Short communication

Antibacterial activity of povidone–iodine against anartificial biofilm of Porphyromonas gingivalis andFusobacterium 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,*aDepartment of Dentistry and Oral Surgery, School of Medicine, Keio University, 35 Shinanomachi, Sinnjuku-ku, Tokyo 160-8582, JapanbDepartment of Periodontology, Tokyo Dental College, Chiba, JapancOral Health Science Center, Tokyo Dental College, Chiba, JapandDepartment of Microbiology, Tokyo Dental College, Chiba, Japan

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 3 6 4 – 3 6 8

a r t i c l e i n f o

Article history:

Accepted 5 September 2011

Keywords:

Povidone–iodine

Periodontopathic bacteria

Biofilm

Antibacterial agent

P. gingivalis

F. nucleatum

a b s t r a c t

Objective: To investigate the antibacterial activity of povidone–iodine (PVP–I) on an artificial

dual species biofilm 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, PVP–I solutions were applied to the biofilm for the time period ranging from 0.5 to

5 min. After addition of a deactivator, each PCM was removed and the biofilm 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 biofilm model, F. nucleatum showed an approximately 200-fold

increase in viable counts when compared with mono-microbial biofilm. In dual species

biofilm, PVP–I with concentration equal to or greater than 2% was required to significantly

reduce P. gingivalis and F. nucleatum. When the contact time of PVP–I was increased to 1 min

or greater, no difference in antibacterial activity of PVP–I was observed in any concentration.

Conclusion: These results suggest that 30 s application of 2% PVP–I would be effective in

suppressing both P. gingivalis and F. nucleatum in dual-species biofilm, and this provides

clinical implication for the control of subgingival biofilm.

# 2011 Elsevier Ltd. All rights reserved.

Available online at www.sciencedirect.com

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

1. Introduction

More than 400 bacterial species have been recorded in samples

taken from the human gingival crevices.1 Dental plaque is a

biofilm consisting of poly-species of oral bacteria and

their metabolic products. Periodontal disease is a plaque

* Corresponding author. Tel.: +81 03 5363 3830; fax: +81 03 3357 1593.E-mail address: tane@z6.keio.jp (T. Nakagawa).

0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2011.09.005

biofilm-induced inflammatory 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 Fuso-

bacterium nucleatum initially adhere to ‘early colonizers’

including gram-positive cocci, and enhance the adherence

of ‘late colonizers’ such as Porphyromonas gingivalis and

d.

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 3 6 4 – 3 6 8 365

Treponema denticola.4,5 Mechanical methods of oral hygiene,

brushing and flossing, are considered by clinicians as the gold

standard methods of plaque (biofilm) control. For the

treatment of periodontitis, mechanical intervention such as

scaling and root planing is necessary in order to effectively

disrupt biofilm 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 biofilm, 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 difficult to

implement a meticulous subgingival debridement. Therefore,

there is a need for the better use of periodontal chemotherapy.

Povidone–iodine (PVP–I) has been used as an oral rinse in

patient home care.10 PVP–I, which possesses an efficient and

broad-spectrum microbicidal property, has also demonstrated

an anti-virus effect.11 Despite long-term use, development of

PVP–I resistance in microorganisms has not been reported.12,13

Although the effect of PVP–I on biofilms other than those in the

oral cavity has been reported,14,15 information regarding its

effect on biofilms organized with periodontal pathogens is

limited.

This study is part of our ongoing effort to develop an

effective antimicrobial regiment against periodontopathic

biofilm. A prototype model of dual-species biofilm of P.

gingivalis and F. nucleatum was used to evaluate the antibacte-

rial activity of PVP–I and to propose an optimal condition for its

use in periodontal treatment.

2. Materials and methods

2.1. Bacterial strains and growth conditions

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

Table 1 – Effect of PVP–I contact time and concentration on P.

Contact time PV

Control 0.23% 0.47%

30 s 3487.1

(1956.0)

1063.3

(1069.3)

2359.5

(2428.3)

1 min 2516.1

(1469.2)

450.0*

(369.7)

2408.8

(512.7)

3 min 2480.9

(1447.2)

509.0*

(268.5)

972.2

(85.0)

5 min 2268.4

(1737.7)

370.8

(95.4)

589.2

(874.5)

Values are the mean CFU (standard deviations) � 104 of triplicate indepe

Significantly different from control, *p < 0.05, **p < 0.01, Kruskal–Wallis t

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 0.8–1.0) at 37 8C under anaerobic conditions.

2.2. Evaluation of biofilm 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

biofilm 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

biofilm formation, 50 ml of P. gingivalis or F. nucleatum was

inoculated onto the insert. For the establishment of dual-

species biofilm, P. gingivalis and F. nucleatum (25 ml each) were

added onto the insert. The plates were incubated anaerobi-

cally for 72 h at 37 8C. P. gingivalis and F. nucleatum were in

stationary phase at 72 h.

2.3. Effect of PVP–I on the mono- and poly-bacterialbiofilm

Biofilms 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 (0.23–7%, 50 ml each) were applied to the biofilm

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.

2.4. Statistical analysis

All experiments were performed in duplicate or triplicate for

each condition and repeated at least three times. Kruskal–

gingivalis mono-biofilm.

P–I concentration

1% 2% 3.5% 5% 7%

125.9**

(108.0)

430.5*

(383.1)

67.7**

(38.6)

6.4**

(7.7)

2.3**

(4.4)

675.0

(512.7)

325.7**

(296.1)

260.3*

(210.0)

28.4**

(38.9)

3.8**

(7.5)

224.8**

(106.2)

140.0**

(23.1)

25.2**

(29.1)

0.03**

(0.1)

0.0**

(0.0)

169.6*

(43.8)

53.3**

(48.5)

43.0**

(54.0)

33.3**

(57.7)

0.0**

(0.0)

ndent determinations from a typical experiment.

est with Dunn post test.

Table 2 – Effect of PVP–I contact time and concentration on F. nucleatum mono-biofilm.

Contact time PVP–I concentration

Control 0.23% 0.47% 1% 2% 3.5% 5% 7%

30 s 8.4

(8.5)

10.7

(5.6)

5.0

(2.4)

2.0

(2.4)

0.3*

(0.4)

0.4*

(1.0)

0.0*

(0.0)

0.0*

(0.0)

1 min 12.1

(14.0)

6.1

(4.3)

17.7

(18.0)

1.2

(1.6)

0.03**

(0.1)

0.3**

(0.7)

0.0**

(0.0)

0.0**

(0.0)

3 min 10.7

(13.7)

6.3

(1.4)

9.1

(6.7)

0.9*

(1.2)

1.1*

(2.0)

0.7*

(0.5)

0.0**

(0.0)

0.0**

(0.0)

5 min 8.1

(6.5)

6.0

(4.8)

2.6

(1.2)

1.2

(2.0)

0.2**

(0.2)

0.6**

(0.9)

0.0**

(0.0)

0.0**

(0.0)

Values are the mean CFU (standard deviations) � 104 of triplicate independent determinations from a typical experiment.

Significantly different from control, *p < 0.05, **p < 0.01, Kruskal–Wallis test with Dunn post test.

Table 3 – Effect of PVP–I concentration on P. gingivalis or F. nucleatum in dual-biofilm (contact time 30 s).

Species PVP–I concentration

Control 0.23% 0.47% 1% 2% 3.5% 5% 7%

P. gingivalis 1070.6

(778.3)

374.0

(113.0)

87.8

(57.9)

124.0

(187.5)

17.5**

(20.6)

110.0*

(194.2)

0.0**

(0.0)

0.0**

(0.0)

F. nucleatum 1638.2

(1288.6)

552.6

(290.5)

201.8

(73.1)

187.0

(203.9)

1.3**

(2.5)

0.2**

(0.4)

3.2**

(6.0)

0.8**

(1.7)

Values are the mean CFU (standard deviations) � 104 of duplicate independent determinations from a typical experiment.

Significantly different from control, *p < 0.05, **p < 0.01, Kruskal–Wallis test with Dunn post test.

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 3 6 4 – 3 6 8366

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

significant.

3. Results

3.1. Effect of PVP–I on mono-microbial biofilm

After 72 h incubation, biofilm formation on PCM was observed

for P. gingivalis and F. nucleatum. P. gingivalis was more efficient

in developing mono-microbial biofilm than F. nucleatum: the

mean total CFUs recovered from P. gingivalis mono-biofilm

were approximately 200–300-fold greater than those from F.

nucleatum (data not shown).

There was a wide variation in the CFUs of P. gingivalis

mono-biofilm treated with PVP–I with concentrations ranging

0.23–0.47%, although a general trend for time-dependent

decrease in the CFUs was observed (Table 1). When the

PVP–I concentration was equal to or greater than 1%, a

statistically significant reduction in the P. gingivalis CFUs was

observed in almost all contact time points.

As for F. nucleatum mono-biofilm, PVP–I with concentra-

tions exceeding 1–2% was needed to significantly reduce the

CFUs (Table 2). Increasing the contact time did not significant-

ly reduce P. gingivalis or F. nucleatum mono-biofilm.

3.2. Effect of PVP–I on dual-species biofilm

In dual-species biofilm, viable counts of F. nucleatum showed

an approximately 200-fold increase when compared with

mono-biofilm (Table 3). More than 2% of PVP–I was required to

significantly reduce both P. gingivalis and F. nucleatum in any

contact time point. PVP–I with concentrations equal to or

greater than 5% completely eradicated both pathogens in dual-

biofilm. Increasing the contact time did not significantly

influence P. gingivalis or F. nucleatum recovery from dual-

biofilm.

4. Discussion

Both P. gingivalis and F. nucleatum are important microbial

constituents in human periodontal biofilm. In our preliminary

experiments, we found that PVP–I with concentrations

indicated for daily oral rinse effectively suppressed periodon-

tal pathogens such as P. gingivalis, Aggregatibacter actinomyce-

temcomitans or F. nucleatum in planktonic cultures (unpublished

observation). In the present study, we sought to delineate the

effect of PVP–I on periodontal pathogens in biofilm. The results

suggest that 30 s application of 2% PVP–I could effectively

suppress P. gingivalis–F. nucleatum dual-species biofilm.

Different methods have been utilized in order to generate

artificial biofilms, including the use of flow-cells and micro-

plates.17 In the present study, we established a dual-species

biofilm model of periodontal pathogens, using inserts of PCM

in a cell culture plate. This method allows us to enumerate

viable bacteria recovered from biofilm. 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-biofilm within control experiments.

Differences in bacterial growth, attachment to PCM and level

of coaggregation between species may be the causes of the

a r c h i v e s o f o r a l b i o l o g y 5 7 ( 2 0 1 2 ) 3 6 4 – 3 6 8 367

data with large standard deviations. P. gingivalis was more

efficient than F. nucleatum in their ability to form mono-biofilm

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 difficult. This may be, at least in part, responsible for

wide variation of the bacterial recoveries in each experiment.

In dual-species biofilm, F. nucleatum exhibited a different

growth characteristic when compared with mono-microbial

biofilm. 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, PVP–I concentrations indicated for

daily oral rinsing (0.23–0.47%) failed to significantly abrogate

biofilm 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 biofilm formation.19 Although PVP–I with concen-

tration of 1% or greater was needed to significantly reduce P.

gingivalis or F. nucleatum in mono- or dual-biofilm, more than

5% were needed to completely kill all bacteria in dual-biofilm.

This study, as well as the previous reports by Nakagawa

et al.20 and Hoang et al.,21 suggests that PVP–I with low

concentrations would be effective when biofilm bacteria are

disrupted by mechanical intervention. Without mechanical

disruption, higher concentrations are necessary to exert its

effect on biofilm.

PVP–I 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 PVP–I 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 PVP–I in high concentrations or log-term use. PVP–I

gargle solution has been known to cause yellowish discolor-

ation 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 PVP–I with the lowest concentration

that would yield a maximum effect against periodontal

biofilm. Our results suggest that short durations of 2% PVP–I

contact with periodontal bacteria in biofilm results in effective

in vitro killing.

Several limitations of this study need to be discussed. First,

we used a dual-species biofilm model. The effect of PVP–I

solutions on polymicrobial biofilm 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 biofilm formation (dental

and periodontal pathogens) via determination of CFU and

fluorescence microscopy allowed fast, unambiguous and repro-

ducible 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 fluid,

an addition of serum into our assay may give us useful

information. Also, the actual effect of the proposed use of

PVP–I in vivo situations needs to be evaluated in further clinical

studies.

Funding

This work was supported by grants-in-aid for scientific

research C-22592317 (to AS) and C-17592164 (to TN) from

Japan Society for Promotion of Science.

Competing interests

The authors have no conflicts of interest to disclose.

Ethical approval

Not required

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