International Journal of Antimicrobial Agents 18 (2001) 575–578
`
`www.ischemo.org
`
`Short communication
`Antibacterial effect of parabens against planktonic and biofilm
`Streptococcus sobrinus
`
`Steinberg Doron a,*, Michael Friedman b, Maher Falach a,b, Ester Sadovnic c,
`Hirschfeld Zvia c
`a Department of Oral Biology, Faculty of Dental Medicine, Hebrew Uni6ersity-Hadassah, P.O. Box 12272, Jerusalem 91120, Israel
`b School of Pharmacy, The Hebrew Uni6ersity, Jerusalem, Israel
`c Department of Restorati6e Dentistry, Faculty of Dental Medicine, Hebrew Uni6ersity-Hadassah, Jerusalem, Israel
`
`Received 30 March 2001; accepted 19 July 2001
`
`Abstract
`
`Tooth decay is an infectious disease caused by bacteria immobilized on the tooth surfaces. Eradication of these bacteria, for
`example Streptococcus sobrinus (S. sobrinus), from the oral cavity is essential in the prevention and treatment of tooth decay. We
`have tested the antimicrobial effect of several paraben derivatives such as methyl (MP), ethyl (EP), propyl (PP) and butyl (BP)
`against immobilized and planktonic S. sobrinus. The antibacterial effect was as follows: MP\EP\PP=BP on immobilized
`bacteria and MP\EP=PP\BP on planktonic bacteria. An antibacterial synergistic effect was found between several
`combinations of parabens on immobilized and planktonic S. sobrinus. Our results indicate that parabens are potential
`antibacterial agents against immobilized or planktonic bacteria found in the oral cavity. © 2001 Elsevier Science B.V. and
`International Society of Chemotherapy. All rights reserved.
`
`Keywords: Paraben; Biofilm; Dental plaque; Streptococcus sobrinus
`
`1. Introduction
`
`Tooth decay (caries) is a worldwide oral disease
`affecting all ages, ethnic groups and genders. Bacterial
`accumulation on the surface of the tooth (dental plaque
`biofilm) is the main precursor of caries. Properties of
`bacteria in the biofilm are unique and may differ from
`planktonic bacteria. It is conceivable that due to these
`differences, the effect of antibacterial agents may differ
`between immobilized bacteria in the biofilm and bacteria
`in suspension [1,2].
`Streptococcus sobrinus (S. sobrinus) is one of the most
`cariogenic bacteria of mutans streptococci
`[3,4] and
`elimination of cariogenic bacteria such as S. sobrinus is
`a fundamental step in preventing and treating dental
`caries. Several antibacterial drugs are being used for
`prevention or treatment of tooth decay [5,6].
`
`* Corresponding author. Tel.: +972-2-6757633;
`6439219.
`E-mail address: dorons@cc.huji.ac.il (S. Doron).
`
`fax: 972-2-
`
`Parabens (hydroxybenzoates), are one of the most
`common preservative agents in the food and pharmaceu-
`tical industries. Parabens possess minimal side effects [7];
`thus, they can act as potential drugs for use in the dental
`field and lately attention has been drawn to their use as
`antibacterial agents in the dental field. For example, it
`was shown that parabens could affect glycolysis of
`Streptococcus mutans by irreversibly inhibiting the phos-
`photransferase system (PTS) [8]. Furthermore, parabens
`were found to be potent inhibitors of arginolysis in
`several oral streptococci [9]. Sissons et al. [10] have shown
`that methyl paraben is effective against immobilized
`dental plaque bacteria in a biofilm model. Steinberg et
`al. [11] reported that parabens had an antibacterial effect
`when used in mouthwashes or when incorporated into
`slow release devices in human volunteers.
`The purpose of this investigation was to assess the
`antimicrobial activity of several derivatives of parabens
`and a possible antibacterial combination against immo-
`bilized and planktonic S. sobrinus, as a step in optimiz-
`
`0924-8579/01/$ - $20 © 2001 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved.
`PII: S 0 9 2 4 - 8 5 7 9 ( 0 1 ) 0 0 4 3 6 - 8
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`576
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`S. Doron et al. /International Journal of Antimicrobial Agents 18 (2001) 575–578
`
`ing the concentration of parabens as antibacterial
`agents in the oral cavity.
`
`2. Materials and methods
`
`2.1. Acti6e agents
`
`Four derivatives of parabens were used in this study:
`methyl paraben (MP), ethyl paraben (EP), propyl
`paraben (PB) and butyl paraben (BP) (Sigma, St. Louis,
`MO, USA).
`
`with bacitracin [15], a selective agar medium for mutans
`streptococci. Following 72 h of incubation, bacterial
`growth on the agar plates was recorded using a colony
`counter (New Brunswick Scientific, New Brunswick,
`USA). Viable bacteria were recorded by calculating the
`number of colony forming units (CFU) and the dilution
`factor. Bacterial growth in biofilm, not exposed to
`parabens, was used to determine the maximal growth
`levels of S. sobrinus. The results are presented as per-
`centage bacterial viability calculated from the maximal
`viability counts. Each experimental set was repeated
`three times.
`
`2.2. Immobilized biofilm bacteria
`
`2.3. Planktonic bacteria
`
`The microorganism used in this study was S. sobrinus
`6715. The in vitro model used for testing the effect of
`parabens on dental plaque was similar to a model
`previously described by Schilling et al. [12], Steinberg et
`al. [13] and Steinberg and Rothman [14].
`
`2.2.1. Bacteria preparation
`S. sobrinus were grown at 37 °C under aerobic condi-
`tions supplemented with 5% CO2. Following 18 h incu-
`bation, the bacterial suspension was centrifuged for 10
`min at 3000×g. The supernatant fluid was then dis-
`carded and the bacterial pellet was resuspended in
`buffered KCl (pH 6.5, 55 mM). This washing procedure
`was repeated three times. The optical density of the
`suspension was adjusted to 1.5 at 540 nm with the
`buffered KCl.
`
`2.2.2. Biofilm formation on hydroxyapatite
`Hydroxyapatite (HA) beads were prepared as fol-
`lows. Forty milligrams of HA beads (Type 1 Bio-Rad
`Hercules, USA) were washed three times with buffered
`KCl. Next, the washed beads were covered with 1 ml of
`the above prepared suspension of S. sobrinus 6715.
`After incubation for 2 h at 37 °C, the beads were
`washed three times with buffered KCl to remove loose
`and unbound bacteria.
`
`2.2.3. Effect of parabens on biofilm bacteria
`The immobilized bacteria on HA, prepared above,
`were exposed to different concentrations of parabens,
`either separately or in combinations of two types of
`parabens, and incubated for 18 h at 37 °C. The
`paraben solution was then discarded and the beads
`were washed three times with buffered KCl. The viabil-
`ity of the surface-bound bacteria was assessed as fol-
`lows: the HA beads were subjected to sonication by a
`probe for three intervals of 1 min each in an ice bath,
`after which aliquots of bacteria from the supernatant
`fluid were serially diluted in PBS. The viability of
`bacteria was determined by plating 0.05 ml of each
`bacterial dilution on mitis salivarius agar supplemented
`
`types of
`the four
`The antibacterial activity of
`parabens was examined for each derivative. Briefly, 0.1
`ml of an overnight culture of S. sobrinus, grown as
`described above, was added to 5.5 ml of TSB supple-
`mented with 0.5 ml of parabens at different concentra-
`tions. The test tubes were incubated at 37 °C in an
`atmosphere enriched with 5% CO2. After 18 h of incu-
`bation, the bacterial suspensions were serially diluted
`and each dilution was plated on four plates of selective
`agar media for mutans streptococci [15]. Viable bacte-
`rial counts were performed as described above for the
`biofilm bacteria. Each experiment was repeated three
`times.
`
`2.4. Combination effect
`
`After establishing the MIC for each of the derivatives
`of the parabens separately, the potential combination
`effect between MP, EP, PP and BP derivatives of
`parabens compounds in solution and in biofilm was
`investigated.
`
`3. Results
`
`Our results demonstrate a dose-dependent antibacte-
`rial effect of parabens against S. sobrinus. The antibac-
`terial effects for the paraben derivatives tested (methyl,
`ethyl, propyl, butyl) against S. sobrinus immobilized in
`biofilm were between 0.5 and 0.062%. (MP\EP\
`PP=BP) (Table 1). Similar trends in antibacterial val-
`ues were also obtained with planktonic bacteria
`(MP\EP=PP\BP).
`The effects of combinations of two different types of
`parabens were tested on immobilized bacteria and on
`planktonic bacteria.
`Bacterial growth on biofilm was affected by different
`combinations of pairs of different parabens (Fig. 1). No
`bacterial growth was recorded in the biofilm when
`either EP or PP or BP was introduced at a concentra-
`tion of 0.03% together with MP at concentrations
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`Table 1
`Antibacterial values (w/v%) of parabens against S. sobrinus
`
`MP
`
`EP
`
`PP
`
`BP
`
`Immobilized
`bacteria
`Planktonic
`bacteria
`
`0.5–0.25
`
`0.25–0.125
`
`0.125–0.062
`
`0.125–0.062
`
`0.5–0.25
`
`0.25–0.125
`
`0.25–0.125
`
`0.125–.062
`
`Range of minimal inhibitory concentrations of methyl paraben (MP),
`ethyl paraben (EP), propyl paraben (PP), butyl paraben (BP) against
`HA-immobilized and planktonic S. sobrinus.
`
`higher than 0.03%. Combinations of parabens showed
`different antibacterial patterns of inhibition of plank-
`tonic bacteria compared with the immobilized bacteria
`(Fig. 2). The occurrence of full
`inhibitory effect for
`planktonic bacteria required higher concentrations of
`parabens compared with immobilized bacteria. A
`stronger antibacterial effect occurred with the combina-
`tions of parabens on immobilized bacteria than on
`planktonic bacteria.
`
`4. Discussion
`
`The debate in the dental field regarding the eradica-
`tion of cariogenic bacteria has not ceased. New drugs
`and drug applications are constantly being tested. The
`use of combinations of parabens has been shown to
`have a synergistic effect on planktonic bacteria [16,17]
`although a complete antibacterial effect is not always
`
`Fig. 1. The antibacterial effect of a combination of methyl paraben
`(MP) with ethyl paraben (EP), propyl paraben (PP), butyl paraben
`(BP) against immobilized S. sobrinus.
`
`Fig. 2. The antibacterial effect of a combination of methyl paraben
`(MP) with ethyl paraben (EP), propyl paraben (PB), butyl paraben
`(BP) against planktonic S. sobrinus.
`
`achieved. The exact antibacterial activity of parabens is
`not fully understood, but appears to be via alteration of
`cell membrane properties [18]. Changes in the integrity
`of the membrane in the presence of parabens, allow
`intercellular solutes to leak from the cells [19]. Ma and
`Marquis [8] have shown that the level of effectiveness of
`parabens in affecting a drop in pH values due to
`bacterial fermentation in an excess of glucose was BP\
`PP\EP\MP. According to our results, the antibacte-
`rial values of these parabens on planktonic S. sobrinus
`were also in this order. Ma and Marquis [8] have further
`shown that BP can irreversibly inhibit F-ATPase of S.
`mutans. Our study and Ma and Marquis’
`[8] study
`indicate that BP has the greatest potential as an antibac-
`terial and anticaries agent
`compared with other
`parabens tested. BP was also found to be superior to
`MP, EP and PP in solution in biofilm for killing
`bacteria. Sissons et al. [10] have tested the duration of
`the effect of MP on immobilized bacteria. They have
`found that MP inhibited the growth of plaque bacteria
`for three days but after this period it had no effect.
`Surprisingly, most of the assays determining the an-
`tibacterial effects of agents against oral bacteria were
`performed in suspension, where it is clear that the most
`important ecological niche of the oral bacteria is the
`dental plaque biofilm. It is conceivable that bacteria
`immobilized in the dental plaque may have a suscepti-
`bility to antibacterial agents which is different from the
`same bacteria in suspension [1,14,20,21]. The difference
`is probably due to environmental and physiological
`differences between planktonic phase and biofilm
`[22,23].
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`578
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`S. Doron et al. /International Journal of Antimicrobial Agents 18 (2001) 575–578
`
`We have tested the effect of several paraben derivatives
`in a planktonic as well as in the immobilized phase and
`found several differences between the activity of
`parabens on planktonic and immobilized S. sobrinus.
`The killing effects of parabens on planktonic bacteria
`or immobilized bacteria were similar. However, differ-
`ent antibacterial effects of combinations of parabens
`were found when testing planktonic bacteria and immo-
`bilized bacteria. The effect of
`the combination of
`parabens on immobilized bacteria was more effective
`than that combination in solution. This enhanced an-
`tibacterial effect on the surface may be due to the
`adsorption properties of the parabens allowing them to
`reach higher local concentrations on the surface com-
`pared with solution, which results in greater antibacte-
`rial efficacy.
`It
`is possible that
`the presence of
`extracellular polysaccharides synthesized in situ by oral
`bacteria will decrease the susceptibility of such bacteria
`to parabens, especially in biofilms [24]. Such conditions
`would require an increase in parabens concentrations to
`produce the same effect.
`Parabens are antibacterial agents that have received
`little attention in the dental field. This study on S.
`sobrinus bacteria, along with other studies on oral
`bacteria, may lead to further tests on the potential
`effect of parabens in this area.
`
`References
`
`[1] Wilson M. Susceptibility of oral bacterial biofilms to antimicro-
`bial agents. Med Microbiol 1996;44:79–87.
`[2] Anwar H, Dasgupta MK, Costerton JW. Testing the susceptibil-
`ity of bacteria in biofilms to antibacterial agents. Antimicrob
`Agents Chemother 1990;34:2043–6.
`[3] Balakrishnan M, Simmonds RS, Tagg JR. Dental caries is a
`preventable infectious disease. Aust Dent J 2000;45:235–45.
`[4] Hanada N. Current understanding of the cause of dental caries.
`Jpn J Infect Dis 2000;53:1.
`[5] Adams D, Addy M. Mouthrinses. Adv Dent Res 1994;8:291–
`301.
`[6] Steinberg D, Friedman M. Dental drug-delivery devices: local
`and sustained release applications. Crit Rev Ther Drug Carrier
`Syst 1999;16:425–59.
`[7] Reynolds JEF. Hydroxybenzoates. In: Martindale, The extra
`pharmacopoeia. London: Royal Pharmaceutical Society, 1996.
`
`pp. 1135–37.
`[8] Ma Y, Marquis RE. Irreversible paraben inhibition of glycolysis
`by
`Streptococcus mutans GS-5. Lett Appl Microbiol
`1996;23:329–33.
`[9] Ma Y, Rutherford GC, Curran TM, Reidmiller JS, Marquis RE.
`Membrane locus and pH sensitivity of paraben inhibition of
`alkali production by oral streptococci. Oral Microbiol Immunol
`1999;14:244–9.
`[10] Sissons CH, Wong L, Cutress TW. Patterns and rates of growth
`of microsom dental plaque biofilms. Oral Microbiol Immunol
`1995;10:160–7.
`[11] Steinberg D, Hirschfeld Z, Taib I, Ben-yosef S, David A, Fried-
`man M. The effect of parabens in a mouth wash and incorpo-
`rated into a sustained release varnish, on salivary bacteria. J
`Dent 1999;27:101–6.
`[12] Schilling KM, Blitzer MH, Bowen WH. Adherence of Strepto-
`coccus mutans to glucans formed in situ in salivary pellicle. J
`Dent Res 1989;68:1678–80.
`[13] Steinberg D, Kopec LK, Bowen WH. Adhesion of actinomyces
`isolates to experimental pellicles. J Dent Res 1993;72:1015–20.
`[14] Steinberg D, Rothman M. Antibacterial effect of chlorhexidine
`on bacteria adsorbed onto experimental dental plaque. Diagn
`Microbiol Infect Dis 1996;26:109–15.
`[15] Tanzer JM, Borjesson AC, Laskowski L, et al. Glucose–su-
`crose–potassium tellurite–bacitracin agar, an alternative to mitis
`salivarius–bacitracin agar for enumeration of Streptococcus mu-
`tans. J Clin Microbiol 1984;20:653–9.
`[16] Gilliland D, Li Wan Po A, Scott E. Kinetic evaluation of
`claimed synergistic paraben combinations using a factorial de-
`sign. J Appl Bacteriol 1992a;72:258–61.
`[17] Gilliland D, Li Wan Po A, Scott E. The bactericidal activity of
`a methyl and propyl parabens combination:
`isothermal and
`non-isothermal studies. J Appl Bacteriol 1992b;72:252–7.
`[18] Sharpell F, Manowitz M. Preservation of cosmetics. In: Block
`SS, editor. Disinfection, sterilization, and preservation. Philadel-
`phia: Lea and Febiger, 1991:887–900.
`[19] Furr JR, Russell AD. Effects of esters of p-hydroxy benzoic acid
`on spheroplasts of Serratia marcescens protoplasts of Bacillis
`megaterium. Microbios 1972;6:47–54.
`[20] Kinniment SL, Wimpenny JW, Adams D, Marsh PD. The effect
`of chlorhexidine on defined, mixed culture oral biofilms grown in
`a novel model system. J Appl Bacteriol 1996;81:120–5.
`[21] Larsen T, Fiehn NE. Resistance of Streptococcus sanguis bi-
`ofilms to antimicrobial agents. APMIS 1996;104:280–4.
`[22] Marsh PD, Bradshaw DJ. Physiological approaches to the con-
`trol of oral biofilms. Adv Dent Res 1997;11:176–85.
`[23] Bowden GH, Hamilton IR. Survival of oral bacteria. Crit Rev
`Oral Biol Med 1998;9:54–85.
`[24] Steinberg D, Poran S, Shapira D. The effect of extracellular
`polysaccharides from Streptococcus mutans on the bactericidal
`activity of human neutrophils. Arch Oral Biol 1999;44:437–44.
`
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