249

Identification of Actinobacillus actinomycetemcomitans by Gold Immunolabeling and Scanning Electron Microscopy A.

Carrassi, * D. Soligo, f G. Vogel, * G. Lambertenghi-Deliliers, f and J.J. Zamboni

Recent

evidence suggests that dental plaque is not a homogeneous bacterial mass the contrary, specific bacterial morphotypes and species may be preferentially located within certain microenvironments. The aim of the present study was to develop combined gold immunolabeling and scanning electron microscopic (SEM) techniques for the identification of periodontal pathogens in subgingival dental plaque and on the root surfaces of extracted teeth. Suspensions of pure A. actinomycetemcomitans cultures or suspensions of A. actinomycetemcomitans mixed together with other oral bacteria were prepared, labeled with goat anti-rabbit IgG conjugated with 5 nm or 40 nm colloidal gold particles, and observed by SEM using both secondary and back-scattered imaging. Scanning electron microscopy demonstrated that the A. actinomycetemcomitans bacterial cell surface was specifically labelled. There was no cross-reaction with any of the other bacterial morphotypes. Only the labeled A. actinomycetemcomitans were visible in mixtures examined by back-scattered imaging scanning electron microscopy. The combined techniques of gold immunolabeling and SEM may, therefore, be useful in identifying A. actinomycetemcomitans in subgingival plaque samples and on the root surfaces of extracted teeth as well as in studies of bacterial ecology in dental plaque, in general. / Periodontol 1990; 61:249-253.

but,

on

Key Words: Dental plaque bacteriology; periodontal disease/pathogenesis; scanning electron

microscopy/methods; immunolabeling/methods; gold,

colloidal.

Scanning electron microscopy (SEM) has been widely used in studies of subgingival dental plaque and periodontallyinvolved root surfaces.1 Recently, the scanning electron microscopic morphology of subgingival plaque associated with different forms of periodontal disease including juvenile Periodontitis,2 chronic adult Periodontitis,3 and rapidly progressing Periodontitis4 has been reported. As a consequence, it has become clear that certain bacterial morphotypes are frequently located in specific areas of subgingival plaque and the associated periodontally-involved root surface. Cocci and filaments, for example, are the bacterial morphotypes most frequently identified on the coronal and middle areas of periodontally-involved root surfaces, while spirochetes, straight and curved rods, and micrococci are associated with the most apical areas of the root surface.4 'Department of

of

Dentistry

Medicine, Milan, Italy.

and

Stomatology, University of Milan,

School

"(Institute of Medical Science.

^Departments

New York at

of Oral

Biology

Buffalo, Buffalo,

and NY.

Periodontology,

State

University of

Unfortunately, SEM studies of subgingival plaque and periodontally-involved root surfaces are limited in that microorganisms can only be classified as to morphology. The ability to identify the species of a given microorganism observed by scanning electron microscopy would constitute a significant improvement in studies of the bacterial ecology in periodontal pockets. A. actinomycetemcomitans is a Gram-negative microorganism which has been implicated in the etiology of certain forms of human periodontal disease including localized juvenile Periodontitis as well as rapidly progressing Periodontitis and refractory Periodontitis in adults.5 Recently, polyclonal antisera and monoclonal antibodies to A. actinomycetemcomitans have been developed which recognize both specific and cross-reactive antigens.6-8 Methods have also been described for immunolabelling cell surfaces using colloidal gold particles.9 Accordingly, the aim of the present study was to utilize these immunologie reagents to develop an immunolabelling technique for the identification of specific bacteria by scanning electron microscopy.

J Periodontol

250

ULTRASTRUCTURAL IDENTIFICATION OF A. ACTINOMYCETEMCOMITANS

MATERIALS AND METHODS

1990

antisera in 0.1 M PBS containing 10% fetal calf and 0.2% sodium azide (NaN3) were added to 50 µ , of bacterial suspension. After 30 minutes incubation at 4°C, bacteria were rinsed twice in PBS/FCS/NaN3, incubated for 30 minutes at 4°C with affinity purified swine anti-rabbit IgG conjugated with fluorescein,* rinsed 3 times in PBS and resuspended in glycerol-PBS (2:1). Samples were observed in a Leitz Laborlux microscope equipped for fluorscence microscopy.

specific

Bacterial Strains and Culture Conditions A. actinomycetemcomitans strains SUNYaB 75, FDC Y4, and SUNYaB 67 were used as representative of serotypes a, b, and c, respectively.6 Fusobacterium nucleatum strain ATCC 23726 and Bacteroides gingivalis strain 381 were used as representative oral bacterial species having distinct cellular morphotypes. The microorganisms were cultured in 2% brain heart infusion broth§ supplemented with 1% yeast extract,11 5mg/liter hemin, and 1 ^g/liter vitamin Is^ at 37°C for 36 hours in an anaerobic chamber containing 85% N2, 10% H2, and 5% C02. The bacterial cells were harvested by centrifugation at 10,000 x g for 20 minutes at 4°C. Suspensions of the individual A. actinomycetemcomitans serotype strains or equal proportions of A. actinomycetemcomitans, F. nucleatum, and B. gingivalis were prepared in phosphate buffered saline (PBS, pH 7.2).

Production of Serotype Specific Antisera Rabbit antisera to each of the A. actinomycetemcomitans strains were prepared and immunosorbed to serotype specificity.6 Bacterial cells of each serotype representative strain of A. actinomycetemcomitans were resuspended at a concentration of 10 mg (wet weight) / ml in sterile saline. One mL aliquots were intravenously injected 3 times per week for 5 weeks via the marginal ear vein into female New Zealand white rabbits. Trial bleedings were obtained from the central ear artery and the antibody titer was determined by 2-fold serial dilution in indirect immunofluorscence assays. Once a satisfactory antibody titer was achieved, the rabbits were exsanguinated by cardiac puncture. Antisera was heated to 56°C for 30 minutes and stored in small portions at 70°C until used. Serotype-specific antisera were produced by immunosorption of whole rabbit antisera to A. actinomycetemcomitans serotype representative strains with whole bacterial cells of the heterologous serotype. One hundred milligrams (wet weight) of bacterial cells was added to 1 ml of rabbit antisera and placed in a shaker at 37°C for 1 hour and then at 4°C for 12 hours. The mixture was centrifuged at 16,000 x g for 30 minutes and the supernatant serum was removed. Each antisera was immunoabsorbed twice with each of the 2 heterologous serotype strains. The serotype specificity of the immunoabsorbed antisera was confirmed by indirect immunofluorescence assays. Each specific A. actinomycetemcomitans antiserum was tested against the heterologous serotypes as well as against 8 additional oral bacterial species. The serotype-specific antisera were stored at -70°C. —

Indirect Immunofluorescence Indirect immunofluorescence assays were performed as previously described.6 Fifty µ of a 1:200 dilution of serotype§DIFCO Laboratories, Detroit, MI.

||BBL Microbiology Systems, Cockeysville,

May

MD.

serum

(FCS)11

Scanning Electron Microscopy Forty \xL of bacterial suspension were pipetted onto poly1-lysine pre-treated glass coverslips and left for 30 minutes. Bacterial samples had been either pre-treated with 0.2% glutaraldehyde in HEPES buffer (pH 7.4) or were processed unfixed. The glass coverslips were then rinsed in PBS and incubated with the homologous serotype specific antisera at 1:200 dilution in PBS/FCS/NaN, for 30 minutes at room temperature. The glass coverslips were rinsed 3 times for

5 minutes with PBS and incubated for 60 minutes with goat anti-rabbit IgG conjugated with 40 nm colloidal gold particles** at 1:10 dilution in Tris-HCl buffer (pH 8.2) or with goat anti-rabbit IgG coupled with 5 nm gold particles (GARG5). In addition, some samples were incubated with biotinylated swine anti-rabbit IgG and then with a streptavidingold (15 nm) conjugate.** After labeling with the gold probe, all the samples were rinsed twice in PBS; post-fixed in 2% glutaraldehyde in PBS; rinsed in distilled water; and dehydrated in sequential 70, 95, and 100% ethanol baths. Before dehydration, samples incubated with 5 nm and 15 nm gold particles (GAR-G5 and SA-15) were treated with the Inten-SE Enhancement Kit.** Dehydrated specimens were critical point dried from C02 using a Balzer Critical Point Drying Apparatus and coated with approximately 100 A of evaporated carbon in an Edwards evaporator. Other samples of the different bacterial strains were prepared for SEM without immunolabelling. In particular, after attachment to the glass coverslip, an aliquot of bacterial suspension was directly fixed in glutaraldehyde, dehydrated, and dried as above and gold-coated using a Polaron 5000 sputter coater. All samples were observed using either a Hitachi 2300 scanning electron microscope or a Jeol 840 scanning electron microscope in both secondary and back-scattered electron imaging modes.

RESULTS Indirect immunofluorescence assays showed strong fluorescence for A actinomycetemcomitans strains Y4, 67, and 75 reacted with working titer concentrations (1:200) of the homologous serotype-specific rabbit antisera. Little crossreactivity was observed for the heterologous reactions with A. actinomycetemcomitans even at the lowest antiserum difGibco, Grand Island, NY. #DakoPatts, Glostrup, Denmark. "Janssen Pharmaceutica, Beerse, Belgium.

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251

Figure 1. An unlabeled culture of . actinomycetemcomitans strain Y4. Typical coccoid and coccobacillary morphology is evident. 60 to 80 nm in diameter vesicles are present on both the bacterial surface and the coverslip. Original magnification: 52,000 X.

Figures 3.A and 3.B. A. actinomycetemcomitans strain 67 mixed with B. gingivalis strain 381. A: This is a secondary electron image showing the uniform distribution of 5 nm gold particles enhanced by silver on the bacterial surface of A. actinomycetemcomitans strain 67. B. Unlabeled B. gingivalis disappear because they are not labeled by the probe. Original magnification: 70,000 X. 2. A culture of A. actinomycetemcomitans strain Y4 labeled with gold particles. This photomicrograph shows the gold markers as light points on the bacterial surface. Original magnification: 53,000 X.

Figure 40

nm

lution (1:10). When cross-reactivity was observed, it disappeared at antiserum dilutions greater than 1:40. No crossreactions were seen at working titer concentrations of antisera toward any of the other microorganisms studied. Unlabeled strains of A. actinomycetemcomitans showed a coccoid and/or coccobacillar morphology by scanning electron microscopy (Fig. 1). Small vesicles, approximately

60 to 80 nm in diameter, were often detected on the bacterial cell wall. Specimens labeled with 40 nm colloidal gold exhibited numerous brilliant points on the bacterial surface (Fig. 2) whereas bacteria labeled with smaller sized gold particles (5 or 15 nm) and subsequently enhanced by the silver method showed uniform surface brightness (Fig. 3A and 3B). In the specimens obtained by mixing A. ac-

tinomycetemcomitans with B. gingivalis,

F. nucleatum, or or were rodfilamentunlabeled (Fig. both, shaped bacteria 4A). When the same sample of pooled bacteria was observed either by secondary electron or back-scattered electron imaging modes, gold markers were localized only on bacteria with coccoid or coccobacillar appearance while rods and filaments were without any surface markers and disappeared completely when observed in back-scattered electron mode (Fig. 4A and 4.B). This observation was particularly striking for the samples treated by silver enhancement (Fig. 3).

DISCUSSION Colloidal gold particles were first proposed as an electrondense marker for transmission electron microscopy by Faulk and Taylor in 1971.10 Later these gold particles were utilized for electron microscopy,11 light microscopy, and X-

252

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ULTRASTRUCTURAL IDENTIFICATION OF A. ACTINOMYCETEMCOMITANS

May

1990

microscopy is not ideal for studying the spatial distrispecific microorganisms on the exposed root surface, scanning electron microscopy is a promising technique for this purpose. One of the main advantages of scanning electron microscopy is the high resolution that can be achieved when examining even very large specimens, such as an extracted tooth. Our results with scanning electron microscopy show that it is possible to specifically detect gold-labeled A. actinomycetemcomitans by both secondary electron image and back-scattered electron image and without cross-reactivity to other common oral bacteria. This is, to our knowledge, the first time that secondary electron imaging and back-scattered electron imaging modes have been employed with gold immunolabeling in microbiologtron

bution of

4.A and 4. . A. actinomycetemcomitans strains 75 mixed with gingivalis (B) and F. nucleatum (F). The distinct cellular morphology allows identification of the different bacteria. A. By secondary electron imaging, the surface of the coccoid cells demonstrate gold labelling evident as points of brightness. Original magnifaction: 19,500 X. B. When the same field is observed by back-scattered electron imaging,

Figures

both B.

both the rod and the filament

shaped microorganisms have disappeared. Original magnification: 19,500 X.

micro-analysis.12 However, colloidal gold has not bevery popular.13 Although gold immunolabeling seems to be a very valuable technique for microbiological investigation in pinpointing certain bacterial species, there are relatively few published articles in this field.14 Recently gold immunolabeling has been introduced into periodontal research. By indirect immunocytochemical labeling and transmission electron microscopy, it has been possible to demonstrate A. actinomycetemcomitans in subgingival plaque samples and, more specifically, to localize A. actinomycetemcomitans to the most apical portion of bacterial plaque on teeth extracted from patients with juvenile Periodontitis.15 However, while transmission elecray

come

ical studies. Back-scattered electron imaging offers the investigator several unique possibilities. Back-scattered electrons may penetrate the specimen to a significant depth before being scattered back to the surface. It is, therefore, possible to see contrast in an image due to a feature which is beneath the surface and, as such, invisible to secondary imaging. In addition, back-scattered electron imaging can be used to rule out the possibility that the brilliant points on the bacterial cell surface are artifacts. When the sample is observed in the back-scattered electron imaging mode, signal originates only from the colloidal gold particles. Therefore, specific identification of the antibody binding sites and, thus, of the labeled microorganism is possible. A. actinomycetemcomitans were also labeled with 5 nm gold particles and subsequently treated by a recently described silver enhancement technique.16'17 Small size gold particles have a greater labeling efficiency due to reduced steric hindrance. The bacteria become completely labeled with the gold-silver particles and provide a much higher, but still specific, back-scattered electron imaging signal. Silver enhancement of small colloidal gold particles should provide better sensitivity of the labeling and better visualization of labeled bacteria even when surrounded by unstained microorganisms or at low magnifications. This new technique of bacterial identification by immunocytochemistry and scanning electron microscopy can provide important information about the spatial distribution of suspected periodontal pathogens on the root surface of extracted teeth which may be useful in illuminating the pathogenesis of periodontal diseases associated with these

microorganisms.

Acknowledgement Some of the

micrographs were obtained with the scanning in microscope Dr. E. de Harver's laboratory, Department of Pathology, University of Toronto, whom we acknowledge with gratitude. Supported by grant C.N.R. 86.0285511 to Dr. Carrassi and USPHS Grant DE 07497 to Dr. Zambón. REFERENCES 1. Carrassi

A, Abati S, Santarelli G.

croscopy in

periodontal

research.

The role of scanning electron miScanning Microsc 1988; 2:1123.

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2. Carrassi A, Weinstein R, Vogel G. The morphology of subgingival plaque in a case of juvenile Periodontitis. A scanning electron microscopy investigation. Mondo Odontostomol 1983; 9:2. 3. Newman HN. Ultrastructure of the apical border of dental plaque. In: Lehner, T, ed. The Borderland Between Caries and Periodontal Disease. Academic Press: London. 1977: 79-103. 4. Carrassi A. The morphology of subgingival plaque in rapidly progressive Periodontitis. In: Lehner T, Cimasoni G, eds. The Borderland Between Caries and Periodontal Disease HI. Geneve: Editions Medicine et Hygiene; 1986. 376-382. 5. Zambón JJ. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol 1985: 12:1. 6. Zambón JJ, Slots J, Genco RJ. Serology of oral Actinobacillus actinomycetemcomitans and serotype distribution in human periodontal disease. Infect Immun 1983;41:19. 7. Place DA, Scidmore NC, McArthur WP. Monoclonal antibodies to Actinobacillus actinomycetemcomitans. Infect Immun 1988;56:1394. 8. Zambón JJ, Umemoto T, Jones C, et al. Actinobacillus actinomycetemcomitans in the pathogeneisis of human periodontal disease. Adv Dent Res 1988;2:269. 9. de Harven E, Soligo D. Scanning electron microscopy of cell surface antigens labeled with colloid gold. Am J Anat 1986; 175:277. 10. Faulk WP, Taylor GM. An immunocolloid method for the electron microscope. Immunochem 1971 ;8:1081.

11.

12.

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Horisberger M, Rosset J, Bauer H. Colloidal gold granules as markers for cell surface receptors in the scanning electron microscope. Ex-

perimentia 1975;31:1147. Hoyer L, Lee J, Bucane C. Scanning immunoelectron microscopy for

the identification and mapping of two or more antigens on cell surfaces. Scanning Electron Microsc 1979;3:629. 13. Horisberger M, Colloidal gold: A cytochemical marker for light and fluorescent microscopy and for transmission and scanning electron microscopy. Scanning Electron Microsc 1981;2:9. 14. Beesley J. Bioapplication of colloid gold in microbiological immu-

nocytochemistry. Scanning Micros 1988;2:1055. 15. Berthold , Listgarten M. Distribution of Actinobacillus actinomycetemcomitans in localized juvenile Periodontitis plaque: An electron immunocytochemical study. J Periodont Res 1986;21:473. 16. Danscher G, Nargard J. Light microscopic visualization of colloid gold on resin embedded tissue. JHistochem Cytochem 1983;31:1394. 17. Scopsi L, Larsson LI, Bastholm L, Nielsen MH. Silver-enhancement colloid gold probe as a marker for scanning electron microscopy. Histochem 1986;86:35.

Send

reprint requests to: Dr. A. Carrassi, Clinica Odontoiatrica, Istitute Biomediche, Ospedale S. Paolo, Via di Rudini 8, 20142 Mil-

di Scienze ano, Italy.

Accepted

for

publication

October 24, 1989.

Identification of Actinobacillus actinomycetemcomitans by gold immunolabeling and scanning electron microscopy.

Recent evidence suggests that dental plaque is not a homogeneous bacterial mass but, on the contrary, specific bacterial morphotypes and species may b...
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