You are on page 1of 5

archives of oral biology 57 (2012) 364368

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

Short communication

Antibacterial activity of povidoneiodine against an articial biolm of Porphyromonas gingivalis and Fusobacterium nucleatum
Yasuo Hosaka a, Atsushi Saito b,c, Ryo Maeda a, Chie Fukaya a, Satoru Morikawa a, Asako Makino b, Kazuyuki Ishihara c,d, Taneaki Nakagawa a,*
a

Department of Dentistry and Oral Surgery, School of Medicine, Keio University, 35 Shinanomachi, Sinnjuku-ku, Tokyo 160-8582, Japan Department of Periodontology, Tokyo Dental College, Chiba, Japan c Oral Health Science Center, Tokyo Dental College, Chiba, Japan d Department of Microbiology, Tokyo Dental College, Chiba, Japan
b

article info
Article history: Accepted 5 September 2011 Keywords: Povidoneiodine Periodontopathic bacteria Biolm Antibacterial agent P. gingivalis F. nucleatum

abstract
Objective: To investigate the antibacterial activity of povidoneiodine (PVPI) on an articial dual species biolm of periodontal pathogens. Design: Porphyromonas gingivalis or Fusobacterium nucleatum grown in broth culture was inoculated on polycarbonate membrane (PCM) tissue culture inserts. After incubation for 72 h, PVPI solutions were applied to the biolm for the time period ranging from 0.5 to 5 min. After addition of a deactivator, each PCM was removed and the biolm on the PCM was serially diluted and plated on blood agar plates and cultured anaerobically for 7 days. Then viable bacteria were enumerated. Results: In the dual species biolm model, F. nucleatum showed an approximately 200-fold increase in viable counts when compared with mono-microbial biolm. In dual species biolm, PVPI with concentration equal to or greater than 2% was required to signicantly reduce P. gingivalis and F. nucleatum. When the contact time of PVPI was increased to 1 min or greater, no difference in antibacterial activity of PVPI was observed in any concentration. Conclusion: These results suggest that 30 s application of 2% PVPI would be effective in suppressing both P. gingivalis and F. nucleatum in dual-species biolm, and this provides clinical implication for the control of subgingival biolm. # 2011 Elsevier Ltd. All rights reserved.

1.

Introduction

More than 400 bacterial species have been recorded in samples taken from the human gingival crevices.1 Dental plaque is a biolm consisting of poly-species of oral bacteria and their metabolic products. Periodontal disease is a plaque

biolm-induced inammatory disease of the supporting tissue of the tooth and includes both gingivitis and periodontitis,2 and can be described as one of the predominant polymicrobial infections of humans.3 Periodontal pathogens such as Fusobacterium nucleatum initially adhere to early colonizers including gram-positive cocci, and enhance the adherence of late colonizers such as Porphyromonas gingivalis and

* Corresponding author. Tel.: +81 03 5363 3830; fax: +81 03 3357 1593. E-mail address: tane@z6.keio.jp (T. Nakagawa). 00039969/$ see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.09.005

archives of oral biology 57 (2012) 364368

365

Treponema denticola.4,5 Mechanical methods of oral hygiene, brushing and ossing, are considered by clinicians as the gold standard methods of plaque (biolm) control. For the treatment of periodontitis, mechanical intervention such as scaling and root planing is necessary in order to effectively disrupt biolm in the subgingival milieu. In the meanwhile, there is a growing interest in using antimicrobial agents as an adjunct therapy.6 In a previous study, we tested the antibacterial activity of several antibiotics against periodontopathic bacteria, and found that a wide variation existed in the susceptibility of monomicrobial cultures to a given antibiotic.7 In order to be effective against biolm, the concentration of a typical antibiotic has to be approximately 100 times higher than that needed for planktonic state,8,9 supporting the need for mechanical intervention in periodontal therapy. Given the recent growing importance for a provision of periodontal care to systemically compromised patients, it is sometimes difcult to implement a meticulous subgingival debridement. Therefore, there is a need for the better use of periodontal chemotherapy. Povidoneiodine (PVPI) has been used as an oral rinse in patient home care.10 PVPI, which possesses an efcient and broad-spectrum microbicidal property, has also demonstrated an anti-virus effect.11 Despite long-term use, development of PVPI resistance in microorganisms has not been reported.12,13 Although the effect of PVPI on biolms other than those in the oral cavity has been reported,14,15 information regarding its effect on biolms organized with periodontal pathogens is limited. This study is part of our ongoing effort to develop an effective antimicrobial regiment against periodontopathic biolm. A prototype model of dual-species biolm of P. gingivalis and F. nucleatum was used to evaluate the antibacterial activity of PVPI and to propose an optimal condition for its use in periodontal treatment.

laboratory). The P. gingivalis and F. nucleatum were grown in tryptic soy broth (TSB) (Becton Dickinson, Sparks, MD) supplemented with hemin (5 mg/ml) and menadione (0.5 mg/ ml). The bacterial cultures were grown to mid-log phase (range at OD 660 nm of 0.81.0) at 37 8C under anaerobic conditions.

2.2.

Evaluation of biolm forming activity

A 150 ml aliquot of TSB was added to each well of a 24-well tissue culture-treated polystyrene plate. For the evaluation of biolm forming activity, a polycarbonate membrane (PCM) insert (Transwell, No. 3413, Corning Life Sciences, Acton, MA, USA) was placed into each well. For the mono-microbial biolm formation, 50 ml of P. gingivalis or F. nucleatum was inoculated onto the insert. For the establishment of dualspecies biolm, P. gingivalis and F. nucleatum (25 ml each) were added onto the insert. The plates were incubated anaerobically for 72 h at 37 8C. P. gingivalis and F. nucleatum were in stationary phase at 72 h.

2.3. Effect of PVPI on the mono- and poly-bacterial biolm


Biolms were formed as described above. After incubation, the inserts were washed twice in sterile phosphate-buffered saline (PBS, pH 7.4) to remove nonadherent cells. Then PVP I (Meiji Seika Kaisha, Tokyo, Japan) solutions with varied concentration (0.237%, 50 ml each) were applied to the biolm for the time period ranging from 0.5 to 5 min. A negative control (PBS) only was included in each assay. After addition of a deactivator containing 10% Tween 80, 3% lecithin and 0.5% sodium thiosulfate16 (50 ml), wells were washed once with PBS. Each PCM was then excised using a surgical blade (No. 15), placed into a tube containing 1 ml of sterile PBS, and mixed thoroughly at the highest setting on a Vortex mixer for 60 s. The mixtures were serially diluted and plated on blood agar plates supplemented with hemin and menadione, and incubated anaerobically at 37 8C for 7 days. Colony-forming units of recovered organisms were then enumerated.

2.
2.1.

Materials and methods


Bacterial strains and growth conditions

2.4.
The following bacterial strains were used; P. gingivalis ATCC 33277 (American Type Culture Collection, Rockville, MD, USA), F. nucleatum #20 (a clinical isolate and working strain in our

Statistical analysis

All experiments were performed in duplicate or triplicate for each condition and repeated at least three times. Kruskal

Table 1 Effect of PVPI contact time and concentration on P. gingivalis mono-biofilm. Contact time Control
30 s 1 min 3 min 5 min 3487.1 (1956.0) 2516.1 (1469.2) 2480.9 (1447.2) 2268.4 (1737.7)

PVPI concentration 0.23%


1063.3 (1069.3) 450.0* (369.7) 509.0* (268.5) 370.8 (95.4)

0.47%
2359.5 (2428.3) 2408.8 (512.7) 972.2 (85.0) 589.2 (874.5)

1%
125.9** (108.0) 675.0 (512.7) 224.8** (106.2) 169.6* (43.8)

2%
430.5* (383.1) 325.7** (296.1) 140.0** (23.1) 53.3** (48.5)

3.5%
67.7** (38.6) 260.3* (210.0) 25.2** (29.1) 43.0** (54.0)

5%
6.4** (7.7) 28.4** (38.9) 0.03** (0.1) 33.3** (57.7)

7%
2.3** (4.4) 3.8** (7.5) 0.0** (0.0) 0.0** (0.0)

Values are the mean CFU (standard deviations) 104 of triplicate independent determinations from a typical experiment. Signicantly different from control, *p < 0.05, **p < 0.01, KruskalWallis test with Dunn post test.

366

archives of oral biology 57 (2012) 364368

Table 2 Effect of PVPI contact time and concentration on F. nucleatum mono-biofilm. Contact time Control
30 s 1 min 3 min 5 min 8.4 (8.5) 12.1 (14.0) 10.7 (13.7) 8.1 (6.5)

PVPI concentration 0.23%


10.7 (5.6) 6.1 (4.3) 6.3 (1.4) 6.0 (4.8)

0.47%
5.0 (2.4) 17.7 (18.0) 9.1 (6.7) 2.6 (1.2)

1%
2.0 (2.4) 1.2 (1.6) 0.9* (1.2) 1.2 (2.0)

2%
0.3* (0.4) 0.03** (0.1) 1.1* (2.0) 0.2** (0.2)

3.5%
0.4* (1.0) 0.3** (0.7) 0.7* (0.5) 0.6** (0.9)

5%
0.0* (0.0) 0.0** (0.0) 0.0** (0.0) 0.0** (0.0)

7%
0.0* (0.0) 0.0** (0.0) 0.0** (0.0) 0.0** (0.0)

Values are the mean CFU (standard deviations) 104 of triplicate independent determinations from a typical experiment. Signicantly different from control, *p < 0.05, **p < 0.01, KruskalWallis test with Dunn post test.

Table 3 Effect of PVPI concentration on P. gingivalis or F. nucleatum in dual-biofilm (contact time 30 s). Species Control
P. gingivalis F. nucleatum 1070.6 (778.3) 1638.2 (1288.6)

PVPI concentration 0.23%


374.0 (113.0) 552.6 (290.5)

0.47%
87.8 (57.9) 201.8 (73.1)

1%
124.0 (187.5) 187.0 (203.9)

2%
17.5** (20.6) 1.3** (2.5)

3.5%
110.0* (194.2) 0.2** (0.4)

5%
0.0** (0.0) 3.2** (6.0)

7%
0.0** (0.0) 0.8** (1.7)

Values are the mean CFU (standard deviations) 104 of duplicate independent determinations from a typical experiment. Signicantly different from control, *p < 0.05, **p < 0.01, KruskalWallis test with Dunn post test.

Wallis test with Dunn post test was used to compare differences. Statistical comparisons were performed using a software package (InStat 3.10, GraphPad Software, La Jolla, CA, USA). p-Values less than 0.05 were considered statistically signicant.

signicantly reduce both P. gingivalis and F. nucleatum in any contact time point. PVPI with concentrations equal to or greater than 5% completely eradicated both pathogens in dualbiolm. Increasing the contact time did not signicantly inuence P. gingivalis or F. nucleatum recovery from dualbiolm.

3.
3.1.

Results
Effect of PVPI on mono-microbial biolm

4.

Discussion

After 72 h incubation, biolm formation on PCM was observed for P. gingivalis and F. nucleatum. P. gingivalis was more efcient in developing mono-microbial biolm than F. nucleatum: the mean total CFUs recovered from P. gingivalis mono-biolm were approximately 200300-fold greater than those from F. nucleatum (data not shown). There was a wide variation in the CFUs of P. gingivalis mono-biolm treated with PVPI with concentrations ranging 0.230.47%, although a general trend for time-dependent decrease in the CFUs was observed (Table 1). When the PVPI concentration was equal to or greater than 1%, a statistically signicant reduction in the P. gingivalis CFUs was observed in almost all contact time points. As for F. nucleatum mono-biolm, PVPI with concentrations exceeding 12% was needed to signicantly reduce the CFUs (Table 2). Increasing the contact time did not signicantly reduce P. gingivalis or F. nucleatum mono-biolm.

3.2.

Effect of PVPI on dual-species biolm

In dual-species biolm, viable counts of F. nucleatum showed an approximately 200-fold increase when compared with mono-biolm (Table 3). More than 2% of PVPI was required to

Both P. gingivalis and F. nucleatum are important microbial constituents in human periodontal biolm. In our preliminary experiments, we found that PVPI with concentrations indicated for daily oral rinse effectively suppressed periodontal pathogens such as P. gingivalis, Aggregatibacter actinomycetemcomitans or F. nucleatum in planktonic cultures (unpublished observation). In the present study, we sought to delineate the effect of PVPI on periodontal pathogens in biolm. The results suggest that 30 s application of 2% PVPI could effectively suppress P. gingivalisF. nucleatum dual-species biolm. Different methods have been utilized in order to generate articial biolms, including the use of ow-cells and microplates.17 In the present study, we established a dual-species biolm model of periodontal pathogens, using inserts of PCM in a cell culture plate. This method allows us to enumerate viable bacteria recovered from biolm. It is, however, impossible to distinguish bacteria with similar colony morphologies. Therefore, we chose P. gingivalis and F. nucleatum since they can easily be distinguished from each other by their colony characteristics. There was a wide variation in the CFUs of each species in both mono- and dual-biolm within control experiments. Differences in bacterial growth, attachment to PCM and level of coaggregation between species may be the causes of the

archives of oral biology 57 (2012) 364368

367

data with large standard deviations. P. gingivalis was more efcient than F. nucleatum in their ability to form mono-biolm on PCM. The abilities of F. nucleatum to attach to PCM and to form aggregation are considered to be lower than those of P. gingivalis. In vivo situations, F. nucleatum initially adhere to early colonizers consisting of gram-positive cocci and enhance the adherence of late colonizers including P. gingivalis.4 In the present study, we did not include the early colonizers such as Streptococcus gordonii, because the adjustment of bacterial growth and differentiation of colonies on the plates would become too difcult. This may be, at least in part, responsible for wide variation of the bacterial recoveries in each experiment. In dual-species biolm, F. nucleatum exhibited a different growth characteristic when compared with mono-microbial biolm. This is consistent with the previous report by Saito et al.,18 indicating the possibility for P. gingivalis to enhance growth of F. nucleatum in co-incubation. In the present study, PVPI concentrations indicated for daily oral rinsing (0.230.47%) failed to signicantly abrogate biolm formation. With contact time points between 30 s and 1 min, we occasionally found an increase in CFUs of P. gingivalis or F. nucleatum when compared with negative PBS controls. This may be related to the survival response, in which antimicrobial agents with low concentration can increase biolm formation.19 Although PVPI with concentration of 1% or greater was needed to signicantly reduce P. gingivalis or F. nucleatum in mono- or dual-biolm, more than 5% were needed to completely kill all bacteria in dual-biolm. This study, as well as the previous reports by Nakagawa et al.20 and Hoang et al.,21 suggests that PVPI with low concentrations would be effective when biolm bacteria are disrupted by mechanical intervention. Without mechanical disruption, higher concentrations are necessary to exert its effect on biolm. PVPI is water-soluble, and its short-term use does not irritate healthy or diseased oral mucosa, and exhibits no adverse side-effects, such as discoloration of teeth and tongue and change in taste.22 It was reported that the concentration of free iodine reaches maximum when PVPI is used at 100-fold dilution.23,24 In vivo, continuous delivery of I2 is necessary since it is inactivated upon contact with bacteria or organic substances. However, there are some concerns involved in the use of PVPI in high concentrations or log-term use. PVPI gargle solution has been known to cause yellowish discoloration of the teeth of people using this solution for more than six months, although this discoloration is reversible after weeks of discontinuation of its use.25 Other potential adverse events include allergy or hypersensitivity to the solution and its component products, and hyperthyroidism.26 Therefore, it may be advisable to use PVPI with the lowest concentration that would yield a maximum effect against periodontal biolm. Our results suggest that short durations of 2% PVPI contact with periodontal bacteria in biolm results in effective in vitro killing. Several limitations of this study need to be discussed. First, we used a dual-species biolm model. The effect of PVPI solutions on polymicrobial biolm with wide variety of periodontal pathogens remains to be elucidated. In some experiments, the data were highly variable. Although we repeated the experiments at least three times, the data with

large SD need to be interpreted with caution. Standar et al.27 reported that the combined analysis of biolm formation (dental and periodontal pathogens) via determination of CFU and uorescence microscopy allowed fast, unambiguous and reproducible results, and scanning electron- and confocal laser scanning microscopy complemented these results. For future analysis, we need to incorporate a different method for analysis. Since P. gingivalis and F. nucleatum resides in the subgingival site and is therefore likely to be exposed to gingival crevicular uid, an addition of serum into our assay may give us useful information. Also, the actual effect of the proposed use of PVPI in vivo situations needs to be evaluated in further clinical studies.

Funding
This work was supported by grants-in-aid for scientic research C-22592317 (to AS) and C-17592164 (to TN) from Japan Society for Promotion of Science.

Competing interests
The authors have no conicts of interest to disclose.

Ethical approval
Not required

references

1. Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000 2006;42(1):807. 2. Pihlstrom BL. Periodontal diseases. Lancet 2005;366(9499):180920. 3. Brogden KA, Guthmiller JM, Taylor CE. Human polymicrobial infections. Lancet 2005;365(9455): 2535. 4. Kolenbrander PE. Oral microbial communities: biolms, interactions, and genetic systems. Annu Rev Microbiol 2000;54:41337. 5. Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer Jr RJ et al. Communication among oral bacteria. Microbiol Mol Biol Rev 2002;66(3):486505. 6. Gunsolley JC. Clinical efcacy of antimicrobial mouthrinses. J Dent 2010;38(Suppl. 1):S610. 7. Maeda R, Ishihara K, Hosaka Y, Nakagawa T. Antibacterial activity of antibiotics against periodontopathic bacteria. J Jpn Soc Periodontol 2005;47(3):14652. [in Japanese]. 8. Wright TL, Ellen RP, Lacroix JM, Sinnadurai S, Mittelman MW. Effects of metronidazole on Porphyromonas gingivalis biolms. J Periodontal Res 1997;32(5):4737. 9. Larsen T. Susceptibility of Porphyromonas gingivalis biolms to amoxicillin, doxycycline and metronidazole. Oral Microbiol Immunol 2002;17(5):26771. 10. Nakagawa T, Saito A, Hosaka Y, Yamada S, Tsunoda M, Sato T, et al. Bactericidal effects on subgingival bacteria of irrigation with a povidoneiodine solution (Neojodin). Bull Tokyo Dent Coll 1990;31(3):199203.

368

archives of oral biology 57 (2012) 364368

11. Sriwilaijaroen N, Wilairat P, Hiramatsu H. Mechanisms of the action of povidoneiodine against human and avian inuenza A viruses: its effects on hemagglutination and sialidase activities. Virol J 2009;6:124. 12. Mycock G. Methicillin/antiseptic-resistant Staphylococcus aureus. Lancet 1985;2(8461):94950. 13. Lacey RW, Catto A. Action of povidoneiodine against methicillin-sensitive and -resistant cultures of Staphylococcus aureus. Postgrad Med J 1993;69(Suppl. 3):S7883. 14. Presterl E, Suchomel M, Eder M, Reichmann S, Lassnigg A, Graninger W, et al. Effects of alcohols, povidoneiodine and hydrogen peroxide on biolms of Staphylococcus epidermidis. J Antimicrob Chemother 2007;60(2):41720. 15. Hill KE, Malic S, McKee R, Rennison T, harding KG, Williams DW, et al. An in vitro model of chronic wound biolms to test wound dressings and assess antimicrobial susceptibilities. J Antimicrob Chemother 2010;65(6):1195206. 16. Rikimaru T, Kondo M, Kajimura K, Hashimoto K, Oyamada K, Sagawa K, et al. Bactericidal activities of commonly used antiseptics against multidrug-resistant Mycobacterium tuberculosis. Dermatol 2002;204(Suppl. 1):1520. 17. Davey ME. Techniques for the growth of Porphyromonas gingivalis biolms. Periodontol 2000 2006;42(1):2735. 18. Saito Y, Fujii R, Nakagawa K-I, Kuramitsu HK, Okuda K, Ishihara K. Stimulation of Fusobacterium nucleatum biolm formation by Porphyromonas gingivalis. Oral Microbiol Immunol 2008;23(1):16. 19. Takahashi N, Ishihara K, Kato T, Okuda K. Susceptibility of Actinobacillus actinomycetemcomitans to six antibiotics

20.

21.

22. 23. 24.

25.

26.

27.

decreases as biolm matures. J Antimicrob Chemother 2007;59(1):5965. Nakagawa T, Hosaka Y, Ishihara K, Hiraishi T, Sato S, Ogawa T, et al. The efcacy of povidoneiodine products against periodontopathic bacteria. Dermatology 2006;212(Suppl. 1):10911. Hoang T, Jorgensen MG, Keim RG, Pattison AM, Slots J. Povidoneiodine as a periodontal pocket disinfectant. J Periodont Res 2003;38(3):3117. Slots J. Selection of antimicrobial agents in periodontal therapy. J Periodontal Res 2002;37(5):38998. Rackur H. New aspects of mechanism of action of povidone iodine. J Hosp Infect 1985;6(Suppl. A):1323. Kebukawa H. Basis of iodine products and its notices for use for the promotion of their appropriate use. Sci Rep Meiji Seika Kaisha 1999;38:144. [in Japanese]. Chua JV, Dominguez EA, Sison CMC, Berba RP. The efcacy of povidoneiodine oral rinse in preventing ventilatorassociated pneumonia: a randomized, double blind, placebo-controlled (VAPOR) trial: Preliminary report. Phil J Microbiol Infect Dis 2004;33(4):15361. Rath T, Meissl G. Induction of hyperthyroidism in burn patients treated topically with povidoneiodine. Burns Incl Therm Inj 1988;14(4):3202. Standar K, Kreikemeyer B, Redanz S, Mu nter WL, Laue M, Podbielski A. Setup of an in vitro test system for basic studies on biolm behavior of mixed-species cultures with dental and periodontal pathogens. PLoS ONE 2010;5(10):e13135.

You might also like