sensitivity pattern was noted. A higher concentration of CHX (500ppm) caused an “inferior” inhibition than lower concentrations of CHX (Fig 5). the inhibition doesn’t manifest dose-dependent, as was the case with the fluoride tests. We observed a close similarity of inhibition in growth for the 500 ppm and 15 ppm CHX concentration, which had the lowest impact in growth inhibition. The most significant inhibition in growth was found for 62 ppm followed by 125 ppm. It can also be mentioned that SpaP B seems to withstand CHX somewhat better in the lowest concentration. For SpaP B in the presence of 15 ppm CHX a slight increase in growth was noted, compared to Cnm+ where a total inhibition of growth happened from >15ppm CHX.
Clinical Biocide concentrations
9 give an idea on how different concentrations of fluoride and CHX differ in effect on bacterial, biofilm and caries level.
DISCUSSION
In the control proliferation of all S. mutans biotypes, without any biocide added, we observed that the growth of all biotypes used followed a similar pattern of growth. The chosen media/broth thus seems to be suitable for the purpose of this study, and that the method used for growing S. mutans was consistent. The higher growth for SpaP B compared to SpaP C after 4h incubation is interesting since SpaP C type coincides with lower caries development compared to SpaP B (Esberg., et al, 2017). One explanation a higher cariogenicity for SpaP B’s could be the faster proliferation rate in planktonic form, which could be biologically significant, in terms of possible higher establishment on tooth surfaces in culturing microcolonies after brushing and with its sucrose-independent adhesion capacity (Nobbs et al. 2009). Which could imply considerable significance in caries development and progression, where it promotes a selection of SpaP B strains. Moreover, since the planktonic growth medium provides fairly good growth conditions compared to in vivo biofilm conditions it could be that further stress on growth like other bacteria in the oral cavity competing for nutrition in the presence of F and CHX exposure could have a more pronounced effect on the SpaP C type.
Our results indicate that there is no difference in sensitivity between biotypes of S. mutans to fluoride. In all the lower concentrations (<112ppm) of fluoride tested we observed a dose dependent decline in growth of the bacteria for all biotypes (Figure 4). With higher concentrations of fluoride we noted a higher inhibition on growth, where we could see a cut-off point for total inhibition of growth around 225ppm and above. In a clinical sense this indicates that fluoride has an antibacterial effect on bacterial growth no matter the biotype of S. mutans bacteria. With these results we can assume that fluoride use is as beneficial to high risk caries patients infected with more cariogenic strains of S. mutans as it is to patients infected with a less cariogenic S. mutans composition.
10 amount is used. The fluoride concentrations in dental products need to be considered in the diluted process that occurs when used in the oral environment (in vivo) and are therefore not directly comparable with our in vitro results, but gives an idea of sufficient fluoride concentrations relevant to receive an antibacterial effect. This can imply an important factor regarding fluoride concentrations in the purpose to have a good antibacterial effect on patients with high cariogenic biotypes of S. mutans, when only using fluoride products. After brushing teeth with fluoride containing toothpaste the fluoride concentration in proximal saliva is significantly decreased after only 5 min (Nordström A., Birkhed D. 2009), which could argue for the use of fluoride varnish. Optimal concentrations used may also hinder the growth of fluoride resistant strains of S. mutans which have been shown to occur with lower concentrations of fluoride, 400-600ppm, used (Liao Y. et al., 2017), but further studies are needed.
Comparing the results in our study to other similar studies, in regard to the antibacterial effect of fluoride on S. mutans, can be difficult since other studies have tested the antibacterial effect in the metabolic/biofilm form on agar plates, counting CFU’s (Colony Forming Units) of S. mutans in general, while we are studying the bacteria in its planktonic form, in solutions, and looking on its immediate effect on the growth capability, and if there is any significant difference between biotypes. We haven’t found any studies looking at S. mutans and its biotypes and its differences in sensitivity to biocides.
In the case of the test runs with chlorhexidine on the more cariogenic S. mutans strains, Cnm+ and SpaP B, we got a non dose-dependent pattern of inhibition and the explanation for this is uncertain. What can be considered, though, is the fact that since chlorhexidine has a non-specific mechanism of action (Fejerskov et al. 2015) one could assume that lysis of bacteria, which occurs in higher concentrations, could explain the declining curve of the growth in the higher CHX concentration. Why the highest concentration of CHX (500ppm) only results in a total inhibition of growth and no indication of lysis in the OD measurements is unclear. Regardless, we can assume a total bacterial inhibition of growth in the CHX concentration of >15ppm CHX in vitro. A dose-dependent pattern of inhibition could be expected in the lower concentrations of CHX, <15ppm. If further studies should be considered, in the purpose of investigating if sensitivity differences exist between different S. mutans biotypes, these concentrations of CHX are recommended.
11 include only one test run, can’t be made and the results should be considered with caution. A statement about the specific sensitivity of the other S. mutans biotypes to chlorhexidine can’t be made since they were not included in our tests and further studies are needed. But what can be concluded is that with the result of having the growth inhibitory effect in the lower concentrations of 15ppm, it can be stated that dental products with the high concentrations of 0.2% CHX likely are sufficient and efficient enough, and this in regard to the diluting factor and the saliva-clearance factor in mind. This can also can be stated regarding the S. mutans in biofilms (and not only in planktonic form) where a single rinse with 0.2% CHX results in an oral antibacterial effect of 80-95% (Fejerskov et al. 2015).
Regarding strengths and weaknesses of our study there are a few considerations. The study was conducted in an in vitro laboratory environment so it can be difficult to imply our results in a clinical sense, in vivo. However, we can still observe how Streptococcus mutans interact with biocidal agents on a microbiological level. These basic interactions are similar in vitro and in vivo but under different circumstances. Our tests were made with planktonic bacteria mixed with biocides in test tubes which gives a long interaction between the two. On the contrary, in vivo, this interaction is temporary with a more rinse-like situation when brushing teeth or rinsing with fluoride/chlorhexidine. The mouth also has other factors affecting the antibacterial effect, e.g. saliva’s diluting and clearance effect. When drawing conclusions from our study one must remember the different circumstances named above. A biocide agent that effectively kills planktonic bacteria might require 2-1000 times the concentration to show the same effect in biofilm (Fejerskov et al. 2015). This complicates clinical implication from our results. We have, nonetheless, studied many different S. mutans biotypes and concluded that no major differences in growth and sensitivity to biocide exist between these. This can be of advantage to further studies in this area.
12 REFERENCES:
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14 FIGURES AND TABLES
Table 1: Presentation of the effect of fluoride and CHX on bacterial growth, biofilm in vivo and caries reduction in different concentrations and in clinical concentrations from different studies.
a Measure abbreviation: Ig, inhibition in growth. CFU, colony forming units. Pf, preventive fraction. Cr, Caries reduction.
b Intervention abbreviation: Fr, fluoride rinse. Fv, fluoride Varnish. CHXr, chlorhexidine rinse. CHXg, chlorhexidine gel. Sp, special protocol.
c Group/subject abbreviation: Lmcr, low to moderate caries risk. Ahcr, active or high caries risk.
In vitro, planktonic form (Our
experimental
concentrations) a
In vivo (clinical biofilm
reduction) a, b Caries reduction or preventive fraction a, b, c Fluoride >500 ppm NaF (225ppm F-)= Total Ig Fr 900ppm 2/day for 2v= 56% mutans CFU red. in saliva (Shailaja 2016) Fv 22 000ppm 2-4/year= 25-45% Cr (Bader 2001; Bonetti 2016) 250 ppm NaF (112ppm F -)= 80-90% Ig Fr 250ppm 2/day for 2v= 51% mutans CFU red. in saliva (Shailaja 2016) Fr 900ppm 6-27 rinses/year=30-59% Pf among Lmcr (Sköld M.U. et al. 2005) 125 ppm NaF (56ppm F-)= 50 % Ig
Fr 180ppm 1/day for 2y= 15%
red. among Ahcr (Bader 2001)
CHX >31ppm= Total Ig Single rinse 0,2%= 85-95%
bacterial reduction (Fejerskov)
0,05% CHXr Sp= 3% Cr among Ahcr (Bader 2001)
Rinse 0,2% CHX
twice/day= Almost complete dental biofilm inhibition. (Fejerskov)
1% CHXg 4/year= 44% Cr among Ahcr (Bader 2001)
0,2% CHXr? (Studies not
20 APPENDIX/SUPPLEMENT
Table 2: Presenting mean values for SpaP B and SpaP C from the three test runs of growth in only Jordan broth, without any biocide present. A complement to Figure 1b.
Mean values Standard deviation Margin of error
Time SpaP B SpaP C SpaP B SpaP C SpaP B SpaP C
