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Evaluation of oxazaborolidine activity on Streptococcus mutans biofilm formation
International Journal of Antimicrobial Agents 26 (2005) 491–496
Evaluation of oxazaborolidine activity on Streptococcus mutans biofilm formation Adel Jabbour a,b , Morris Srebnik b , Batia Zaks a , Valerie Dembitsky b , Doron Steinberg a,∗ b
a Institute of Dental Sciences, Faculty of Dentistry, Hebrew University-Hadassah, P.O.B 12272, Jerusalem, 91120 Israel Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University, Jerusalem, 91120 Israel
Received 6 June 2005; accepted 7 August 2005
Abstract Dental diseases are among the most prevalent afflictions of humankind. These diseases are associated with the formation of biofilms harbouring pathogenic bacteria. Eight different derivatives of oxazaborolidines were synthesised and evaluated for their affect on Streptococcus mutans adhesion and biofilm formation. Structure–activity relationship was observed. The B-butyl moiety of the oxazaborolidines contributed an anti-adhesion effect for all derivatives, whilst its effect diminished when the boron atom was incorporated in a fused heterocyclic ring. The B-phenyl group induced bacterial adhesion in all tested compounds Oxazaborolidines may serve as novel agents for affecting oral biofilm formation. Moreover, the ability to alter the oxazaborolidine molecule and thus affect biofilms offers an excellent opportunity to study biofilm formation. © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Oxazaborolidines; Biofilm; Dental; Streptococcus mutans
1. Introduction Dental diseases, including tooth decay, gingivitis and periodontitis, are among the most prevalent afflictions of humankind. Oral biofilms harbouring pathogenic bacteria are the major contributing virulence factors associated with these diseases [1,2]. Adhesion of oral bacteria to the tooth surface is facilitated by physical, chemical and biological mechanisms [3,4]. Streptococcus mutans is one of bacterial species that plays a key role in dental biofilm formation and dental caries [1,5]. Controlling the dental biofilm is one of the major approaches to reducing dental caries and periodontal diseases [6,7]. Antibacterial agents are the most common means of affecting the viability of bacteria in biofilms [8]. Although effective, this approach has many clinical disadvantages, primarily the development of secondary infections, and the emergence of resistant bacteria. Possible alternative means of
∗
Corresponding author. Tel.: +972 2 675 8633; fax: +972 2 675 8561. E-mail address: [email protected] (D. Steinberg).
antibacterial therapy for controlling infectious diseases have recently focused on affecting biofilm formation and bacterial accumulation [9]. Boron-containing compounds play a role in several biologically-active compounds. The presence of boron is essential for the activity of a variety of enzymatic systems in plants and animals [10]. Diazaborines have been shown to be active against malaria [11]. Naturally-occurring borate complexes are used as topical antibiotics [12]. Recently, an ␣amido boronic acid proteosome inhibitor, Velcade® , has been approved for use as an antineoplastic agent [13,14]. Compounds containing the unique B N bond have been shown to possess biological efficacy in anticancer, hypolipidaemic and antifungal activities [15]. Oxazaborolidines are compounds possessing a B N bond and are readily obtained from an amino alcohol and boronic acid. Nevertheless, despite their ubiquity in organic synthesis [16,17], the effect of oxazaborolidines on bacterial adhesion, biofilm formation, or any other pharmacological activity has never been reported. We reasoned that since oxazaborolidines contain a five-member boron heterocycle, they may possess other biological activities in addition to their effect
0924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2005.08.018
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on bacterial viability in the plankton environment, as reported recently [18]. Alternative means of therapy have recently focused on affecting bacterial adhesion and biofilm formation rather than bacterial viability. In the present study we investigated the influence of oxazaborolidines on adhesion of S. mutans to a hydroxyapatite (HA) surface as a new generation of compounds for controlling infectious diseases by affecting bacterial adhesion.
3000 × g for 10 min (Universal 16; Hettich, Kirchlengern, Germany) at room temperature. After three washes, the obtained bacterial pellet was re-suspended in buffered KCl. The turbidity of the bacterial suspension was adjusted to an optical density of 1.2 ± 0.1 at 540 nm (Uvikon 810P; Kontron, Basel, Switzerland), following dechaining and dissociation for 10 min in an ultrasonic water-bath (Transonic 460; Elma, Pforzheim, Germany). 2.3. Bacterial adhesion
2. Materials and methods 2.1. Synthesis and design of oxazaborolidine derivatives All compounds (Fig. 1) were synthesised and characterised as described by Jabbour et al. [18], by reacting either phenyl or butyl boronic acid with an amino alcohol. Phenyl and butyl boronic acid were selected as representative structures for non-binding and -complex interactions. The amino alcohols selected for the synthesis represent various degrees of steric hindrance and aqueous solubility. Compounds BNOO1 and BNOO2 represent highly rigid and charged structures compared with the other synthesised compounds that are neutral and not as rigidly structured. These compounds were synthesised to study electrostatic interactions. 2.2. Bacterial growth and radioactive labelling Streptococcus mutans ATCC 27351 was grown overnight in Brain–Heart Infusion (BHI) medium at 37 ◦ C under aerobic conditions enriched with 5% CO2 . Cells were radioactively labelled by supplementation of the growth medium with 7.5 Ci/mL of 3 [H]-thymidine (Amersham, Arlington Heights, IL) [19]. Following incubation, the bacteria were washed three times in buffered KCl (50.0 mmol/L KCl, 0.65 mmol/L KH2 PO4 , 0.35 mmol/L K2 HPO4 , 1.0 mmol/L CaCl2 and 0.1 mmol/L MgCl2 , pH 6.5) and centrifuged at
Bacterial adhesion to HA was performed using a method similar to Schilling and Bowen [20]. Briefly, 40 mg samples of HA beads (ceramic hydroxyapatite type I, 80 m; BioRad Laboratories, Hercules, CA) (surface area, 0.63 cm2 /mg) were equilibrated with three washes of buffered KCl to which 250 L of labelled radioactive bacteria (prepared as described above), 400 L of the tested oxazaborolidine and 50 L of 700 mM sucrose (in buffered KCl solution) were added. The culture medium was incubated for 60 min at 37 ◦ C with gentle rotation. At the end of the incubation period, the beads were washed three times with buffered KCl and then rinsed with 2 mL ethanol into vials containing 10 mL scintillation fluid (Ecoscint A; National Diagnostics, Manville, NJ). The amount of radioactively labelled bacteria adsorbed onto the HA beads was measured by scintillation counter (BETAmatic scintillation counter; Kontron, Basel, Switzerland). Results are expressed as percent of bacterial adhesion compared with a control group containing no agent. 2.4. Preparation of biofilms Homo-species biofilms were constructed in sterile microculture dishes containing multiple wells (Nunclon; Nunc, Roskilde, Denmark). Each well contained 140 L BHI medium, 20 L of an overnight suspension of S. mutans, 20 L of 40% sucrose solution and 20 L of the tested compound. Each oxazaborolidine derivative was tested at three different concentrations (0.2 mM, 0.9 mM and 9.0 mM) in triplicate. Cultures were incubated at 37 ◦ C in a 5% CO2 enriched atmosphere for 24 h. 2.5. Biofilm bacterial viability counts
Fig. 1. Chemical structures of oxazaborolidine derivatives.
Bacteria in biofilms were stained with fluorescent dyes to distinguish between dead and live bacteria (LIVE\DEAD BacLightTM Bacterial Viability Kits; Molecular Probes, Eugene, OR) [21]. Each well was treated with 5 L of freshly prepared stain containing equal volumes of SYTO 9 and propidium iodide diluted (1:50) in distilled deionised water. The biofilms were incubated at room temperature in the dark for 15 min, washed twice with physiological saline and fixed with 2% formaldehyde for 10 min and finally washed with physiological saline. The stained biofilms were examined by a Zeiss LSM 410 confocal laser scanning system attached to a Zeiss Axiovert
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inverted microscope with 40×/1.3 Plan-Neofluar lens. The green fluorescence stain represents live bacteria. In each experiment, exciting laser intensity, background level, contrast and electronic zoom size were maintained at the same level. At least three random fields for each experiment were taken and scored from each sample. Biofilm structure was analysed as a series of horizontal optical sections. Z-series of optical sections were acquired at spacing intervals of 6.75 m from the biofilm base through the specimen’s vertical axis by a computer-controlled motor drive. Image processing of the stained biofilms was performed by the Image-Pro plus program (Media Cybernetics, Silver Spring, MD) to calculate the relative amount of live (greenstained) bacteria. 2.6. Statistical analysis Statistical analyses were performed using the Student’s t-test. The level of significance was determined at P < 0.05.
3. Results All compounds were synthesised and characterised as previously described by Jabbour et al. [18]. The ability of the oxazaborolidines (Fig. 1) to affect bacterial adhesion to HA was tested at a range of concentrations below and above the minimum inhibitory concentration (MIC). At low concentrations up to 1 mM, BNO3, BNO5 and BNOO1 had a minor effect on bacterial adhesion (less than 10% effect) (Fig. 2). BNO1, BNO2 and BNO4 lowered bacterial adhesion by ca. 20%. BNOO2 promoted adhesion by 22% and BNO6 promoted adhesion by 50% (P < 0.05). At higher concentrations of oxazaborolidines (between 6 mM and 120 mM), a dose–response and structure–activity relationship between the different derivatives of the synthesised oxazaborolidines and their effect on bacterial adhesion was observed. In general, compounds that contained a Bbutyl group (BNO3, BNO5, BNO6 and BNOO1) showed a significant (P < 0.05) anti-adhesion effect of 21–73% at their
Fig. 3. Effect of oxazaborolidine derivatives on bacterial adhesion above minimum inhibitory concentrations.
maximum tested concentration (Fig. 3). Replacing the butyl group by a phenyl group (BNO1, BNO2, BNO4, BNOO2) created an adverse effect of increased adhesion of 18–62%. We chose BNO2 (a phenyl derivative of oxazaborolidine) and BNO3 (butyl derivative) as representatives of the oxazaborolidine series for further examination of their influence on the biofilm environment. Using a confocal laser scanning microscopy technique, we have demonstrated that the biofilm formed in the presence of BNO2 was thicker than the biofilm formed in the presence of BNO3 (Fig. 4). The antibacterial effect of BNO2 and BNO3 on S. mutans assembled in biofilm indicated that at 0.2 mM BNO2 maintained the same amount (percentage) of live bacteria compared with the control, while a partial antibacterial effect was observed at 0.9 mM, 9 mM and 90 mM. BNO3 reduced bacterial survival in biofilm to 40% at 0.2 mM, and at 90 mM no biofilm growth was detected (Fig. 5).
4. Discussion
Fig. 2. Effect of oxazaborolidine derivatives on bacterial adhesion at subminimum inhibitory concentrations.
Taking into account the vast possibilities of chemical structures incorporating boron, the use of boron-containing compounds in medicine has barely been utilised [22]. Oxazaborolidines represent a novel class of boron-containing compounds that are highly stable and water-soluble. However, their biological activity has been largely ignored. This omission prompted us to undertake an evaluation of this group of compounds. Oxazaborolidines at concentrations below the MIC showed no statistically significant anti-adhesion effect on S. mutans, except for BNO6 and BNOO2, which enhanced
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Fig. 4. Confocal laser scanning microscopy image gallery of biofilm that was grown in the presence of (a) 9 mM BNO2, (b) 9 mM BNO3 and (c) control (no oxazaborolidine added) (green colour indicates live bacteria).
bacterial adhesion. In contrast to the low concentrations, at higher concentrations of oxazaborolidines a dose–response and structure–activity relationship between the synthesised oxazaborolidines and their effect on bacterial adhesion was observed. In general, compounds that contained a B-butyl group (BNO3, BNO5, BNO6 and BNOO1) showed a significant anti-adhesion effect, whilst replacing the butyl group by a phenyl group (BNO1, BNO2, BNO4 and BNOO2) created an adverse effect of increased bacterial adhesion. However, BNOO2 did not significantly affect sucrose-dependent
bacterial adhesion and showed no structure–activity relationship, even when the phenyl or butyl groups were replaced. This seems to indicate that for an anti-adhesion effect, a fivemember heterocyclic compound that incorporates a B-butyl group is desirable rather than a fused five-member ring with a B-butyl group. Owing to the adverse effects of antibacterial agents, the use of anti-biofilm agents has been proposed as a means to affect bacterial pathogenicity. BNO3 acted as an anti-biofilm agent at concentrations higher than its MIC, whilst BNO2
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their local concentration and reaching relatively higher concentrations that lead to the antibacterial effect. In this study we have shown that oxazaborolidines are capable of affecting biofilm formation. The B-butylcontaining derivatives significantly decreased bacterial adhesion and affected bacterial viability in biofilms. Thus, oxazaborolidines have the ability in vitro to act as novel agents in affecting biofilm formation and represent a step in preventing biofilm-associated diseases. Since the use of oxazaborolidines as described here is novel, it offers further elucidation of the mechanism of anti-adhesion. Fig. 5. Percent of viable bacteria in biofilm exposed to different concentrations of BNO2 and BNO3.
Acknowledgments had less impact on the biofilm. It may not be possible to simultaneously affect bacterial viability and prevent adhesion and biofilm formation. Some agents may kill bacteria but not promote their desorption from the biofilm structure, whilst others may do the opposite. Affecting viable counts of bacteria in the biofilm without affecting the biofilm mass itself may lead to further virulence, as dead bacteria may be considered pathogenic triggers that evoke a host defence reaction. Furthermore, excretion of toxins during bacterial lysis may also affect the host’s surrounding environment. Therefore, the basic objective of affecting the virulence of biofilms is to minimise bacterial adherence also at concentrations higher than MIC. Some of the proposed oxazaborolidine derivatives we have synthesised affected bacterial vitality, whilst others also reduced biofilm formation. There are clear clinical advantages of an agent that exhibits both characteristics. Chlorhexidine, one of the most effective antibacterial agents in dental medicine, has a strong antibacterial effect; however, because of its highly positively charged domains, it may promote bacterial adhesion at concentration higher than its MIC value [23]. Similarly, BNO2 affects bacterial viability but does not diminish biofilm formation, indicating its effect more as an antibacterial agent than as an anti-biofilm agent. It has been proposed that agents in the biofilm environment have less of an effect than in a planktonic environment, mainly owing to hindered diffusion across the biofilm layers. We have also observed a difference in killing concentrations of oxazaborolidines in planktonic versus biofilm conditions. At concentrations as high as 90 mM, BNO2 did not have a full antibacterial effect on S. mutans in biofilm, whilst its planktonic MIC was recorded at 6 mM [18]. BNO3 eliminated bacterial viability at concentrations of 90 mM, which is almost 30 times greater that its planktonic MIC [18]. This effect is due to partial exploitation of the potential antibacterial capacity of antibacterial agents in biofilms [24,25]. Both BNO2 and BNO3 have shown partial antibacterial effects in biofilms at concentrations below the planktonic MIC. This may indicate that in addition to their high diffusion characteristics (data not shown), oxazaborolidines may also accumulate in the outer layer of the biofilm, thereby increasing
This research was funded in part by the Israel Science Foundation. This study is part of the PhD dissertation of Adel Jabbour.
References [1] Liljemark WF, Bloomquist C. Human oral microbial ecology and dental caries and periodontal diseases. Crit Rev Oral Biol Med 1996;7:180–98. [2] Steinberg D. Studying plaque biofilms on various dental surfaces. In: An YH, Friedman RJ, editors. Handbook of bacterial adhesion: principles, methods, and applications. Totowa, NJ: Humana Press; 2000. p. 353–70. [3] Bowden GH, Hamilton IR. Survival of oral bacteria. Crit Rev Oral Biol Med 1998;9:54–85. [4] Gibbons RJ. Bacterial adhesion to oral tissues: a model for infectious diseases. J Dent Res 1998;68:750–60. [5] Hamada S, Slade HD. Biology, immunology and cariogenicity of Streptococcus mutans. Microbiol Rev 1980;44:331–84. [6] Marsh PD. Plaque as a biofilm: pharmacological principles of drug delivery and action in the sub- and supragingival environment. Oral Dis 2003;9(Suppl. 1):16–22. [7] Socransky SS, Haffajee AD. Dental biofilms: difficult therapeutic targets. Periodontol 2002;28:12–55. [8] Baehni PC, Takeuchi Y. Anti-plaque agents in the prevention of biofilm-associated oral diseases. Oral Dis 2003;9(Suppl 1):23– 9. [9] Ofek I, Sharon N. A bright future for anti-adhesion therapy of infectious diseases. Cell Mol Life Sci 2002;59:1666–7. [10] Dell B, Brown PH, Bell RW. Boron in soils and plants. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1997. p. 219. [11] Surolia N, RamachandraRao SP, Surolia A. Paradigm shifts in malaria parasite biochemistry and anti-malarial chemotherapy. Bioessays 2002;24:192–6. [12] Gupta UC. Boron and its role in crop production. Boca Raton, FL: CRC Press; 1993. p. 53–85. [13] Broos PF, Kane R, Farrell AT, et al. Approval summary for bortezomib for injection in the treatment of multiple myeloma. Clin Cancer Res 2004;10:3954–64. [14] Cusack JC, Liu R, Houston M, et al. Enhanced chemosensitivity to cpt-11 with proteasome inhibitor ps-341: implications for systemic nuclear factor-kB inhibition. Cancer Res 2001;61:3535–40. [15] Dembitsky VM, Srebnik M. Synthesis and biological activity of ␣amino boronic acids, amine-carboxyboranes and their derivatives. Tetrahedron 2003;59:579–93. [16] Corey EJ, Helal C. Reduction of carbonyl compounds with chiral oxazaborolidine catalysts: a new paradigm for enantioselective
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A. Jabbour et al. / International Journal of Antimicrobial Agents 26 (2005) 491–496 catalysis and a powerful new synthetic method. Angew Chem Int Ed Engl 1988;37:1986–2012. Srebnik M, Deloux L. Asymmetric boron-catalyzed reactions. Chem Rev 1993;93:763–84. Jabbour A, Steinberg D, Valery MD, Moussaieff A, Zaks B, Srebnik M. Synthesis and evaluation of oxazaborolidines for antibacterial activity against Streptococcus mutans. J Med Chem 2004;47: 2409–10. Steinberg D, Sela MN, Kilnger A, Kohavi D. Adhesion of periodontal bacteria to titanium and titanium alloy powders. Clin Oral Implants Res 1998;9:67–72. Schilling KM, Bowen WH. The activity of glucosyltransferase adsorbed onto saliva-coated hydroxyapatite. J Dent Res 1988;67:2–8.
[21] Zaura-Arite E, van Marle J, ten Cate JM. Confocal microscopy study of undisturbed and chlorhexidine-treated dental biofilm. J Dent Res 2001;80:1436–40. [22] Morin C. The chemistry of boron analogues of biomolecules. Tetrahedron 1994;50:12521–69. [23] Maleckas M, Daubaras V, Vatikus V, et al. Increased postoperative peritoneal adhesion formation after the treatment of experimental peritonitis with chlorhexidine. Langenbecks Arch Surg 2004;389:256–60. [24] Gilbert P, Das J, Foley I. Biofilm susceptibility to antimicrobials. Adv Dent Res 1997;11:160–7. [25] Wilson M. Susceptibility of oral bacterial biofilms to antimicrobial agents. J Med Microbiol 1996;44:79–87.
Evaluation of oxazaborolidine activity on Streptococcus mutans biofilm formation Adel Jabbour a,b , Morris Srebnik b , Batia Zaks a , Valerie Dembitsky b , Doron Steinberg a,∗ b
a Institute of Dental Sciences, Faculty of Dentistry, Hebrew University-Hadassah, P.O.B 12272, Jerusalem, 91120 Israel Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University, Jerusalem, 91120 Israel
Received 6 June 2005; accepted 7 August 2005
Abstract Dental diseases are among the most prevalent afflictions of humankind. These diseases are associated with the formation of biofilms harbouring pathogenic bacteria. Eight different derivatives of oxazaborolidines were synthesised and evaluated for their affect on Streptococcus mutans adhesion and biofilm formation. Structure–activity relationship was observed. The B-butyl moiety of the oxazaborolidines contributed an anti-adhesion effect for all derivatives, whilst its effect diminished when the boron atom was incorporated in a fused heterocyclic ring. The B-phenyl group induced bacterial adhesion in all tested compounds Oxazaborolidines may serve as novel agents for affecting oral biofilm formation. Moreover, the ability to alter the oxazaborolidine molecule and thus affect biofilms offers an excellent opportunity to study biofilm formation. © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Oxazaborolidines; Biofilm; Dental; Streptococcus mutans
1. Introduction Dental diseases, including tooth decay, gingivitis and periodontitis, are among the most prevalent afflictions of humankind. Oral biofilms harbouring pathogenic bacteria are the major contributing virulence factors associated with these diseases [1,2]. Adhesion of oral bacteria to the tooth surface is facilitated by physical, chemical and biological mechanisms [3,4]. Streptococcus mutans is one of bacterial species that plays a key role in dental biofilm formation and dental caries [1,5]. Controlling the dental biofilm is one of the major approaches to reducing dental caries and periodontal diseases [6,7]. Antibacterial agents are the most common means of affecting the viability of bacteria in biofilms [8]. Although effective, this approach has many clinical disadvantages, primarily the development of secondary infections, and the emergence of resistant bacteria. Possible alternative means of
∗
Corresponding author. Tel.: +972 2 675 8633; fax: +972 2 675 8561. E-mail address: [email protected] (D. Steinberg).
antibacterial therapy for controlling infectious diseases have recently focused on affecting biofilm formation and bacterial accumulation [9]. Boron-containing compounds play a role in several biologically-active compounds. The presence of boron is essential for the activity of a variety of enzymatic systems in plants and animals [10]. Diazaborines have been shown to be active against malaria [11]. Naturally-occurring borate complexes are used as topical antibiotics [12]. Recently, an ␣amido boronic acid proteosome inhibitor, Velcade® , has been approved for use as an antineoplastic agent [13,14]. Compounds containing the unique B N bond have been shown to possess biological efficacy in anticancer, hypolipidaemic and antifungal activities [15]. Oxazaborolidines are compounds possessing a B N bond and are readily obtained from an amino alcohol and boronic acid. Nevertheless, despite their ubiquity in organic synthesis [16,17], the effect of oxazaborolidines on bacterial adhesion, biofilm formation, or any other pharmacological activity has never been reported. We reasoned that since oxazaborolidines contain a five-member boron heterocycle, they may possess other biological activities in addition to their effect
0924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2005.08.018
A. Jabbour et al. / International Journal of Antimicrobial Agents 26 (2005) 491–496
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on bacterial viability in the plankton environment, as reported recently [18]. Alternative means of therapy have recently focused on affecting bacterial adhesion and biofilm formation rather than bacterial viability. In the present study we investigated the influence of oxazaborolidines on adhesion of S. mutans to a hydroxyapatite (HA) surface as a new generation of compounds for controlling infectious diseases by affecting bacterial adhesion.
3000 × g for 10 min (Universal 16; Hettich, Kirchlengern, Germany) at room temperature. After three washes, the obtained bacterial pellet was re-suspended in buffered KCl. The turbidity of the bacterial suspension was adjusted to an optical density of 1.2 ± 0.1 at 540 nm (Uvikon 810P; Kontron, Basel, Switzerland), following dechaining and dissociation for 10 min in an ultrasonic water-bath (Transonic 460; Elma, Pforzheim, Germany). 2.3. Bacterial adhesion
2. Materials and methods 2.1. Synthesis and design of oxazaborolidine derivatives All compounds (Fig. 1) were synthesised and characterised as described by Jabbour et al. [18], by reacting either phenyl or butyl boronic acid with an amino alcohol. Phenyl and butyl boronic acid were selected as representative structures for non-binding and -complex interactions. The amino alcohols selected for the synthesis represent various degrees of steric hindrance and aqueous solubility. Compounds BNOO1 and BNOO2 represent highly rigid and charged structures compared with the other synthesised compounds that are neutral and not as rigidly structured. These compounds were synthesised to study electrostatic interactions. 2.2. Bacterial growth and radioactive labelling Streptococcus mutans ATCC 27351 was grown overnight in Brain–Heart Infusion (BHI) medium at 37 ◦ C under aerobic conditions enriched with 5% CO2 . Cells were radioactively labelled by supplementation of the growth medium with 7.5 Ci/mL of 3 [H]-thymidine (Amersham, Arlington Heights, IL) [19]. Following incubation, the bacteria were washed three times in buffered KCl (50.0 mmol/L KCl, 0.65 mmol/L KH2 PO4 , 0.35 mmol/L K2 HPO4 , 1.0 mmol/L CaCl2 and 0.1 mmol/L MgCl2 , pH 6.5) and centrifuged at
Bacterial adhesion to HA was performed using a method similar to Schilling and Bowen [20]. Briefly, 40 mg samples of HA beads (ceramic hydroxyapatite type I, 80 m; BioRad Laboratories, Hercules, CA) (surface area, 0.63 cm2 /mg) were equilibrated with three washes of buffered KCl to which 250 L of labelled radioactive bacteria (prepared as described above), 400 L of the tested oxazaborolidine and 50 L of 700 mM sucrose (in buffered KCl solution) were added. The culture medium was incubated for 60 min at 37 ◦ C with gentle rotation. At the end of the incubation period, the beads were washed three times with buffered KCl and then rinsed with 2 mL ethanol into vials containing 10 mL scintillation fluid (Ecoscint A; National Diagnostics, Manville, NJ). The amount of radioactively labelled bacteria adsorbed onto the HA beads was measured by scintillation counter (BETAmatic scintillation counter; Kontron, Basel, Switzerland). Results are expressed as percent of bacterial adhesion compared with a control group containing no agent. 2.4. Preparation of biofilms Homo-species biofilms were constructed in sterile microculture dishes containing multiple wells (Nunclon; Nunc, Roskilde, Denmark). Each well contained 140 L BHI medium, 20 L of an overnight suspension of S. mutans, 20 L of 40% sucrose solution and 20 L of the tested compound. Each oxazaborolidine derivative was tested at three different concentrations (0.2 mM, 0.9 mM and 9.0 mM) in triplicate. Cultures were incubated at 37 ◦ C in a 5% CO2 enriched atmosphere for 24 h. 2.5. Biofilm bacterial viability counts
Fig. 1. Chemical structures of oxazaborolidine derivatives.
Bacteria in biofilms were stained with fluorescent dyes to distinguish between dead and live bacteria (LIVE\DEAD BacLightTM Bacterial Viability Kits; Molecular Probes, Eugene, OR) [21]. Each well was treated with 5 L of freshly prepared stain containing equal volumes of SYTO 9 and propidium iodide diluted (1:50) in distilled deionised water. The biofilms were incubated at room temperature in the dark for 15 min, washed twice with physiological saline and fixed with 2% formaldehyde for 10 min and finally washed with physiological saline. The stained biofilms were examined by a Zeiss LSM 410 confocal laser scanning system attached to a Zeiss Axiovert
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inverted microscope with 40×/1.3 Plan-Neofluar lens. The green fluorescence stain represents live bacteria. In each experiment, exciting laser intensity, background level, contrast and electronic zoom size were maintained at the same level. At least three random fields for each experiment were taken and scored from each sample. Biofilm structure was analysed as a series of horizontal optical sections. Z-series of optical sections were acquired at spacing intervals of 6.75 m from the biofilm base through the specimen’s vertical axis by a computer-controlled motor drive. Image processing of the stained biofilms was performed by the Image-Pro plus program (Media Cybernetics, Silver Spring, MD) to calculate the relative amount of live (greenstained) bacteria. 2.6. Statistical analysis Statistical analyses were performed using the Student’s t-test. The level of significance was determined at P < 0.05.
3. Results All compounds were synthesised and characterised as previously described by Jabbour et al. [18]. The ability of the oxazaborolidines (Fig. 1) to affect bacterial adhesion to HA was tested at a range of concentrations below and above the minimum inhibitory concentration (MIC). At low concentrations up to 1 mM, BNO3, BNO5 and BNOO1 had a minor effect on bacterial adhesion (less than 10% effect) (Fig. 2). BNO1, BNO2 and BNO4 lowered bacterial adhesion by ca. 20%. BNOO2 promoted adhesion by 22% and BNO6 promoted adhesion by 50% (P < 0.05). At higher concentrations of oxazaborolidines (between 6 mM and 120 mM), a dose–response and structure–activity relationship between the different derivatives of the synthesised oxazaborolidines and their effect on bacterial adhesion was observed. In general, compounds that contained a Bbutyl group (BNO3, BNO5, BNO6 and BNOO1) showed a significant (P < 0.05) anti-adhesion effect of 21–73% at their
Fig. 3. Effect of oxazaborolidine derivatives on bacterial adhesion above minimum inhibitory concentrations.
maximum tested concentration (Fig. 3). Replacing the butyl group by a phenyl group (BNO1, BNO2, BNO4, BNOO2) created an adverse effect of increased adhesion of 18–62%. We chose BNO2 (a phenyl derivative of oxazaborolidine) and BNO3 (butyl derivative) as representatives of the oxazaborolidine series for further examination of their influence on the biofilm environment. Using a confocal laser scanning microscopy technique, we have demonstrated that the biofilm formed in the presence of BNO2 was thicker than the biofilm formed in the presence of BNO3 (Fig. 4). The antibacterial effect of BNO2 and BNO3 on S. mutans assembled in biofilm indicated that at 0.2 mM BNO2 maintained the same amount (percentage) of live bacteria compared with the control, while a partial antibacterial effect was observed at 0.9 mM, 9 mM and 90 mM. BNO3 reduced bacterial survival in biofilm to 40% at 0.2 mM, and at 90 mM no biofilm growth was detected (Fig. 5).
4. Discussion
Fig. 2. Effect of oxazaborolidine derivatives on bacterial adhesion at subminimum inhibitory concentrations.
Taking into account the vast possibilities of chemical structures incorporating boron, the use of boron-containing compounds in medicine has barely been utilised [22]. Oxazaborolidines represent a novel class of boron-containing compounds that are highly stable and water-soluble. However, their biological activity has been largely ignored. This omission prompted us to undertake an evaluation of this group of compounds. Oxazaborolidines at concentrations below the MIC showed no statistically significant anti-adhesion effect on S. mutans, except for BNO6 and BNOO2, which enhanced
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Fig. 4. Confocal laser scanning microscopy image gallery of biofilm that was grown in the presence of (a) 9 mM BNO2, (b) 9 mM BNO3 and (c) control (no oxazaborolidine added) (green colour indicates live bacteria).
bacterial adhesion. In contrast to the low concentrations, at higher concentrations of oxazaborolidines a dose–response and structure–activity relationship between the synthesised oxazaborolidines and their effect on bacterial adhesion was observed. In general, compounds that contained a B-butyl group (BNO3, BNO5, BNO6 and BNOO1) showed a significant anti-adhesion effect, whilst replacing the butyl group by a phenyl group (BNO1, BNO2, BNO4 and BNOO2) created an adverse effect of increased bacterial adhesion. However, BNOO2 did not significantly affect sucrose-dependent
bacterial adhesion and showed no structure–activity relationship, even when the phenyl or butyl groups were replaced. This seems to indicate that for an anti-adhesion effect, a fivemember heterocyclic compound that incorporates a B-butyl group is desirable rather than a fused five-member ring with a B-butyl group. Owing to the adverse effects of antibacterial agents, the use of anti-biofilm agents has been proposed as a means to affect bacterial pathogenicity. BNO3 acted as an anti-biofilm agent at concentrations higher than its MIC, whilst BNO2
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their local concentration and reaching relatively higher concentrations that lead to the antibacterial effect. In this study we have shown that oxazaborolidines are capable of affecting biofilm formation. The B-butylcontaining derivatives significantly decreased bacterial adhesion and affected bacterial viability in biofilms. Thus, oxazaborolidines have the ability in vitro to act as novel agents in affecting biofilm formation and represent a step in preventing biofilm-associated diseases. Since the use of oxazaborolidines as described here is novel, it offers further elucidation of the mechanism of anti-adhesion. Fig. 5. Percent of viable bacteria in biofilm exposed to different concentrations of BNO2 and BNO3.
Acknowledgments had less impact on the biofilm. It may not be possible to simultaneously affect bacterial viability and prevent adhesion and biofilm formation. Some agents may kill bacteria but not promote their desorption from the biofilm structure, whilst others may do the opposite. Affecting viable counts of bacteria in the biofilm without affecting the biofilm mass itself may lead to further virulence, as dead bacteria may be considered pathogenic triggers that evoke a host defence reaction. Furthermore, excretion of toxins during bacterial lysis may also affect the host’s surrounding environment. Therefore, the basic objective of affecting the virulence of biofilms is to minimise bacterial adherence also at concentrations higher than MIC. Some of the proposed oxazaborolidine derivatives we have synthesised affected bacterial vitality, whilst others also reduced biofilm formation. There are clear clinical advantages of an agent that exhibits both characteristics. Chlorhexidine, one of the most effective antibacterial agents in dental medicine, has a strong antibacterial effect; however, because of its highly positively charged domains, it may promote bacterial adhesion at concentration higher than its MIC value [23]. Similarly, BNO2 affects bacterial viability but does not diminish biofilm formation, indicating its effect more as an antibacterial agent than as an anti-biofilm agent. It has been proposed that agents in the biofilm environment have less of an effect than in a planktonic environment, mainly owing to hindered diffusion across the biofilm layers. We have also observed a difference in killing concentrations of oxazaborolidines in planktonic versus biofilm conditions. At concentrations as high as 90 mM, BNO2 did not have a full antibacterial effect on S. mutans in biofilm, whilst its planktonic MIC was recorded at 6 mM [18]. BNO3 eliminated bacterial viability at concentrations of 90 mM, which is almost 30 times greater that its planktonic MIC [18]. This effect is due to partial exploitation of the potential antibacterial capacity of antibacterial agents in biofilms [24,25]. Both BNO2 and BNO3 have shown partial antibacterial effects in biofilms at concentrations below the planktonic MIC. This may indicate that in addition to their high diffusion characteristics (data not shown), oxazaborolidines may also accumulate in the outer layer of the biofilm, thereby increasing
This research was funded in part by the Israel Science Foundation. This study is part of the PhD dissertation of Adel Jabbour.
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