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Morphology of gram-positive filamentous bacteria identified in dental plaque by fluorescent antibody technique
Arch orul Viol. V&.12,pp.12691273, 1967. Pcrsanron Press Ltd. Printed in Gt.
Britain.
MORPHOLOGY OF GRAM-POSITIVE FILAMENTOUS BACTERIA IDENTIFIED IN DENTAL PLAQUE BY FLUORESCENT ANTIBODY TECHNIQUE M. L. SNYDER, W. W. BULLOCK and R. B. PARKER Department of Bacteriology, University of Oregon Dental School, Portland, Oregon, U.S.A. Summary-In a preliminary study of specimens of dental plaque from fourteen adults, it was possible to identify by the indirect immunofluorescent method the presence and incidence of organisms reacting speciiically with antisera prepared against the following species: Actinomyces israelii (14/14), Actinomyces naeslundii (8/14), Actinomyces oa?mtoIyticus (4/14), Nocardia akntocariosus (6/14), Odontomyces viscosus (7/14), Bacterionema matruchotii (14/14), and Leptotrichia buccalis (10/14). Of these, only Leptotrichia buccalis had identical morphology in plaque material and in culture. Bacterionema matruchotii appeared only as atypically long filaments without branching, while all the others were identified as coccobacillary forms in contrast to the filamentous, branching forms observed in culture media. INTRODUCTION THE RECOGNITIONthat some of the gram-positive filamentous organisms of oral origin especially Actinomyces and Nocardia, exist in a coccal as well as filamentous state has led to questioning the reliability of staining to distinguish these particular forms from the true coccal flora of the dental plaque. This differentiation has not been possible to resolve until the specificity of the fluorescent antibody technique became available. Thus, RITZ (1963) has reported on the identification of Nocardiu species in dental plaque by this method. As part of a project aimed at studying the role filamentous bacteria play in formation and composition of dental plaque, we have by this technique extended his observations to the identification of other filamentous species in this material. Our results indicate feasibility and are presented in a preliminary way.
MATERIALS
AND METHODS
Antibody production Antibodies were prepared using disrupted whole cells of the following species: Actinomyces israelii (C. 959 Howell), Actinomyces naeslundii (ATCC 1247), Actinomyces odontolyticus (NTCC 9931), Nocardia dentocariosus (ATCC 14190), Odontomyces viscosus (ATCC 15987), Bacterionema matruchotii (ATCC 14266), kptotrichia buccalis (#5). New Zealand White rabbits were inoculated with the respective antigens intramuscularly and intravenously on 2 successive days followed by 5-day rest periods covering at least 3 weeks until satisfactory titres ( > 1: 160) were demonstrable. A.
1269 D
1270
M. L. SNYDER, W. W. BULLOCKAND
R. B. PARKER
The animals were bled by intracardiac puncture and the sera obtained. preserved with l-10000 merthiolate and stored at 4°C until used.
These were
B. Plaque
Samples of plaque were taken from the lingual surfaces of mandibular premolars and molars of each of fourteen members of the research staff and the respective specimen immediately placed in 2-O ml cold buffered saline (PBS: 0.85 % NaCl, 0.005M KPH POI, pH 7.5). Adequate dispersion of the plaque material in the PBS was accomplished by 30 set agitation in a Serval Blender. C. Staining
Each suspension of plaque material was spread on approximately twenty slides which were dried, fixed in 95 % ethyl alcohol for 5 min, rinsed with distilled water and again dried. A specific antibacterial serum (rabbit), diluted 1: 30 with cold PBS, was flooded onto a slide preparation where it remained for 30 min, after which it was washed in two changes of PBS for 30 min each and once with distilled water for 1 min. The procedure was repeated for the respective antisera. Each of these preparations was then flooded with fluorescent labeled anti-rabbit-globulin globulin from sheep. This labeled globulin (Sylvana) had been previously absorbed with rabbit kidney and sheep liver tissue powders, refrigerated, and diluted 1: 30 with cold PBS just prior to use. D. Microscopy The stained preparations for all species tested were examined with a Leitz Labolux microscope fitted for U.V. observation as follows: BG 38 heat filter, BG 12 exciting filter, HBO 200 U.V.source kept above 50 per cent maximum output, and EUPHOS barrier filters for eyepieces and camera lens. The slides were read on a scale of (O-4 +) staining: 0 1-t 2+ 3+ 4+
= = = = =
Negative or barely visible dull yellow Dull green stain Definite green staining, walls visibly stained Bright yellow-green walls completely stained Intense yellow-green walls, centre of cell is dark by contrast
The l-30 dilutions of test antisera and FA antiglobulin sera gave minimal (O-l +) staining with heterologous cultures, and brightly staining (3-4 +) cells withhomologous organisms. Thus, the criteria for identification was the occurrence of brightly staining (3-4 +) cells in the plaque material stained with respective specific antisera. RESULTS
The incidence of the seven species and their dominant morphological features in the dental plaque material studied and in culture are listed in Table 1. Table 1 shows that only Bacterionema matruchotii and Actinomyces israelii were demonstrable in all fourteen plaque samples examined, Leptotrichia buccaZisnext more
MORPHOLOGY OFGRAM-POWWE FHAMENTOUS BACTERIA IDENTIFIED IN DENTAL
1271
PLAQUE
TABLE~.THEOGCURRENGE ANDPREDOMINANTMORPHOLOGYOFTHEGRAM-POSITIVEORALFILAMENTOUS BACTERIA MPURE CULTURBAND in SPECIMENS OF DENTAL
Organism
situ
AS OBSERVED BY FLUORESCENT-ANTIBODYSTAINING PLAQUBFROM EACH OF POURTEENADULTS
Number of plaques in which organism occurred
In vitro morphology
14114
Long branching rods
Actinomyces naeslundii
8/14
Long branching rods
Actinomyces odontolyticus
4114
Long branching rods
Nocardia dentocariosus
6114
Long branching rods
Odontomyces viscosus
7114
Long branching rods
Bacterionema matruchotii
14114
Leptotrichia buccalis
10/14
Long branching rods -bulb ends Large non-branching rods
Actinomyces israelii
IN
In situ
morphology Short rods and coccoidal forms Short rods and coccoidal forms Short rods and coccoidal forms Short rods and coccoidal forms Short rods and coccoidal forms Long tangled uniform diameter filaments Large non-branching rods
frequent, and the others irregularly present in these specimens. While this is a simple listing and reveals no information about frequence, in our experience Bacterionema matruchotii was numerically the most frequent filamentous bacterial form observed in the material studied. This finding would confirm the earlier observations of DAVIS and BAIRD-PARKER (1959) who used the name Leptotrichia dent&m for this species. It is interesting to note that this appears to be the first recognition of Odontomyces viscosus (1965) in or derived from human plaque. The appearance of some of these filamentous species as they occur in plaque material and in culture is exemplified in Fig. 1. Here it is seen that Leptotrichia (and only this species) has identical morphology in plaque material and in culture: large, long, straight rods with blunted end occurring singly or in chains of two to three cells (Fig. le and f). Bacterionema matruchotii forms atypically long filaments of uniform diameter compared with the branching and enlarged branch endings observed in culture (Fig. la and b); it also occurs frequently in tangled masses to form a matrix throughout which the smaller rods and cocci of the other species are scattered. The difference of an organism appearing in coccoidal form in plaque material and as a branching or filamentous form in culture is exemplified by coccobacillary elements identifiable as Actinomyces israelii in plaque material compared with the growth of a strain of this species in broth culture (Fig. lc and d). DISCUSSION
Gram-staining of dental plaque, or for that matter any other specimen, yields information only in respect to stain reaction, morphological types present, and to some extent numbers and distribution of microbial forms observed; little is gained for precise identification of genus and species. Despite these shortcomings, considerable information has been gained about the microscopic composition of dental plaque as reported by BIBBY (1938), ENNEVER, ROBINSON and KITCHEN (1951), BLAYNEY(1961), MORRIS (1954), MACDOUGALL (1963), and others. In respect to filamentous forms,
1272
M. L. SNYDER, W. W. BULLOCK AND R. B. PARKER
DAVISand BAIRD-PARKER (1959) concluded that their Leptotrichia dentium (or Bacterionema matruchotii) and Leptotrichia buccaiis formed the majority of filamentous bacteria present in materia alba; HOWELL, STEPHANand PAUL (1962) found Actinomyces israelii to be the most commonly isolated of Actinomyces israelii, Actinomyces naeslundii and Bacterionema matruchotii from selected areas of the oral cavity, and MCCARTHY,SNYDERand PARKER(1965) found Actinomyces species to be the second most prevalent anaerobe type in infants by the end of the first year of life. On the other hand, filamentous organisms growing as such are not specifically described by GIBBONS et al. (1964) and mentioned only as probably present in their aerobic and anaerobic “diphtheroid” groups totalling 41 per cent of the cultivable microbiota of five specimens of plaque. In our hands these gram-positive filamentous species were consistently present in dental plaque taken from a small group of adults and these organisms were for the most part to be observed in both coccoidal and filamentous states. This information could then provide an answer to DAVISand BAIRD-PARKER (1959) who questioned whether the apparent absence of bacteria other than Bacterionema matruchotii in materia alba was due to their scarcity, or presence in an unrecognizable form. Similarly, it is clear that classification of bacteria in dental plaque as coccoids, rods or filaments on the basis of direct microscopic observation or by gram-staining could give an erroneous respesentation of the incidence of these filamentous species. An explanation can also be provided to account for the discrepancy between HOWELL’S finding that Actinomyces israelii was the dominant culturable filamentous species in dental plaque and the observations of DAVISand BAIRD-PARKER about Bacterionema matruchotii in materia alba. This would be the inherent inability of plating techniques to differentiate between the large multicellular filaments of Bacterionema matruchotii and numerous smaller single coccoids formed by Actinomyces israelii in the plaque environment. On the other hand, it is recognized that care must be taken in interpretation of immunofluorescent reactions when it is known that extensive crossreactions exist either in this group with specific reference to Actinomyces (SNYDER, PARKERand BULLOCK,1967) or any other group under study. Within these limitations, fluorescent antibody technique should prove invaluable in the search for increased understanding of the complex ecology of the dental plaque with specific reference to the filamentous species. It is also predictable that the appearance of these forms in respect to chronological age can be determined more accurately by fluorescent antibody than by culture. Acknowledgement-This project supported in part by a grant, DE-02234, awarded by the United States Public Health Service. Rksum6-Au tours d’une Etude prkliminaire de plaques dentaires de 14 adultes, il a ttC possible d’identifier et de dknombrer, par une m&hode d’immunofluorescence indirecte, les organismes rkagissant spkcifiquement aux antis&a prkparks contre les es&es suivantes: Actinomyces israelii (14/14), Actinomyces naeslundii (8/14), Actinomyces odontolyticus (4/14), Nocardia dentocariosus (6/14), Odontomyces viscosus (7/14), Bacterionema matruchotii (14/14), and Leptotrichia buccalis (10/14). De tous ces
MORPHOLOGYOF GRAM-POSITIVE FILAMENTOUS BACTERIAIDENTIFIED IN DENTALPLAQUE
1273
organismes, seul Leprorrichia buccalis presente une morphologie identique dans la plaque et en culture. Bucterionema matruchotii se prbente comme des filaments atypiquement allonges, sans ramification, et toutes les autres esp&es ont une forme cocco-bacillaire, contrairement aux formes filamenteuses, ramifiees, observ&s en culture. Zusannnenfassung-Bei einer vorllufigen Untersuchung an Plaquesproben von 14 Erwachsenen war es moglich, durch indirekte Immunofluoreszenztechnik die Anwesenheit und die Haufigkeit von Organismen zu bestimmen, die spezifisch mit Antisercn gegen folgende Spezies reagieren: Actinomyces israelii (14/14), Actinomyces naeslundii (8/14), Actinomyces odontolyticus (4/14), Nocardia dentocariosus (6/14), Odontomyces viscosus (7114) Bacterionema matruchotii (14114) und Leptotrichia buccalis (10/14). Von diesen wiesen lediglich die Leptotrichia bucculis eine in den Plaquesproben und in der Kultur identische Morphologie auf. Eucterionema mcztruchotii trat lediglich als atypische lange Faserchen ohne Verzweigung auf, wahrend alle anderen im Gegensatz zu den faserartigen, verzweigten Formen in den Kulturmedien als Kokkenformen identifiziert wurden.
REFERENCES BIBBY, B. G. 1938. The bacterial flora in different parts BLAYNEY, R. J. 1961. Personal experiences in research.
of the mouth. J. dent. Res. 17,471-476. Dent. Prog. 2, 12-20. DAVIS. G. H. G. and BAIRD-PARKER.A. C. 1959. The bacterial elements of materia alba. Br. dent. J. 106, 142-146. ENNEVER,J., ROBINSON,H. B. G. and KITCHM, P. C. 1951. Actinomyces and the dentobacterial plaque. J. dc:zt. Res. 30, 88-96. GIBBONS, R. J., SOCRANSKY, S. S., DE ARAUJO, W. C. and VAN HOUTE, J. 1964. Studies on the predominant cultivable microbiota of dental plaque. Archs oral Biol. 9,365-370. HOWELL, A., JORDAN, H. V., GEORG, L. K. and PINE, L. 1965. Odontomyces viscosus, gen. nov. spec. nov. A filamentous microorganism isolated from periodontal plaque in hamster. Sabouraudiu 4, 65-68. MACDOUGALL, W. A. 1963. Studies on the dental plaque II. The histology of the developing interproximal plaque, Aust. dent. J. 8, 398-407. MCCARTHY, C. M., SNYDER, M. L. and PARKER, R. B. 1965. The indigenous oral flora of man I.
The newborn to the l-year-old infant. Archs oral Biol. 10, 61-70. MORRIS, E. 0. 1954. The bacteriology of the oral cavity. V. Corynebucterium and gram-positive fllamentous bacteria. Br. dent. J. 97, 29-36. RITZ, H. L. 1963. Localization of Nocardiu in dental plaque by immuno-fluorescence. Proc. Sot. Exp. Biol. Med. 113,925-929. SNYDER, M. L., PARKER, R. B. and BULLOCK, W. 13. 1967. Studies on oral filamentous bacteria. Serological relationships within the genera Actinomyces, Nocardia, Bacterionema and Leptotrichia.
J. Infect. Dis. In press.
PLATE 1 OVERLEAF
M. L. SNYDER, W. W. BULLOCK AND R. B. PARKER
FIG. 1.
a. b. c. d. e. f.
B. matruchotii in culture, nigrosin strain. B. mafruchotii, fluorescent antibody preparation from dental plaque. A. israelii in culture, nigrosin stain. A. israelii, fluorescent antibody preparation from dental plaque. L. buccalis in culture, nigrosin stain. L. burcalis, fluorescent antibody preparation from dental plaque.
PLATE
1
A.O.B. f.p. 1274
Britain.
MORPHOLOGY OF GRAM-POSITIVE FILAMENTOUS BACTERIA IDENTIFIED IN DENTAL PLAQUE BY FLUORESCENT ANTIBODY TECHNIQUE M. L. SNYDER, W. W. BULLOCK and R. B. PARKER Department of Bacteriology, University of Oregon Dental School, Portland, Oregon, U.S.A. Summary-In a preliminary study of specimens of dental plaque from fourteen adults, it was possible to identify by the indirect immunofluorescent method the presence and incidence of organisms reacting speciiically with antisera prepared against the following species: Actinomyces israelii (14/14), Actinomyces naeslundii (8/14), Actinomyces oa?mtoIyticus (4/14), Nocardia akntocariosus (6/14), Odontomyces viscosus (7/14), Bacterionema matruchotii (14/14), and Leptotrichia buccalis (10/14). Of these, only Leptotrichia buccalis had identical morphology in plaque material and in culture. Bacterionema matruchotii appeared only as atypically long filaments without branching, while all the others were identified as coccobacillary forms in contrast to the filamentous, branching forms observed in culture media. INTRODUCTION THE RECOGNITIONthat some of the gram-positive filamentous organisms of oral origin especially Actinomyces and Nocardia, exist in a coccal as well as filamentous state has led to questioning the reliability of staining to distinguish these particular forms from the true coccal flora of the dental plaque. This differentiation has not been possible to resolve until the specificity of the fluorescent antibody technique became available. Thus, RITZ (1963) has reported on the identification of Nocardiu species in dental plaque by this method. As part of a project aimed at studying the role filamentous bacteria play in formation and composition of dental plaque, we have by this technique extended his observations to the identification of other filamentous species in this material. Our results indicate feasibility and are presented in a preliminary way.
MATERIALS
AND METHODS
Antibody production Antibodies were prepared using disrupted whole cells of the following species: Actinomyces israelii (C. 959 Howell), Actinomyces naeslundii (ATCC 1247), Actinomyces odontolyticus (NTCC 9931), Nocardia dentocariosus (ATCC 14190), Odontomyces viscosus (ATCC 15987), Bacterionema matruchotii (ATCC 14266), kptotrichia buccalis (#5). New Zealand White rabbits were inoculated with the respective antigens intramuscularly and intravenously on 2 successive days followed by 5-day rest periods covering at least 3 weeks until satisfactory titres ( > 1: 160) were demonstrable. A.
1269 D
1270
M. L. SNYDER, W. W. BULLOCKAND
R. B. PARKER
The animals were bled by intracardiac puncture and the sera obtained. preserved with l-10000 merthiolate and stored at 4°C until used.
These were
B. Plaque
Samples of plaque were taken from the lingual surfaces of mandibular premolars and molars of each of fourteen members of the research staff and the respective specimen immediately placed in 2-O ml cold buffered saline (PBS: 0.85 % NaCl, 0.005M KPH POI, pH 7.5). Adequate dispersion of the plaque material in the PBS was accomplished by 30 set agitation in a Serval Blender. C. Staining
Each suspension of plaque material was spread on approximately twenty slides which were dried, fixed in 95 % ethyl alcohol for 5 min, rinsed with distilled water and again dried. A specific antibacterial serum (rabbit), diluted 1: 30 with cold PBS, was flooded onto a slide preparation where it remained for 30 min, after which it was washed in two changes of PBS for 30 min each and once with distilled water for 1 min. The procedure was repeated for the respective antisera. Each of these preparations was then flooded with fluorescent labeled anti-rabbit-globulin globulin from sheep. This labeled globulin (Sylvana) had been previously absorbed with rabbit kidney and sheep liver tissue powders, refrigerated, and diluted 1: 30 with cold PBS just prior to use. D. Microscopy The stained preparations for all species tested were examined with a Leitz Labolux microscope fitted for U.V. observation as follows: BG 38 heat filter, BG 12 exciting filter, HBO 200 U.V.source kept above 50 per cent maximum output, and EUPHOS barrier filters for eyepieces and camera lens. The slides were read on a scale of (O-4 +) staining: 0 1-t 2+ 3+ 4+
= = = = =
Negative or barely visible dull yellow Dull green stain Definite green staining, walls visibly stained Bright yellow-green walls completely stained Intense yellow-green walls, centre of cell is dark by contrast
The l-30 dilutions of test antisera and FA antiglobulin sera gave minimal (O-l +) staining with heterologous cultures, and brightly staining (3-4 +) cells withhomologous organisms. Thus, the criteria for identification was the occurrence of brightly staining (3-4 +) cells in the plaque material stained with respective specific antisera. RESULTS
The incidence of the seven species and their dominant morphological features in the dental plaque material studied and in culture are listed in Table 1. Table 1 shows that only Bacterionema matruchotii and Actinomyces israelii were demonstrable in all fourteen plaque samples examined, Leptotrichia buccaZisnext more
MORPHOLOGY OFGRAM-POWWE FHAMENTOUS BACTERIA IDENTIFIED IN DENTAL
1271
PLAQUE
TABLE~.THEOGCURRENGE ANDPREDOMINANTMORPHOLOGYOFTHEGRAM-POSITIVEORALFILAMENTOUS BACTERIA MPURE CULTURBAND in SPECIMENS OF DENTAL
Organism
situ
AS OBSERVED BY FLUORESCENT-ANTIBODYSTAINING PLAQUBFROM EACH OF POURTEENADULTS
Number of plaques in which organism occurred
In vitro morphology
14114
Long branching rods
Actinomyces naeslundii
8/14
Long branching rods
Actinomyces odontolyticus
4114
Long branching rods
Nocardia dentocariosus
6114
Long branching rods
Odontomyces viscosus
7114
Long branching rods
Bacterionema matruchotii
14114
Leptotrichia buccalis
10/14
Long branching rods -bulb ends Large non-branching rods
Actinomyces israelii
IN
In situ
morphology Short rods and coccoidal forms Short rods and coccoidal forms Short rods and coccoidal forms Short rods and coccoidal forms Short rods and coccoidal forms Long tangled uniform diameter filaments Large non-branching rods
frequent, and the others irregularly present in these specimens. While this is a simple listing and reveals no information about frequence, in our experience Bacterionema matruchotii was numerically the most frequent filamentous bacterial form observed in the material studied. This finding would confirm the earlier observations of DAVIS and BAIRD-PARKER (1959) who used the name Leptotrichia dent&m for this species. It is interesting to note that this appears to be the first recognition of Odontomyces viscosus (1965) in or derived from human plaque. The appearance of some of these filamentous species as they occur in plaque material and in culture is exemplified in Fig. 1. Here it is seen that Leptotrichia (and only this species) has identical morphology in plaque material and in culture: large, long, straight rods with blunted end occurring singly or in chains of two to three cells (Fig. le and f). Bacterionema matruchotii forms atypically long filaments of uniform diameter compared with the branching and enlarged branch endings observed in culture (Fig. la and b); it also occurs frequently in tangled masses to form a matrix throughout which the smaller rods and cocci of the other species are scattered. The difference of an organism appearing in coccoidal form in plaque material and as a branching or filamentous form in culture is exemplified by coccobacillary elements identifiable as Actinomyces israelii in plaque material compared with the growth of a strain of this species in broth culture (Fig. lc and d). DISCUSSION
Gram-staining of dental plaque, or for that matter any other specimen, yields information only in respect to stain reaction, morphological types present, and to some extent numbers and distribution of microbial forms observed; little is gained for precise identification of genus and species. Despite these shortcomings, considerable information has been gained about the microscopic composition of dental plaque as reported by BIBBY (1938), ENNEVER, ROBINSON and KITCHEN (1951), BLAYNEY(1961), MORRIS (1954), MACDOUGALL (1963), and others. In respect to filamentous forms,
1272
M. L. SNYDER, W. W. BULLOCK AND R. B. PARKER
DAVISand BAIRD-PARKER (1959) concluded that their Leptotrichia dentium (or Bacterionema matruchotii) and Leptotrichia buccaiis formed the majority of filamentous bacteria present in materia alba; HOWELL, STEPHANand PAUL (1962) found Actinomyces israelii to be the most commonly isolated of Actinomyces israelii, Actinomyces naeslundii and Bacterionema matruchotii from selected areas of the oral cavity, and MCCARTHY,SNYDERand PARKER(1965) found Actinomyces species to be the second most prevalent anaerobe type in infants by the end of the first year of life. On the other hand, filamentous organisms growing as such are not specifically described by GIBBONS et al. (1964) and mentioned only as probably present in their aerobic and anaerobic “diphtheroid” groups totalling 41 per cent of the cultivable microbiota of five specimens of plaque. In our hands these gram-positive filamentous species were consistently present in dental plaque taken from a small group of adults and these organisms were for the most part to be observed in both coccoidal and filamentous states. This information could then provide an answer to DAVISand BAIRD-PARKER (1959) who questioned whether the apparent absence of bacteria other than Bacterionema matruchotii in materia alba was due to their scarcity, or presence in an unrecognizable form. Similarly, it is clear that classification of bacteria in dental plaque as coccoids, rods or filaments on the basis of direct microscopic observation or by gram-staining could give an erroneous respesentation of the incidence of these filamentous species. An explanation can also be provided to account for the discrepancy between HOWELL’S finding that Actinomyces israelii was the dominant culturable filamentous species in dental plaque and the observations of DAVISand BAIRD-PARKER about Bacterionema matruchotii in materia alba. This would be the inherent inability of plating techniques to differentiate between the large multicellular filaments of Bacterionema matruchotii and numerous smaller single coccoids formed by Actinomyces israelii in the plaque environment. On the other hand, it is recognized that care must be taken in interpretation of immunofluorescent reactions when it is known that extensive crossreactions exist either in this group with specific reference to Actinomyces (SNYDER, PARKERand BULLOCK,1967) or any other group under study. Within these limitations, fluorescent antibody technique should prove invaluable in the search for increased understanding of the complex ecology of the dental plaque with specific reference to the filamentous species. It is also predictable that the appearance of these forms in respect to chronological age can be determined more accurately by fluorescent antibody than by culture. Acknowledgement-This project supported in part by a grant, DE-02234, awarded by the United States Public Health Service. Rksum6-Au tours d’une Etude prkliminaire de plaques dentaires de 14 adultes, il a ttC possible d’identifier et de dknombrer, par une m&hode d’immunofluorescence indirecte, les organismes rkagissant spkcifiquement aux antis&a prkparks contre les es&es suivantes: Actinomyces israelii (14/14), Actinomyces naeslundii (8/14), Actinomyces odontolyticus (4/14), Nocardia dentocariosus (6/14), Odontomyces viscosus (7/14), Bacterionema matruchotii (14/14), and Leptotrichia buccalis (10/14). De tous ces
MORPHOLOGYOF GRAM-POSITIVE FILAMENTOUS BACTERIAIDENTIFIED IN DENTALPLAQUE
1273
organismes, seul Leprorrichia buccalis presente une morphologie identique dans la plaque et en culture. Bucterionema matruchotii se prbente comme des filaments atypiquement allonges, sans ramification, et toutes les autres esp&es ont une forme cocco-bacillaire, contrairement aux formes filamenteuses, ramifiees, observ&s en culture. Zusannnenfassung-Bei einer vorllufigen Untersuchung an Plaquesproben von 14 Erwachsenen war es moglich, durch indirekte Immunofluoreszenztechnik die Anwesenheit und die Haufigkeit von Organismen zu bestimmen, die spezifisch mit Antisercn gegen folgende Spezies reagieren: Actinomyces israelii (14/14), Actinomyces naeslundii (8/14), Actinomyces odontolyticus (4/14), Nocardia dentocariosus (6/14), Odontomyces viscosus (7114) Bacterionema matruchotii (14114) und Leptotrichia buccalis (10/14). Von diesen wiesen lediglich die Leptotrichia bucculis eine in den Plaquesproben und in der Kultur identische Morphologie auf. Eucterionema mcztruchotii trat lediglich als atypische lange Faserchen ohne Verzweigung auf, wahrend alle anderen im Gegensatz zu den faserartigen, verzweigten Formen in den Kulturmedien als Kokkenformen identifiziert wurden.
REFERENCES BIBBY, B. G. 1938. The bacterial flora in different parts BLAYNEY, R. J. 1961. Personal experiences in research.
of the mouth. J. dent. Res. 17,471-476. Dent. Prog. 2, 12-20. DAVIS. G. H. G. and BAIRD-PARKER.A. C. 1959. The bacterial elements of materia alba. Br. dent. J. 106, 142-146. ENNEVER,J., ROBINSON,H. B. G. and KITCHM, P. C. 1951. Actinomyces and the dentobacterial plaque. J. dc:zt. Res. 30, 88-96. GIBBONS, R. J., SOCRANSKY, S. S., DE ARAUJO, W. C. and VAN HOUTE, J. 1964. Studies on the predominant cultivable microbiota of dental plaque. Archs oral Biol. 9,365-370. HOWELL, A., JORDAN, H. V., GEORG, L. K. and PINE, L. 1965. Odontomyces viscosus, gen. nov. spec. nov. A filamentous microorganism isolated from periodontal plaque in hamster. Sabouraudiu 4, 65-68. MACDOUGALL, W. A. 1963. Studies on the dental plaque II. The histology of the developing interproximal plaque, Aust. dent. J. 8, 398-407. MCCARTHY, C. M., SNYDER, M. L. and PARKER, R. B. 1965. The indigenous oral flora of man I.
The newborn to the l-year-old infant. Archs oral Biol. 10, 61-70. MORRIS, E. 0. 1954. The bacteriology of the oral cavity. V. Corynebucterium and gram-positive fllamentous bacteria. Br. dent. J. 97, 29-36. RITZ, H. L. 1963. Localization of Nocardiu in dental plaque by immuno-fluorescence. Proc. Sot. Exp. Biol. Med. 113,925-929. SNYDER, M. L., PARKER, R. B. and BULLOCK, W. 13. 1967. Studies on oral filamentous bacteria. Serological relationships within the genera Actinomyces, Nocardia, Bacterionema and Leptotrichia.
J. Infect. Dis. In press.
PLATE 1 OVERLEAF
M. L. SNYDER, W. W. BULLOCK AND R. B. PARKER
FIG. 1.
a. b. c. d. e. f.
B. matruchotii in culture, nigrosin strain. B. mafruchotii, fluorescent antibody preparation from dental plaque. A. israelii in culture, nigrosin stain. A. israelii, fluorescent antibody preparation from dental plaque. L. buccalis in culture, nigrosin stain. L. burcalis, fluorescent antibody preparation from dental plaque.
PLATE
1
A.O.B. f.p. 1274