JAC Advance Access originally published online on March 20, 2006
Journal of Antimicrobial Chemotherapy 2006 57(5):865-871; doi:10.1093/jac/dkl085
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Effect of different iodine formulations on the expression and activity of Streptococcus mutans glucosyltransferase and fructosyltransferase in biofilm and planktonic environments
1 Institute of Dental Sciences, Faculty of Dentistry, Hebrew University-Hadassah, Jerusalem, Israel; 2 Department of Pharmacology, School of Pharmacy, Faculty of Medicine, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel; 3 Department of Pharmacology and School of Pharmacy, Ben Gurion University of the Negev, Beer-Sheva, Israel
* Corresponding author. Tel: +972-2-6757633; Fax: +972-2-6758561; E-mail: dorons{at}cc.huji.ac.il
Received 19 January 2006; returned 10 February 2006; revised 15 February 2006; accepted 21 February 2006
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Abstract |
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Objectives: The glucosyltransferase (GTF) and fructosyltransferase (FTF) enzymes play a pivotal role in dental biofilm formation as they synthesize polysaccharides that act as the extracellular matrix of the biofilm. Iodine is a unique antibacterial agent that has distinct properties from other conventional antibacterial agents. In this study we have examined the effect of iodine and povidone iodine (PI) on gtf and ftf expression in biofilm and planktonic environments and on immobilized and unbound GTF and FTF activity.
Methods: Real-time reverse transcription–PCR was used to investigate the effect of iodine and PI on ftf, gtfB and gtfC expression. The effect of iodine and PI on GTF and FTF activity was tested using radioactive assays.
Results: Our results indicate that iodine and PI in a tetraglycol carrier cause enhancement of expression of gtfB in Streptococcus mutans in biofilms but not in planktonic bacteria. PI in water induced expression of gtfB and gtfC in planktonic bacteria. However, iodine and PI strongly inhibit polysaccharide production by GTF and to a lesser extent by FTF activity. The inhibitory effect on GTF activity was similar in solution compared to its activity in the immobilized environment. This unique effect may be attributed to the distinct chemical properties of iodine compared with other antibacterial agents.
Conclusions: This study indicates that iodine at sub-bactericidal concentrations demonstrates molecular and enzymatic effects that are highly associated with biofilm formation.
Keywords: povidone iodine , gene expression , enzymatic activity , S. mutans
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Introduction |
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Dental caries is a microbial disease that continues to pose a worldwide health problem. Streptococcus mutans, harbouring the dental biofilm, is the principal aetiological factor of this disease. Its ability to adhere to teeth surfaces is paramount for the progression of the disease.1 The bacterial adhesion mechanism is mediated by several means of which the synthesis of extracellular polysaccharides such as glucans and fructans is cardinal in dental biofilm formation. The above polysaccharides are synthesized by extracellular enzymes glucosyltransferase (GTF) and fructosyltransferase (FTF).2,3
Iodine has long been known as an antibacterial agent.4,5 Several clinical studies have also shown the efficacy of iodine (I2) and povidone iodine (PI) in oral hygiene.6–8 However, limited studies have been performed on iodine's effect on dental biofilm constituents
One of the drawbacks of using iodine is its low solubility in water, as well as its potential staining of teeth. One avenue to overcome these disadvantages is changing the drug delivery of iodine. Iodine complexed with polyvinyl pyrrolidone (PVP), to form PI, increases water solubility, reduces irritation and decreases the staining caused by pure iodine. Apart from their antibacterial activity, PI and iodine are effective in protecting skin damage against chemical9 and thermal10 stimuli. It was shown that iodine or PI formulated in tetraglycol (TG) was more effective than the water-based formulations.
Since formulation plays a crucial role in the pharmacological activity of a drug, the purpose of this study was to investigate the effect of iodine and PI, in a novel carrier, on enzymatic and molecular factors associated with dental biofilm formation.
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Materials and methods |
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Materials
We have formulated several iodine- and PI-containing pharmaceutical formulations: (i) PI/H2O, 10% PI dissolved in water; (ii) PI/TG, 10% PI dissolved in TG; (iii) I2/TG, 2% iodine dissolved in TG; (iv) I2 + KI/H2O, 2% iodine + 2.4% KI dissolved in water. Each formulation was compared with a control (the formulation without the iodine or PI). These formulations served as stock solutions from which PI or iodine were diluted with the reaction medium to form the concentrations indicated in the text.
Bacterial strains and culture conditions
S. mutans UA 159, used in the present study, was grown overnight at 37°C in brain heart infusion (BHI) (Difco, MD, USA) in an atmosphere enriched with 5% CO2.
For biofilm generation, 20 µL of S. mutans culture was placed in 20 mm diameter, 15 mm deep polystyrene multidishes (six wells) and cultivated with 5 mL of BHI supplemented with 2% sucrose at 37°C under anaerobic conditions enriched with 5% CO2. After 18 h of incubation, the spent medium was aspirated from the wells and the biofilm was incubated again in fresh BHI with 2% sucrose, supplemented with the tested formulations (iodine formulations at concentrations of 0.007% iodine or the equivalent iodine in 0.035% PI). After 4 h of incubation, the cells of the biofilms were dislodged into a 2 mL microcentrifuge tube containing 40 mg of glass beads (106 µm diameter; Sigma-Aldrich, St Louis, MO, USA) and 1 mL of TRI Reagent (Sigma).
For planktonic experiments, S. mutans UA 159 was inoculated in BHI supplemented with 2% sucrose. The tubes were incubated in a 5% CO2 atmosphere at 37°C for 18 h supplemented with the above-tested iodine solutions. After incubation, the suspension was centrifuged and the pellet was placed in a 2 mL microcentrifuge tube containing 0.4 mL of glass beads and 1 mL of TRI Reagent (Sigma).
Extraction of total RNA
The above bacterial cells obtained after incubation were disrupted with the aid of a Fast Prep Cell Disrupter (Bio 101; Savant Instruments, Inc., NY, USA), centrifuged and RNA-containing supernatant was supplemented with 1-bromo-3-chloropropane (BCP) (Molecular Research Center, Cincinnati, OH, USA). The upper aqueous phase was precipitated with isopropanol. After centrifugation, the resulting RNA pellet was washed with 75% ethanol and resuspended in diethyl pyrocarbonate (DEPC)-treated water (Invitrogen, Carlsbad, CA, USA). Because of the sensitivity of PCR, residual contaminating DNA was eliminated by incubation of the sample with RNase-free DNase (Promega, Madison, WI, USA). The DNase was then inactivated by incubation at 65°C for 10 min, and the RNA was precipitated with ethanol and suspended in DEPC-treated water. The RNA concentration was determined spectrophotometrically with the aid of a Nanodrop Instrument (ND-1000, Nanodrop Technologies, Wilmington, DE, USA). The integrity of the RNA was assessed by agarose-gel electrophoresis (data not shown).
Reverse transcription
A reverse transcription (RT) reaction mixture (20 µL) containing 50 ng of random hexamers, 10 mM dNTPs mix and 2 µg of total RNA sample was incubated at 65°C for 5 min to remove any secondary structure and placed on ice. Then 10x RT buffer, 25 mM MgCl2, 0.1 M DTT, 40 U of RNaseOUT Recombinant Ribonuclease Inhibitor and 50 U of Super Script II RT (Invitrogen, Life Technologies, Carlsbad, CA, USA) were added to each reaction mixture. After incubation at 25°C for 10 min, the mixture was incubated at 42°C for 50 min. The reaction was terminated by heating the mixture at 70°C for 15 min, and the cDNA samples were stored at 4°C until they were used.
Real-time quantitative PCR
Amplification, detection and analysis of mRNA were performed using the ABI-Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with an SYBR Green PCR Master Mix (Applied Biosystems). All primers were designed using the algorithms provided by Primer Express (Applied Biosystems) for uniformity in size (
100 bp) and melting temperature. For each set of primers, a standard amplification curve was plotted and only those with slope 
–3 were considered reliable primers. Primers and sequences are provided in Table 1.
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The reaction mixture (20 µL) contained 1x SYBR Green PCR Master Mix (Applied Biosystems), 1 µL of the cDNA sample and the appropriate forward (0.5 µM) and reverse PCR primers. PCR conditions included an initial denaturation at 95°C for 10 min, followed by a 40 cycle amplification consisting of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. All primer pairs were checked for primer–dimer formation by using the two-step protocol described above without the addition of RNA template. As an additional control for each primer pair and each RNA sample, the cDNA synthesis reaction was carried out in the absence of reverse transcriptase in order to identify whether the RNA samples were contaminated by residual genomic DNA. The critical threshold cycle (Ct) was defined as the cycle in which fluorescence becomes detectable above the background fluorescence and is inversely proportional to the logarithm of the initial number of template molecules. A standard curve was plotted for each primer set with Ct values obtained from amplification of known quantities of S. mutans cDNA. The standard curves were used for transformation of the Ct values to the relative number of cDNA molecules. The contamination of genomic DNA was determined from control reactions, devoid of reverse transcriptase. The same procedure was repeated for all of the primers.
Effect of iodine formulations on the activity of immobilized GTF and FTF
The effect of iodine formulations on the activity of cell-free GTF and FTF immobilized on hydroxyapatite (HA) was conducted according to an assay described previously11,12 Briefly, 40 mg of HA beads (diameter 80 µm, surface area 40 m2/g; Bio-Rad Laboratories, Hercules, CA, USA) was equilibrated by three washes in buffered KCl, pH = 6.5. The beads were incubated with GTF or FTF prepared as described by Steinberg et al.11 After 2 h of incubation, the HA beads were washed with KCl buffer. The enzyme-coated HA beads were incubated with 200 mM sucrose supplemented with 0.25 µCi/mL [3H-fructose]sucrose for FTF activity, or with [14C-glucose]sucrose (American Radiolabeled Chemicals, Inc., St Louis, MO, USA) for GTF activity, as described above, for 3 h in the absence and presence of various concentrations of the tested iodine formulations. Fructans or glucans synthesized on the HA beads were washed three times with KCl buffer, dried with 4 mL of EtOH and measured in a scintillation counter (Beta-counter, Kontron Basel, Switzerland). Results are presented as percentage enzymatic activity with respect to control (absence of iodine).
Effect of iodine formulations on the activity of unbound GTF and FTF
The effect of iodine formulations on cell-free unbound GTF, prepared as described previously, was tested as follows. The isolated GTF was incubated with 200 mM sucrose, supplemented with [14C-glucose]sucrose (American Radiolabeled Chemicals, Inc.) in 10 mM phosphate buffer (pH 6.5). Iodine formulations at tested concentrations were added to the GTF solution. The reaction was terminated after 3 h of incubation at 37°C by adding ice-cold ethanol to a final concentration of 70%. The ethanol-insoluble polysaccharides were allowed to precipitate overnight at 4°C. The precipitate was collected and washed over a glass fibre filter (GF/C, Whatman, Maidstone, UK) using a multi-sample vacuum manifold (Millipore Corporation, Bedford, MA, USA). The filters were dried, and the radioactively labelled glucans collected on the glass filter were counted in a scintillation counter (Beta-counter, Kontron). Results are presented as percentage enzymatic activity with respect to control (absence of iodine).
The effect of iodine formulations on FTF activity in solution was studied as described above but changing the enzymatic substrate to [3H-fructose]sucrose (NEN, Boston, MA, USA) at 0.25 µCi/mL.
Statistical analysis
Student's t-test was used to calculate the significance of the difference between the mean effect of a given formulation of iodine or PI compared with a placebo. A P value of <0.05 was considered statistically significant.
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Effect of iodine on gtf and ftf expression
All isolated RNA samples contained negligible amounts of double-stranded DNA. An equal amount of total RNA (2 µg) from each phase culture was used for quantification of the transcript levels of the tested genes. Dissociation curves revealed that there were no non-specific products in any amplification reaction. The expression levels of all genes were normalized by using amplification of the 16S rRNA gene of S. mutans as an internal standard.
Real-time PCR was used to quantify the effect of iodine on gtfB, gtfC and ftf gene expression (Figures 1 and 2). In the biofilm environment; PI/TG and I2/TG significantly induced gtfB expression (P < 0.05). Their effect on gtfC was much less and no effect on ftf expression was demonstrated. PI/H2O had little effect on expression of gtfB and no effect on gtfC and ftf expression in biofilm (Figure 1). A different expression profile was observed with planktonic S. mutans. The most profound effect on gene expression in the planktonic environment was demonstrated by PI/H2O, which significantly increased expression of gtfC (P < 0.05), but had less influence on gtfB, while the effect on ftf expression was minor. The other iodine formulations had a minute influence on gtfB, gtfC and ftf expression (Figure 2).
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Effect of iodine formulations on GTF and FTF activity
Iodine and PI in aqueous solutions caused a sharp decrease in the activity of the unbound GTF in solution and of HA-immobilized GTF (P < 0.05) (Figure 3a and b). Both iodine and PI in TG carrier also demonstrated a very sharp inhibitory effect on the immobilized and unbound GTF in solution (P < 0.05) (Figure 4a and b). At the lowest tested concentration of each formulation, the effect on immobilized GTF activity was minor compared with the effect on the unbound GTF in solution.
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However, compared with the effect on GTF, the effect of aqueous iodine and PI formulations on FTF activity was less profound. In general, I2/H2O had a marked inhibitory effect on unbound FTF activity in solution (P < 0.05) but its effect was less than on the HA-immobilized FTF. The same trend was also observed for PI/H2O where the effect was less profound at higher concentrations of iodine (Figure 5). A similar mode of action was also recorded with iodine or PI in TG carrier (Figure 6), although the inhibition of unbound FTF in the presence of iodine in TG was less than in iodine in water.
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Discussion |
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Most of the work on enzymatic inhibitors associated with dental plaque has focused on their effect in the unbound state in a solution environment. However, studying the influence of inhibitors on these biofilm-building enzymes on a surface is of great interest, as this immobilized environmental condition reflects more closely the one on the tooth surface.
Iodine is a well-known antibacterial agent.4 In this study we explored potential effects of iodine on gene expression and the activity of enzymes that are associated with dental biofilm formation.
Both formulations of iodine and PI in TG carrier induced expression of gtfB and gtfC in biofilm, while no effect on ftf expression was observed. However, while PI/H2O formulations induced expression of gtfB and gtfC in planktonic S. mutans, the TG-based iodine and PI compounds had a minor effect on ftf, gtfB and gtfC expression. These differences in the effect of iodine on the tested gene expression imply that bacteria in the immobilized environment of the biofilm are less sensitive to induction by PI/H2O but are most sensitive to this iodine formulation in the planktonic environment. In addition, the selectivity of the effect of iodine on gene expression may also be attributed to the different pharmaceutical carrier in which the iodine is formulated.
The sharp increase in gtf expression, especially gtfB, indicates that iodine may have an adverse effect on the adhesion process. On the one hand iodine inhibits GTF and FTF activity, but on the other hand it may also indirectly induce adhesion by enhancing expression of gtf and ftf. However, it should be noted that expression of GTF and FTF is not an indication of an adhesion process, because their activity requires the presence of sucrose, and without this substrate the adhesion process is strongly reduced.
Clearly, the effect of agents on expression of genes that are heavily involved in biofilm formation is of interest. Using real-time reverse transcription–PCR13 has shown that sucrose, which is the obligate substrate of GTF, affects expression of gtf by enhancing gtfD expression, whereas it reduces expression of gtfB and gtfC. Sato et al.14 have shown that xylitol activates the expression of gbp, a gene encoding the glucans binding protein, an enzyme involved in the glucan adhesion pathway of oral bacteria. According to the results, xylitol, a sugar alcohol not involved in sugar metabolism and pH reduction of oral bacteria, may have an indirect effect on the cariogenic pathway.
It is conceivable that the effect of anti-plaque agents on GTF and FTF activity in solution may differ from their effect on immobilized enzymes.15–17 Most studies on the enzymatic inhibitors of GTF or FTF were conducted using unbound enzymes. In most studies that did compare the effect of an agent on the unbound GTF or FTF activity in solution with the immobilized state it was found that the inhibitory effect was much more pronounced in solution than in the immobilized state.18–21 This difference in activity between the unbound and the immobilized states was attributed mostly to the low diffusion/permeability capability of the agents into the immobilized enzymes and to the change of enzymatic conformation due to the adsorption process.
Our study shows that the inhibitory effect of iodine on unbound GTF activity was similar in the immobilized state. This surprising finding may be attributed to the unique molecular size of iodine compared with all of the other agents reported above. Owing to its small molecular weight, the diffusion capability of iodine is high; therefore, the effective concentration of the iodine in situ in the biofilm microenvironment may be similar to that in solution. This results in a similar inhibitory effect on GTF in solution and in the immobilized state. In addition, iodine is a unique molecule, which, unlike many other antibacterial agents, does not possess a positive charge; thus it may bind to different sites from the cationic antibacterial agents. Binding of an enzyme to the surface causes a conformational change. As a result, additional sites that may act as targets for active agents may be exposed, facilitating easier binding of the iodine to the enzyme and therefore preventing GTF activity. Similarly, an amino alcohol molecule, delmopinol, has been shown to have an even more profound inhibitory effect on immobilized GTF compared with GTF in solution.12
The effect of iodine and PI on FTF activity was not similar to their effect on GTF activity. Unlike GTF, the inhibitory effect of iodine and PI on FTF activity was less profound and their effect on immobilized FTF was not similar to the unbound FTF. This result indicates the specificity of action of iodine and PI on enzymatic activity.
Clearly, agents that possess the same inhibitory effect on immobilized enzymes as on enzymes in solution bear a strong potential antibiofilm effect. The carrier in which the drug is embedded may also affect the bioavailability of iodine and thus may alter its biological activity. Comparison between the effects of PI dissolved in water and PI in TG revealed that the MIC of the latter formulation was four times lower than that of the former (data not shown). The superiority of the TG-containing formulation was also observed in the counter-irritation effect of iodine against chemical9 and thermal10 burns. The TG-related effects may be due to its ability to dissolve molecular iodine in the presence of water, while ethanolic solutions of iodine precipitate in the presence of water unless iodide salt is added to the solution to form the water soluble I3– ion. The association between the physical and pharmacological properties of the TG-containing iodine formulation is under investigation.
Environmental stress conditions may trigger expression of genes.22–24 It may be assumed that iodine may act as a stress factor for bacteria, which in turn evokes a molecular response of high expression of genes such as gtfB, gtfC and ftf. This effect is perceived by a counter effect of iodine and PI that inhibits the activity of those enzymes.
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None to declare.
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Acknowledgements |
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This study was partially sponsored by the Horowitz Applied Research Foundation of the Hebrew University. This study is part of A. T.'s MSc studies.
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References |
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