439

Isolation and Purification of Bacteriocin From Prevotella intermedia (Bacteroides

intermedius)* Kazuko Takada, Masatomo Hirasawa, and Tadashi Ikeda

Thirty-three out of 46 strains of Prevotella intermedia (Bacteroides intermedius) isolated from gingival deposits of patients with adult Periodontitis possessed the ability to inhibit growth of P. intermedia strain ATCC 25611, and 6 of the 33 showed strong inhibitory activity. Thirteen isolates did not have the ability to inhibit the growth of P. intermedia strain ATCC 25611. An inhibition factor from one of the isolates, P. intermedia TH14 which had strong inhibition, was purified and characterized. Bacteriocin TH14 activity was effective against only P. intermedia ATCC 25611 and Fusobacterium nucleatum, but not against other crevicular bacteria, including P. intermedia ATCC

33563, Porphyromonas gingivalis (Bacteroides gingivalis), Capnocytophaga sputigena

and Actinobacillus actinomycetemcomitans. Streptococci, actinomyces, and veillonellas not inhibited by bacteriocin TH14. Bacteriocin TH14 was heat sensitive, proteinaceous with molecular weight of 1,700 daltons and inhibited the growth of strain ATCC 25611 in 15 minutes. The growth inhibition of P. intermedia ATCC 25611 by bacteriocin TH14 was found to be due to the bactericidal activity of the bacteriocin. / Periodontol

were

1991; 62:439-444.

Key Words: Periodontitis/microbiology; Prevotella (Bacteroides) intermedia; Fusobacterium nucleatum; Porphyromonas (Bacteroides) gingivalis; Capnocytophaga sputigena; Actinobacillus actinomycetemcomitans; bacteriocin.

Certain species of black-pigmented oral bacteria (Prevotella1 and Porphyromonas2) may play an important role in the development of adult periodontal diseases. Among these species, Prevotella intermedia has been considered as one of the important pathogens for advancing the periodontal disease including adult Periodontitis,3-4 acute necrotizing ulcerative gingivitis,5 and pregnancy gingivitis.6 It has been shown that bacteriocins produced by cariogenic bacterium Streptococcus mutans group inhibit the growth of some species and prevent the development of dental caries in rat model.7-11 On the other hand, very few studies have been performed to isolate and characterize bacteriocin produced by periodontopathic bacteria.12 Recent studies have provided evidence that Actinobacillus actinomycetemcomitans associated with juvenile Periodontitis

produces bacteriocin.13-14

Periodontal diseases can be prevented by eliminating periodontopathic bacteria from the gingival crevice and plaque. The development of bacteriocin which specifically inhibits the growth of causative bacterium could be a beneficial therapeutic method in the regulation of plaque microflora.

'Department

of

Microbiology,

Matsudo, Matsudo, Japan

Nihon

University

School of

Dentistry

at

The bacteriocin which possesses activity against P. intermedia ATCC 25611, periodontitis-associated strain,15 must be considered as a possible epidemiologie tool and potential source of anti-periodontitis agents. Thus, the aim of this study was to isolate and purify bacteriocin which specifically affect P. intermedia ATCC 25611.

MATERIALS AND METHODS

Microorganisms and Growth Media Laboratory stock strains of Prevotella and Porphyromonas

spp and other oral bacteria used in this study are shown in Table 1. Forty-six P. intermedia strains were isolated from gingival crevice deposits of 23 adult Periodontitis subjects. P. intermedia ATCC 25611 was used as the indicator strain for the assay of bacteriocin activity. Microorganisms were maintained in brain heart infusion* agar supplemented with hemin (1 mg/1), menadione (1 mg/1) and defibrinated rabbit blood (5% v/v). Microorganisms were grown in GAM broth* containing hemin (1 mg/1) and menadione (1 mg/1) (modified GAM) for experimental purposes. For the production fDifco Laboratories, Detroit, MI. *Nissui Pharmaceutical Co., Japan.

440

J Periodontol July 1991

BACTEKJOCIN FROM PREVOTELLA INTERMEDIA

Table 1.

Sensitivity of Various Oral

Bacteria to Bacteriocin TH14

Organisms Prevotella P. intermedia P. intermedia P. melaninogenica P. loescheii P. denticola

Strain ATCC ATCC ATCC ATCC ATCC

Porphyromonas P. gingivalis Capnocytophaga C. sputigena

Fusobacterium F. nucleatum Veillonella V. alcalescens Actinobacillus A. actinomycetemcomitans

Actinomyces

A. naeslundii A. viscosas

Sensitivity

25611 33563 25845 15930 33185

+

*

381 NUD NUD

NUD ATCC 29522

ATCC 12104 ATCC 19246

Streptococcus S. S. S. S. S.

sanguis

salivarius mitior mutans mutans

ATCC 10556 HHT NCTC 11391 NCTC 10449

Ingbritt

measuring greater than indicating sensitivity to the bacteriocin. *Zone of inhibition

for 24 hours. Analyses of sensitivity to various oral bacteria also assayed by well-plate technique using 15 units of bacteriocin TH14 per each oral bacterium. The experimental manipulation of cultures was always performed in an anaerobic chamber. Units of bacteriocin activity were determined by wellplate technique. One unit of the activity was defined as the amount of bacteriocin 14 which gave a clear inhibition zone of 0.6 cm in diameter around the indicator strain. were

6

mm

in diameter

was

taken

of bacteriocin,

partially defined medium with glucose (PDwith 0.001% hemin, 0.001% menasupplemented glc)16 dione, and 0.5% -globulin was used (modified PD-glc). Cultures were incubated at 37°C for 24 hours in an anaerobic chamber with an atmosphere of 80% N2, 10% H2 and

10%CO2.

Assay for Bacteriocin In this study, two methods were employed to measure the activity of the inhibitory factor. In the first, for isolation of bacteriocin-producing strains, isolates from gingival fluid

patients with adult Periodontitis were stab cultured in plates of enriched trypticase soy (ETS)17 agar with 5% horse serum medium (modified ETS) and then incubated at 37°C for 24 hours. Ten ml of molten ETS containing 0.75% agar (ca 45°C), with 0.3 ml (absorbance of 1.5) of P. intermedia of

ATCC 25611 grown in modified GAM broth at 37°C for 24 hours, was carefully layered onto the incubated plate. After 24 hours of additional incubation at 37°C, the zone of inhibition surrounding the isolates was measured. This method was termed as the overlay technique according to the original procedure of Fredericq.18 In the second method, preparations of purification steps were assayed by well-plate technique as described previously19 using modified ETS agar and modified GAM broth instead of BHI agar and BHI broth, respectively. Plates were prepared by inoculating 20 ml of molten modified ETS agar with 0.3 ml (absorbance of 1.5) of P. intermedia ATCC 25611 grown in modified GAM broth. Samples were added to individual wells (0.4 cm diameter) in the agar plates, and the plates were incubated at 37°C

Purification of Bacteriocin Three liters of a 24-hour culture of P. intermedia 14 grown in modified PD-glc was centrifuged (10,000 x g, 30 minutes), and the culture supernatant fluid was concentrated by vacuum rotator at below 30°C to a volume of 300 ml. The concentrated material was then applied to an ion retardation resin column, Retardion 11A8,§ (10 by 50 cm) to remove salts. The column was washed with distilled water and fractions which emerged in the void volume which did not precipitate by AgN03 solution were pooled and concentrated by vacuum rotator at below 30°C. The concentrated material was then applied to an Ultrogel AcA-3411 (linear fractionation range of 3.5 x 10s to 2.0 x 104 daltons column (5 by 200 cm). The column was washed with 0.01 M phosphate buffer, pH 7.0, and column fractions were measured at absorbance of 280 nm and monitored for bacteriocin activity by the well-plate technique. All fractions containing inhibitory activity were pooled and concentrated by vacuum rotator at below 30°C. The concentrate was further applied to a Bio-gel P61 (linear fractionation range of 6 x 103 to 1 x 103 daltons) column (7 by 80 cm), followed by a Bio-gel P21 (linear fractionation range of 1.8 x 103 to 1 x 103 daltons) column (3 by 120 cm). The monitoring for bacteriocin activity was performed by the same manner as described above. After concentration by vacuum rotator, the fraction containing bacteriocin activity was applied to a Retardion 11A8 column (2 by 10 cm) in order to remove salts. The bacteriocin fractions (designated bacteriocin 14) which emerged in the void volume were pooled and lyophilized. Measurement of Molecular Weight The molecular weight of bacteriocin TH14 was determined by Bio-gel P2 and P6 gel filtrations. The molecular weight standards11 which were used are glutathione, disulfide (600 daltons), vitamin B-12 (1,350), myoglobulin III (2,556), and ACTH (3,500).

Bactericidal Activity of Bacteriocin TH14 P. intermedia ATCC 25611 was grown in modified GAM broth, harvested, washed, and suspended using saline to an optical density of 0.1 at 540 nm. From these suspensions, §Muromachi Chemical Co.,

Japan.

'LKB Instruments, Inc., Rockville, MD. 'Bio-Rad Laboratories, Richmond, CA.

Volume 62 Number 7 Table 2.

Activity

TAKADA,

Screening of 46 Strains of intermedia

Activity

Indicator P. intermedia ATCC 25611

+ +

No. of Strains 6 27 13

*

++ -*

*Zone of clear inhibition with

for Antibacterial

sharp edge.

tZone of turbid inhibition with hazy edge. tNo inhibition.

0.1 ml of material

added to 0.9 ml of 0.05 M trishydrochloride buffer, pH 7.0, containing 15 units of bacteriocin TH14. The mixture was incubated at room temperature for 15, 30, and 45 minutes, and then a sample was washed with the same buffer and was immediately diluted and spread on ETS agar plates. In studies where increasing concentrations of bacteriocin were used, the mixtures containing different amounts of bacteriocin ranging from 2 to 10 units were incubated at room temperature for 15 minutes. These were washed, diluted, and spread on ETS agar plates. The plates were incubated and after 48 hours incubation, the number of colony forming units (CFU) was counted, and the percentage killed was calculated from a control culture not treated with bacteriocin. All the procedures were always performed in an anaerobic chamber. was

Analytical Methods

Protein was determined by a protein dye binding assay with the standard provided by Bio-Rad.1 The identification of clinical isolates was performed both in the Minitek system* and by serological analysis using the double immunodiffusion method with antibody against P. intermedia ATCC 25611.

Survey of Bacteriocin Production by P. intermedia

clinical isolates of P. intermedia strains were screened by the overlay technique for their ability to inhibit the growth of the indicator strain. As shown in Table 2, 33 isolates produced growth inhibitors which were effective against P. intermedia ATCC 25611. Among these strains, 6 clearly showed strong inhibitory activity of the indicator strain. Thirteen isolates had no ability to inhibit the growth

Forty-six

Microbiology Systems, Cockeysville,

of P. intermedia ATCC 25611. One clinical isolated strain, designated P. intermedia 14, spontaneously released bacteriocin in media. The concentrate of the culture supernatant fluid of P. intermedia 14 was capable of causing a clear and wide zone of inhibition against P. intermedia ATCC 25611. The bacteriocin from strain TH14 was, therefore, purified and characterized. Purification and General Properties of the Bacteriocin A summary of the purification of bacteriocin TH14 is presented in Table 3. A 348-fold purification was achieved after a second Retardion 11A8 chromatography, and a single peak of activity was obtained after chromatography on both Bio-gel P2 and Retardion 11A8 (Fig. 1). A loss of the activity of the bacteriocin was found by 24 hour dialysis using membrane molecular weight cutoff of 10,000 daltons. The reduction of the activity during the 24 hour dialysis was observed with membrane molecular weight cutoff of 3,500 daltons." From the gel filtration studies, the molecular weight was estimated to be approximately 1,700 daltons. The bacteriocin activity was stable when kept in the crude state. The purified bacteriocin was relatively stable for a period of a few months when it was lyophilized or stored in aqueous solution at pH 7.0 at 20°C. However, the inhibitory activity was inactivated at 90°C for 5 minutes. A significant reduction in inhibitory activity occurred when the bacteriocin was kept at room temperature for several weeks. The inhibition activity of bacteriocin 14 was removed by protasen at pH 7.5 at 37°C for 18 hours. -

Sensitivity of Various Bacteria to Bacteriocin TH14

Representative strains of various oral bacteria were tested sensitivity to bacteriocin 14 (Table 1). The sensitivity to the bacteriocin was observed when Fusobac-

for their

was used as indicator strain instead of P. intermedia ATCC 25611. P. intermedia ATCC 33563, the other species of Prevotella and Porpyhromonas, and other oral bacteria including representatives of the genera Veil-

loneüa, Actinobacillus, Actinomyces, Capnocytophaga, and

Streptococcus were not sensitive to the bacteriocin.

Antibacterial Effect To obtain information

bacteriocin TH14,

Step

Protein

Sp act (U/mg of

Purification

0.5 26.2 59.5 165.9 173.8

52.4 119.0 331.8 347.6

Recovery

(fraction)_(mg)_(U)_protein)_(fold)_(%)

First 11A8 AcA34 P6 P2 Second 11A8

17,466.0 286.2 84.0 4.8 4.2

8,950.0 7,500.0 5,000.0 793.6 730.1

the nature of the inhibition by P. intermedia

"Spectrum Medical Industries, Inc., CA. "Sigma Chemical Co., St. Louis, MO.

MD.

Activity

on

exponentially growing

Table 3: Purification of Bacteriocin TH14

Purification

441

terium nucleatum

RESULTS

*BBL

HI RASAWA, IKEDA

100 83.8 55.9 8.9 8.2

442

J Periodontol July 1991

BACTERIOCIN FROM PREVOTELLA INTERMEDIA 100.0

10.0

Fraction number

1. Retardion 11A8 elation profile teriocin TH14.

Figure

i(4

of a Bio-gel P2 fraction of bac-

treatment

•

no

O

treatment

2

4

Concentration

6 ,

10

U

Figure 3.

Bactericidal activity of bacteriocin TH14. P. intermedia ATCC 25611 cells were treated with various bacteriocin TH14 concentrations at 37°C for 15 minutes.

1(4 Ü

o

0

15 30 45 Incubation time,min of bacteriocin TH14-induced lethality. P.

Figure 2. Kinetics ATCC 25611 cells were treated with 15 units at 3TC for various incubation times.

intermedia

(o) or without (*) bacteriocin

ATCC 25611 cell suspension was treated at various times with increasing concentrations of the bacteriocin and the number of surviving CFU was determined. Addition of 15 units (the killing concentration) of the bacteriocin to cell suspensions resulted in a rapid death in 15 minutes (Fig. 2). Figure 3 suggests that the inhibitory activity of bacteriocin TH14 is a result of its bactericidal activity. The number of units of bacteriocin needed to kill 50% of the resting cells of P. intermedia ATCC 25611 was 1.5 units, and more than 10 units of bacteriocin were sufficient to completely eliminate the growth of indicator cells (Fig. 3). DISCUSSION The pathogenesis of periodontal diseases is extremely complex due to direct and indirect effects on the periodontal tissues of bacterial plaque deposits and host defense mech-

anisms. Several Gram-negative microorganisms including P. gingivalis, P. intermedia, and A. actinomycetemcomitans have been considered as possible pathogens for various forms of periodontal diseases.4 A number of these organisms that colonize the oral cavity have been shown to possess strongly adhesive properties for binding to soft tissue, salivary pellicle, or other bacteria.20 This binding initiates the process of periodontal disease. One possible approach to eliminate these pathogens at local disease sites could be through the use of bacteriocin against periodontopathic bacteria. Toward this effort studies to identify, isolate, and characterize bacteriocin with activity against such bacteria are being done. Bacteriocins specifically inhibited only species belonging to taxonomically close bacteria. Bacteriocinogeny has been extensively characterized by using S. mutans group. Several reports have showed that more than 80% of the strains from the 5. mutans group have the ability to produce a bacteriocin.8'11-21 With regard to bacteriocin produced by periodontopathic microorganisms, Nakamura et al.12 were the first to show that strains of Prevotella melaninogenica could produce bacteriocin. They showed that 13 strains out of 23 isolates (57%) produce growth inhibitors which were effective against at least one spectrum of strains. Our survey of P. intermedia strains for bacteriocin production has shown that 72% of the isolates have inhibitory ability to the growth of P. intermedia ATCC 25611 (Table 2). In this report, the inhibitory factor against P. intermedia ATCC 25611 was purified and characterized. We have shown that the bacteriocin 14 is a dialyzable substance with a

Volume 62 Number 7

TAKADA, HIRASAWA, IKEDA

443

bactericidal mode of action which can be inactivated by heating. This heat instability is not unusual in light of the fact that bacteriocin is proteinaceous in nature. The bacteriocins from Bacteroides and Prevotella species vary in size. The molecular weight of bacteriocin from the fecal Bacteroides strains was reported to be more than 300,000 daltons.22 The bacteriocin from P. melaninogenica

associated P. intermedia ATCC 25611, but not P. intermedia ATCC 33563 which is related with a healthy status. The bacteriocin TH14 might be a useful tool for prevention of Periodontitis. In the future, adaptation of modem molecular biotechnology may be able to produce mass quantities of this bacteriocin TH14 for usage in daily treatment.

was

REFERENCES 1. Shah HN, Collins

estimated to be approximately 105,000 daltons.12 On the other hand, bacteriocin from Bacteroides fragilis was reported to have a rather low molecular weight which ranged from 13,500 to 18,700.23 Our studies show that the purified bacteriocin TH14 had a molecular weight of only 1,700 daltons. A unique feature of TH14 is its low molecular weight. Although we have no explanation for this finding, an unusually small size bacteriocin (1,000 daltons) has been isolated as mutacin from 5. mutans serotype c.9 The bacteriocin 14 is similar in size to mutacin from S. mutans than the bacteriocin from other Bacteroides and Prevotella

species. A previous report

has shown that the bacteriocin of A. actinomycetemcomitans also had a relatively narrow antibiotic spectrum, when its activity was demonstrated against other A. actinomycetemcomitans, Actinomyces viscosus, Streptococcus sanguis, and Streptococcus uberis.13 Bacteriocin TH14 has a very narrow antibiotic spectrum, and it was active against two other taxonomically remote species, P. intermedia ATCC 25611 and Fusobacterium nucleatum (Table 1). It was interesting to note that the bacteriocin was not active against P. intermedia ATCC 33563 and other taxonomically close Prevotella and Porphyromonas species (Table 1). P. intermedia is separated into two groups genetically. One group is represented by ATCC 25611 (VPI 4197) and the other by ATCC 33563 (VPI 8944).24·25 The former strain has been associated with Periodontitis, while the latter strain was related to health.15 Bacteriocin 14 can be considered as a useful agent for prevention of Periodontitis which is induced by P. intermedia. Bacteriocin 14 was shown to be lethal for P. intermedia ATCC 25611 (Fig. 3). From the kinetics of inactivation, we show that the cells were completely killed in 15 minutes (Fig. 2). The interaction of a single molecule of bacteriocin with a target cell may occur directly and rapidly. The rate at which bacteriocins from B. fragilis, A. actinomycetemcomitans, or S. mutans serotype c hit target cells is usually fast. The inactivation is complete in approximately 30 minutes.10,13'23 On the other hand, the bacteriocin from feces Bacteroides strain requires at least 1 to 2 hours incubation time for the elimination of indicator organisms.22 Figure 3 also shows that the death curves have a shoulder and were curvilinear. The presence of a shoulder suggests that the action of the bacteriocin showed multiple hit kinetics, and that more than one type of sensitive cell may be present. In summary, we found that bacteriocin 14 isolated from P. intermedia TH14 possesses a very narrow antibiotic spectrum. This bacteriocin specifically affects periodontitis-

2.

3. 4.

5. 6. 7.

8. 9.

10.

11.

12.

13. 14.

15.

16.

17. 18. 19.

20.

21.

22.

DM. Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol 1990; 40:205. Shah HN, Collins MD. Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int J Syst Bacteriol 1988; 38:128. Mayrand D, Holt SC. Biology of asaccharolytic black-pigmented Bacteroides species. Microbio! Rev 1988; 52:134. Slots J, Listgarten MA. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol 1988; 15:85. Loesche WJ, Syed SA, Laughon BE, Stoll J. The bacteriology of acute necrotizing ulcerative gingivitis. J Periodontol 1982; 53:223. Kornman K, Loesche WJ. The subgingival microbial flora during pregnancy. J Periodont Res 1980; 15:111. Fukushima H, Fukushima S, Umemoto T, Fukuhara H, Sagawa H. Purification and chemical analysis of a bacteriocin from oral bacterium Streptococcus mutans RM-10. Arch Oral Biol 1982; 27:721. Hamada S, Ooshima T. Production and properties of bacteriocin (mutacins) from Streptococcus mutans. Arch Oral Biol 1975; 20:641. Hillman JD, Johnson KP, Yaphe BI. Isolation of a Streptococcus mutans strain producing a novel bacteriocin. Infect Immun 1984; 44:141. Ikeda T, Iwanami T, Hirasawa M, Watanabe C, McGhee JR, Shiota T. Purification and certain properties of a bacteriocin from Streptococcus mutans. Infect Immun 1982; 35:861. Rogers AH. Bacteriocin patterns of strains belonging to various serotypes of Streptococcus mutans. Arch Oral Biol 1976; 21:243. Nakamura T, Fujimura S, Obata , Yamazaki . Bacteriocin-like substance (melaninocin) from oral Bacteroides melaninogenicus. Infect Immun 1981; 31:28. Hammond BF, Lillard SE, Stevens RH. A bacteriocin of Actinobacillus actinomycetemcomitans. Infect Immun 1987; 55:686. Stevens RH, Lillard SE, Hammond BF. Purification and biochemical properties of a bacteriocin from Actinobacillus actinomycetemcomitans. Infect Immun 1987; 55:692. Moore LVH, Moore WEC, Cato EP, et al. Bacteriology of human gingivitis. J Dent Res 1987; 66:989. Ikeda T, Shiota T, McGhee JR, et al. Virulence of Streptococcus mutans: Comparison of the effects of a coupling sugar and sucrose on certain metabolic activities and cariogenicity. Infect Immun 1978; 19:477. Syed SA. Characteristics of Bacteroides asaccharolyticus from dental plaques of beagle dogs. / Clin Microbiol 1980; 11:522. Fredericq P. Colicins. Ann Rev Microbiol 1957; 11:7. Takada K, Ikeda T, Mitsui I, Shiota T. Mode of inhibitory action of a bacteriocin produced by Streptococcus mutans C3603. Infect Immun 1984; 44:370. Slots J, Gibbons RJ. Attachment of Bacteroides melaninogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets. Infect Immun 1978; 19:254. Fabio U, Bondi M, Manicardi G, Mossi , Neglia R. Production of bacteriocin-like substances by human oral streptococci. Microbiologica 1987; 10:363. Booth SJ, Johnson JL, Wilkins TD. Bacteriocin production by strains of Bacteroides isolated from human feces and the role of these strains

444

BACTERIOCIN FROM PREVOTELLA INTERMEDLA

in the bacterial ecology of the colon. Antimicrob Agents Chemother 1977; 11:718. 23. Mossis KG, Jones DT, Robb FT, Woods DR. Characterization and mode of action of a bacteriocin produced by a Bacteroides fragilis strain. Antimicrob Agents Chemother 1979; 16:724. 24. Johnson JL, Holdeman LV. Bacteroides intermedias umb. nov. and descriptions of Bacteroides corporis sp. nov. and Bacteroides levii sp. nov. Int J Syst Bacteriol 1983; 33:15. 25. Holdeman LV, Kelley RW, Moore WEC. Anaerobic Gram-negative

J Periodontol July 1991

straight, curved and helical rods. Family I Bacteroidacese Genus I Bacteroides. In: Krieg NR, Holt JG eds. Bergey's Manual of Systematic Bacteriology, vol. 1. Baltimore: Williams & Wilkins; 1984:602. Send reprint requests to: Dr. Kazuko Takada, Department of Microbiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan. Accepted for publication January 30, 1991.