Archs oral B&l. Vol. 35, No. 9, pp. 775-177,1990 Printed in Great Britain. At1rights reserved
ooo3-9969/90$3.00+ 0.64 copyright @ 1990Pergamon Press pie
SI-IORT COMMUNICATION rNHIB~rORY EFFECTS OF SYNTHETIC HISTIDINE-RICH PEPTIDES ON HAEMAGGLUTINATION BY BACTEROIDES GINGIVALIS 381 Y. M~~RAKA~,’T. TAKESHITA,’S. SHIZUKUISHI,’ A. TSUNEMITSU’*and S. AIMOTO* ‘Department of Preventive Dentistry, Osaka University Faculty of Dentistry and ‘Institute for Protein Research, Osaka University, l-8 Yamadaoka, Suita, Osaka 565, Japan (Accepted 26 April 1990) haemagglutinating activity of Bacteroidesgingiualis381 was significantly inhibited by the synthetic peptide, Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-GIu-Lys-His-HisSet-His-Arg-Gly-Tyr. However, bradykinin potentiator C, which does not contain cationic amino acids such as L-histidine, t-arginine and L-lysine, had no inhibitory effect.
Summary-T!le
Key words: hzcteroides gingiualis,haemagglutination,
It is well known th.at the haema~utinating activity of Bacteroides gingivalis is inhibited by human whole saliva, especially high molecular-weight components of saliva (Slots and Gibbons, 1978; Okuda, Slots and Genco, 1981). Howlever, little attention has been paid to the inhibitory substances such as low molecularweight proteins aon haemagglutinating activity. Inoshita et al. (1986) have pointed out that the haemagglutinating activity of the exohaemagglutinin from the culture medium of Bact. gingiualis 381 was inhibited by angio-tensin I peptide in which histidine and arginine residues are involved. In saliva, numerous polypeptides and proteins have been isolated and recognized as strongly binding to hydroxyapatite molecules. Concerning the adsorption properties, the most studied histidine-rich protein was described by Hay (1975) and Baum et al. (1976). MacKay et al. (1984) and Pollock et al. (1984) have shown that histidine-rich proteins have antimicrobial effects including growth inhibition of the Candida al&cans and streptococcus mutans. More recently, Op~nheim et al. (1988) have established that there are three major histidine-rich proteins in human parotid saliva and have clarified their structural interrelationships. These three proteins have been completely purified to apparent homogeneity. Their primary structures have been determined, and their anti-Candidal activity measured. Based on the different primary structures of three proteins, the major salivary histidine-rich proteins were named histatins 1, 3 and 5. Oppenheim et al. have suggested that histatins 1 and 3 are derived from different structural genes, whereas histatin 5 is a proteolytic product of histatin 3. We had a strong interest in each histatin having cationic amino acids such as L-histidine, L-arginine and L-lysine in its to: Dr A. Tsunemitsu, Department of Preventive Dentistry, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565, Japan.
*Address correspondence
synthetic histidine-rich peptide.
peptide chains. We now describe the inhibitory effwt of the synthetic peptide of histatin 5 alone, which is a potent inhibitor of C. a&cans germination, compared to that of histatins 1 and 3, on haemagglutinating activity of Bact. gingiualis. The inhibitory effects on haemagglutination of two shorter synthetic peptides of histatin 5 which are not known exist in human parotid saliva were also examined. Bact. gingiuah 381 was a gift from stock strains at the Research Laboratory of Oral Biology, Sunstar Inc., Osaka, Japan. The strain was maintained by weekly transfer on plates containing trypticase soy agar (BBL Microbiology Systems, Cockeysville, MD) supplemented with 5% sheep blood, 1 mg of yeast extract (BBL) per ml, 5 pg of haemin per ml, 1 @g of menadione per ml. The organism was grown in 10 ml of trypticase soy broth (BBL) supplemented with 1 mg of yeast extract per ml, 5 pg of haemin per ml, 1 fig of menadione per ml. The first series of cultures was transferred into 100 ml of the same type of broth medium used for pre-culture. Cultures were incubate in an anaerobic system 1024 (Forma, Morietta, OH) in an N,-H&O, (85: 10:5) atmosphere at 37°C. The haemagglutinating activity was determined at room temperature with a microtitre plate (Limbro Division Flow Laboratories Inc., Hamden, CT). Because the haemaggiutination of Butt. gingivaris was nonspecific for blood-group ABO types in our preli~nary check, human O-type erythrocytes were used for the haemagglutination assay. The sample was diluted in a 2-fold series in isotonic saline, and an equal volume of a 2% suspension of human erythrocytes was added. The mixtures were left to stand for 90 min, and haemagglutination was determined by observing the mixture with the naked eye. Titre was expressed as the reciprocal of the highest dilution showing positive agglutination. The haemagglutination inhibition test for the synthetic peptides was by the method of Inoshita
Y. MURAKAMIet al.
776
E
3
Table 2. Inhibitory effect on haemagglutination of Bact. gingivalis by synthetic peptides using the spectrophotometric method
-------------
$ in z
3
Peptide concentration (M)
II 2
‘72
El T)
1x 5x 1x 5x 1x 5x 1x 5x
t\
k
q 0
2.5
5.0
z5
Time (mini
10-d 10-s 10-S 10-S 10-6 10-7 10-r 10-s
Inhibition (%) Control
I
0 0 0 0 0 0 0 0
100 97 86 47 10 0 0 0
II
III
100 100 100 98 29 8 0 0
100 100 100 100 67 12 6 0
Fig. 1. Inhibitory haemagglutinating curve by synthetic peptides of peptide III. Reaction mixture contained 5 x 10’ erythrocytes, 3 x IO*bacterial cells and 10 mM PBS, pH 7.0 in a final volume of 2 ml (-). Added the synthetic peptide III to the reaction mixture at lo-’ M concentration (----).
The values in the table show the mean in duplicate experiments. Bradykinin potentiator C was used as a control. The sequence of peptides I, II and III are indicated in Table 1.
et al. (1986). The haemagglutinating activity was also determined by a spectrophotometric method. The reaction mixture for assay contained 5 x 10’ human O-type erythrocytes, 3 x 10’ bacterial cells and
on a YMC-gel ODS S-S AM type column (YMC, Kyoto, Japan). The peptide thus obtained was lyophilized with distilled water. Amino acid composition after 24 h hydrolysis at 110°C with constant-boiling hydrochloric acid was as follows. Peptide I: Ser,,, Gly,.,, Tyr,,, His,,, Lysi,, Arg,,,. Peptide II: Ser,,, Glu,.,, Glyr.,, Tyr,.,, Phei,, His,,, L~sr.~, Arg,,. Peptide III: Asp,.,, Serr.e, Glui.r, GIY,.~, Alai.,, Tyrr.i,
10 mM phosphate-buffered saline, pH 7.0, in a final volume of 2ml. Haemagglutinating activity was spectrophotometrically monitored with a decreased optical density at 542 nm. Based on the change of optical density 5 min after start of reaction, percentage inhibition was calculated. Three peptides, Lys-HisHis-Ser-His-Arg-Gly-Tyr (‘peptide I: shortest fragment of histatin 5) Gly-Tyr-Lys-Arg-Lys-Phe-His-GluLys-His-His-Ser-His-Arg-Gly-Tyr (‘peptide II: shorter fragment of histatin 5), and Asp-Ser-His-Ala-LysArg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-LysHis-His-Ser-His-Arg-Gly-Tyr (peptide III: same as histatin 5) were synthesized by a solid-phase technique using an Applied Biosystems Inc. (Foster, CA) peptide synthesizer 430A with a Boc-amino acid anhydride method. After the completion of a peptide chain assembly, a peptide-linked resin was treated with anhydrous hydrogen fluoride containing 10% anisole at 0°C for 75 min. The crude peptide was purified
Phei,a, His,,,
Lyss.9, Args.c.
The haemagglutinating curve is shown in Fig. 1. When the synthetic peptide III was added to the reaction mixture, no change of optical density at 542 nm was found in the haemagglutinating curve. The results of haemagglutination inhibition by some peptides are shown in Tables 1 and 2. The haemagglutinating activity was markedly inhibited by peptide III, moderately by peptide II, and weakly by peptide I, but bradykinin potentiator C (Peptide Institute Inc., Minoh, Osaka, Japan) was not inhibitory. These results suggest that the cationic peptides containing histidine, arginine and lysine residues may function electrostatically as the binding domains for the Bact. gingivalis adhesin and/or erythrocyte surfaces during
Table 1. Inhibition of haemagglutination by Bact. gingivalis with synthetic peptides by means of microtitre plate assay Peptides Control I II
III
Primary structure 1 5 10 Pyr-Gly-Leu-Pro-Pro-Gly-Pro-Pro-Ile-Pro-Pro 1 5 Lys-His-His-Ser-His-Arg-Gly-Tyr 1 5 10 Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His15 His-Ser-His-Arg-Gly-Tyr
1 5 10 Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg15 20 Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr
Lowest inhibitory concentration (PM) > 420.0 12.5 1.6
0.8
Bradykinin potentiator C was used as a control. The sequence of peptide III is the same as that of histatin 5 named by Oppenheim et al. (1988). Peptide I and II are shorter fragments of histatin 5.
Inhibitors of haemag~utinating activity agglutination, although the exact mechanism is not clear. In our experiments, bradykinin potentiator C alone was used as a control. Additional peptides in which cationic amino acids are not included may be needed for a control experiment. The Bact. girrgiu&s that colonize thie mouth have often been shown to have adhesive properties that enable them to bind to epithelial cells, salivary pellicle, as well as to Gram-positive bacterial species (Slots and Gibbons, 1978; Okuda et al., 1981; Gibbons and Etherdern, 1983; Boyd and McBride, 1984). Based on the observed inhibition of haema~lutination, it is quite possible that this histatin 5 might play an inhibitor roie in the colonization of Bact. gingivalis in the mouth. REFERENCES Baum B. J., Bird J. L., Millar D. B. and Longton R. W. (1976) Studies on hi~.tidine-richpolypeptides from human
parotid saliva. Ar&r B&hem.
Riophys. 177, 427-436.
Boyd J. F. and McBride B. C. (1984) Fractionation hemagglutinating and bacterial binding adhesins
of of
Bacteroides gingivahs. Infect. Immun. 45, 403-409.
Gibbons R. J. and Etherdern I. (1983) Comparative hydrophobicities of oral bacteria and their adherence to salivary pellicles. htfect. ~rnn~~. 41, 1190-I 196.
777
Hay D. I. (1975) Fractionation of human parotid salivary proteins and the isolation of an histidine-rich acidic peptide which shows high affinity for hydroxyapatite surfaces. Archs oral Biol. 20, 553-558. Inoshita E., Amano A., Hanioka T., Tamagawa H., Shizukuishi S. and Tsunemitsu A. (1986) Isolation and some properties of exohemag~utinin from the culture medium of Bacteroides gingjvaI~ 381. Injkt. Immun. 52, 421-427.
MacKay B. J., Denepitiya L., Iacono V. J., Krost S. B. and Pollock J. J. (1984) Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich peptides on Streptococcus mutans. Infect. Immun. 44, 695-101. Okuda K., Slots J. and Genco R. J. (1981) Bacteroides gingivalis. ~a~teroides ~a~charo~yti~s and Bacteroides melaninogeni~us subspecies: cell surface morphology and adherence to erythrocytes and human buccal epithelial cells. Curr. Microbial. 6, 7-12. Oppenheim F. G., Xu T., McMillian F. M., Levitz S. M., Diamond R. D., Offner G. D. and Troxler R. F. (1988) Histatins, a novel family of histidine-rich proteins in human parotid secretion. .J. biol. Chem. 263, 7472-7476. Pollock J. J., Denepitiya L., MacKay B. J. and Iacono V. J, (1984) Fungistatic and fungicidal activity of human parotid salivary histidine-rich peptides on Candida albicans. Infect. Immun. 44, 102-101.
Slots J. and Gibbons R. J. (1978) Attachment of Bacteroides meianinogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets. Infect. Immun. 19, 254-264.
ooo3-9969/90$3.00+ 0.64 copyright @ 1990Pergamon Press pie
SI-IORT COMMUNICATION rNHIB~rORY EFFECTS OF SYNTHETIC HISTIDINE-RICH PEPTIDES ON HAEMAGGLUTINATION BY BACTEROIDES GINGIVALIS 381 Y. M~~RAKA~,’T. TAKESHITA,’S. SHIZUKUISHI,’ A. TSUNEMITSU’*and S. AIMOTO* ‘Department of Preventive Dentistry, Osaka University Faculty of Dentistry and ‘Institute for Protein Research, Osaka University, l-8 Yamadaoka, Suita, Osaka 565, Japan (Accepted 26 April 1990) haemagglutinating activity of Bacteroidesgingiualis381 was significantly inhibited by the synthetic peptide, Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-GIu-Lys-His-HisSet-His-Arg-Gly-Tyr. However, bradykinin potentiator C, which does not contain cationic amino acids such as L-histidine, t-arginine and L-lysine, had no inhibitory effect.
Summary-T!le
Key words: hzcteroides gingiualis,haemagglutination,
It is well known th.at the haema~utinating activity of Bacteroides gingivalis is inhibited by human whole saliva, especially high molecular-weight components of saliva (Slots and Gibbons, 1978; Okuda, Slots and Genco, 1981). Howlever, little attention has been paid to the inhibitory substances such as low molecularweight proteins aon haemagglutinating activity. Inoshita et al. (1986) have pointed out that the haemagglutinating activity of the exohaemagglutinin from the culture medium of Bact. gingiualis 381 was inhibited by angio-tensin I peptide in which histidine and arginine residues are involved. In saliva, numerous polypeptides and proteins have been isolated and recognized as strongly binding to hydroxyapatite molecules. Concerning the adsorption properties, the most studied histidine-rich protein was described by Hay (1975) and Baum et al. (1976). MacKay et al. (1984) and Pollock et al. (1984) have shown that histidine-rich proteins have antimicrobial effects including growth inhibition of the Candida al&cans and streptococcus mutans. More recently, Op~nheim et al. (1988) have established that there are three major histidine-rich proteins in human parotid saliva and have clarified their structural interrelationships. These three proteins have been completely purified to apparent homogeneity. Their primary structures have been determined, and their anti-Candidal activity measured. Based on the different primary structures of three proteins, the major salivary histidine-rich proteins were named histatins 1, 3 and 5. Oppenheim et al. have suggested that histatins 1 and 3 are derived from different structural genes, whereas histatin 5 is a proteolytic product of histatin 3. We had a strong interest in each histatin having cationic amino acids such as L-histidine, L-arginine and L-lysine in its to: Dr A. Tsunemitsu, Department of Preventive Dentistry, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565, Japan.
*Address correspondence
synthetic histidine-rich peptide.
peptide chains. We now describe the inhibitory effwt of the synthetic peptide of histatin 5 alone, which is a potent inhibitor of C. a&cans germination, compared to that of histatins 1 and 3, on haemagglutinating activity of Bact. gingiualis. The inhibitory effects on haemagglutination of two shorter synthetic peptides of histatin 5 which are not known exist in human parotid saliva were also examined. Bact. gingiuah 381 was a gift from stock strains at the Research Laboratory of Oral Biology, Sunstar Inc., Osaka, Japan. The strain was maintained by weekly transfer on plates containing trypticase soy agar (BBL Microbiology Systems, Cockeysville, MD) supplemented with 5% sheep blood, 1 mg of yeast extract (BBL) per ml, 5 pg of haemin per ml, 1 @g of menadione per ml. The organism was grown in 10 ml of trypticase soy broth (BBL) supplemented with 1 mg of yeast extract per ml, 5 pg of haemin per ml, 1 fig of menadione per ml. The first series of cultures was transferred into 100 ml of the same type of broth medium used for pre-culture. Cultures were incubate in an anaerobic system 1024 (Forma, Morietta, OH) in an N,-H&O, (85: 10:5) atmosphere at 37°C. The haemagglutinating activity was determined at room temperature with a microtitre plate (Limbro Division Flow Laboratories Inc., Hamden, CT). Because the haemaggiutination of Butt. gingivaris was nonspecific for blood-group ABO types in our preli~nary check, human O-type erythrocytes were used for the haemagglutination assay. The sample was diluted in a 2-fold series in isotonic saline, and an equal volume of a 2% suspension of human erythrocytes was added. The mixtures were left to stand for 90 min, and haemagglutination was determined by observing the mixture with the naked eye. Titre was expressed as the reciprocal of the highest dilution showing positive agglutination. The haemagglutination inhibition test for the synthetic peptides was by the method of Inoshita
Y. MURAKAMIet al.
776
E
3
Table 2. Inhibitory effect on haemagglutination of Bact. gingivalis by synthetic peptides using the spectrophotometric method
-------------
$ in z
3
Peptide concentration (M)
II 2
‘72
El T)
1x 5x 1x 5x 1x 5x 1x 5x
t\
k
q 0
2.5
5.0
z5
Time (mini
10-d 10-s 10-S 10-S 10-6 10-7 10-r 10-s
Inhibition (%) Control
I
0 0 0 0 0 0 0 0
100 97 86 47 10 0 0 0
II
III
100 100 100 98 29 8 0 0
100 100 100 100 67 12 6 0
Fig. 1. Inhibitory haemagglutinating curve by synthetic peptides of peptide III. Reaction mixture contained 5 x 10’ erythrocytes, 3 x IO*bacterial cells and 10 mM PBS, pH 7.0 in a final volume of 2 ml (-). Added the synthetic peptide III to the reaction mixture at lo-’ M concentration (----).
The values in the table show the mean in duplicate experiments. Bradykinin potentiator C was used as a control. The sequence of peptides I, II and III are indicated in Table 1.
et al. (1986). The haemagglutinating activity was also determined by a spectrophotometric method. The reaction mixture for assay contained 5 x 10’ human O-type erythrocytes, 3 x 10’ bacterial cells and
on a YMC-gel ODS S-S AM type column (YMC, Kyoto, Japan). The peptide thus obtained was lyophilized with distilled water. Amino acid composition after 24 h hydrolysis at 110°C with constant-boiling hydrochloric acid was as follows. Peptide I: Ser,,, Gly,.,, Tyr,,, His,,, Lysi,, Arg,,,. Peptide II: Ser,,, Glu,.,, Glyr.,, Tyr,.,, Phei,, His,,, L~sr.~, Arg,,. Peptide III: Asp,.,, Serr.e, Glui.r, GIY,.~, Alai.,, Tyrr.i,
10 mM phosphate-buffered saline, pH 7.0, in a final volume of 2ml. Haemagglutinating activity was spectrophotometrically monitored with a decreased optical density at 542 nm. Based on the change of optical density 5 min after start of reaction, percentage inhibition was calculated. Three peptides, Lys-HisHis-Ser-His-Arg-Gly-Tyr (‘peptide I: shortest fragment of histatin 5) Gly-Tyr-Lys-Arg-Lys-Phe-His-GluLys-His-His-Ser-His-Arg-Gly-Tyr (‘peptide II: shorter fragment of histatin 5), and Asp-Ser-His-Ala-LysArg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-LysHis-His-Ser-His-Arg-Gly-Tyr (peptide III: same as histatin 5) were synthesized by a solid-phase technique using an Applied Biosystems Inc. (Foster, CA) peptide synthesizer 430A with a Boc-amino acid anhydride method. After the completion of a peptide chain assembly, a peptide-linked resin was treated with anhydrous hydrogen fluoride containing 10% anisole at 0°C for 75 min. The crude peptide was purified
Phei,a, His,,,
Lyss.9, Args.c.
The haemagglutinating curve is shown in Fig. 1. When the synthetic peptide III was added to the reaction mixture, no change of optical density at 542 nm was found in the haemagglutinating curve. The results of haemagglutination inhibition by some peptides are shown in Tables 1 and 2. The haemagglutinating activity was markedly inhibited by peptide III, moderately by peptide II, and weakly by peptide I, but bradykinin potentiator C (Peptide Institute Inc., Minoh, Osaka, Japan) was not inhibitory. These results suggest that the cationic peptides containing histidine, arginine and lysine residues may function electrostatically as the binding domains for the Bact. gingivalis adhesin and/or erythrocyte surfaces during
Table 1. Inhibition of haemagglutination by Bact. gingivalis with synthetic peptides by means of microtitre plate assay Peptides Control I II
III
Primary structure 1 5 10 Pyr-Gly-Leu-Pro-Pro-Gly-Pro-Pro-Ile-Pro-Pro 1 5 Lys-His-His-Ser-His-Arg-Gly-Tyr 1 5 10 Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His15 His-Ser-His-Arg-Gly-Tyr
1 5 10 Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg15 20 Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr
Lowest inhibitory concentration (PM) > 420.0 12.5 1.6
0.8
Bradykinin potentiator C was used as a control. The sequence of peptide III is the same as that of histatin 5 named by Oppenheim et al. (1988). Peptide I and II are shorter fragments of histatin 5.
Inhibitors of haemag~utinating activity agglutination, although the exact mechanism is not clear. In our experiments, bradykinin potentiator C alone was used as a control. Additional peptides in which cationic amino acids are not included may be needed for a control experiment. The Bact. girrgiu&s that colonize thie mouth have often been shown to have adhesive properties that enable them to bind to epithelial cells, salivary pellicle, as well as to Gram-positive bacterial species (Slots and Gibbons, 1978; Okuda et al., 1981; Gibbons and Etherdern, 1983; Boyd and McBride, 1984). Based on the observed inhibition of haema~lutination, it is quite possible that this histatin 5 might play an inhibitor roie in the colonization of Bact. gingivalis in the mouth. REFERENCES Baum B. J., Bird J. L., Millar D. B. and Longton R. W. (1976) Studies on hi~.tidine-richpolypeptides from human
parotid saliva. Ar&r B&hem.
Riophys. 177, 427-436.
Boyd J. F. and McBride B. C. (1984) Fractionation hemagglutinating and bacterial binding adhesins
of of
Bacteroides gingivahs. Infect. Immun. 45, 403-409.
Gibbons R. J. and Etherdern I. (1983) Comparative hydrophobicities of oral bacteria and their adherence to salivary pellicles. htfect. ~rnn~~. 41, 1190-I 196.
777
Hay D. I. (1975) Fractionation of human parotid salivary proteins and the isolation of an histidine-rich acidic peptide which shows high affinity for hydroxyapatite surfaces. Archs oral Biol. 20, 553-558. Inoshita E., Amano A., Hanioka T., Tamagawa H., Shizukuishi S. and Tsunemitsu A. (1986) Isolation and some properties of exohemag~utinin from the culture medium of Bacteroides gingjvaI~ 381. Injkt. Immun. 52, 421-427.
MacKay B. J., Denepitiya L., Iacono V. J., Krost S. B. and Pollock J. J. (1984) Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich peptides on Streptococcus mutans. Infect. Immun. 44, 695-101. Okuda K., Slots J. and Genco R. J. (1981) Bacteroides gingivalis. ~a~teroides ~a~charo~yti~s and Bacteroides melaninogeni~us subspecies: cell surface morphology and adherence to erythrocytes and human buccal epithelial cells. Curr. Microbial. 6, 7-12. Oppenheim F. G., Xu T., McMillian F. M., Levitz S. M., Diamond R. D., Offner G. D. and Troxler R. F. (1988) Histatins, a novel family of histidine-rich proteins in human parotid secretion. .J. biol. Chem. 263, 7472-7476. Pollock J. J., Denepitiya L., MacKay B. J. and Iacono V. J, (1984) Fungistatic and fungicidal activity of human parotid salivary histidine-rich peptides on Candida albicans. Infect. Immun. 44, 102-101.
Slots J. and Gibbons R. J. (1978) Attachment of Bacteroides meianinogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets. Infect. Immun. 19, 254-264.
