Bacterial pellicle-like substances andpolyphosphate formation by enamel-adherent oral microorganisms*N. Tinanoff, D.D.S., M.S.J. M. Tanzer, D.M.D., Ph.D.
AbstractAssociation with enamel o[ strains of streptococci andactinomycetes was studied both visually and electronmicroscopically. Microorganisms that were notedto form visible plaque on enamel exhibited electronmicroscopically cell attachment apparently mediated by abacterially derived "pellicle." The ability to produce thisstructure may be a critical determinant of initial stages ofbacterial attachment to enamel. Electron-lucent "holes"were also noted in cells of all the strains observed. Theseholes are compatible with the ultrastructural appearanceof bacterial polyphosphates.
Introduction
Dental plaque, the predominantly bacterial massthat adheres to tooth surfaces, has been of interestdue to its causal relationship with two of the mostprevalent diseases in man--caries and periodontal dis-ease. The bacteria that initially colonize teeth arederived from the indigenous microbiota suspended inthe oral fluids. Yet, only certain of the many speciespresent in the mouth can be found on the teeth, ~ andthose organisms which colonize smooth tooth surfacescab only do so because they are capable of tenaciousand essentially irreversible attachment to this hardsurface.
After the initial adherence of bacteria to enamel,organisms divide and cohere, and thus bacterial
* This study was supported by U.S. Army Contract DAMD 17-77-C-7058 and U.S. Public Health Service Grant DE-03758-03 fromthe National Institute of Dental Research.
masses, termed plaque, enlarge. Despite the wide-spread occurrence of bacterial attachment to hardsurfaces in many natural environments, remarkablylittle is known about the mechanisms responsible forthese events.2
Some models which have been used to observebacterial attachment to surfaces involve growth ofbacteria on plastic 3 or on plastic-embedded hydroxy-apatite powder.4 However, the recent development ofa technique for thin sectioning plaque on enamel insitu 5 has enabled ultrastructural study of the interac-tions of plaque-forming bacteria with enamel. We havepreviously described some of the parameters involvedin early plaque formation by Streptococcus mutans onenamel in vitro.6
The purpose of this investigation was to observeelectron microscopically the characteristic features ofthe attachment of other plaque-forming species toenamel, and to compare these features to our previousfindings for S. mutans.
Methods and materials
Enamel Specimen Preparation
Enamel specimens used as a substratum for bacte-rial attachment were prepared by cutting blocks (40mm2) from the smooth surfaces of extracted humanmolars which had been stored in distilled water. Ahole to accept a 20 gauge nichrome wire was thenmade through the specimens. The surface enamel waspolished with fine pumice and washed with water inan ultrasonic cleaner. The specimens were then sus-
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pended by wire, placed in stoppered test tubes, andautoclaved prior to plaque growth.
Microorganisms, Media, and Growth
The following microorganisms were studied withrespect to their attachment to enamel: Streptococcussanguis ATCC 10558, S. sanguis UCHC-FG, S. sali-varius ATCC 13419(rough) S. salivarius13419(smooth), S. salivarius UCHC-FL(r0ugh), salivarius UCHC-FL(smooth), S. salivariusTOVE(rough), S. salivarius UCHC-EL(smooth), salivarius UCHC-LL(smooth), S. salivarius UCHC-DL(smooth), S. mutans 6715-13 WT, S. mutans NCTC10449, S. faecalis ATCC 9790, Group D streptococcus,Actinomyces viscosus M-100, A. viscosus UCHC,R3A,A. viscosus TO4, A. naeslundii ATCC 12104, A. naes-lundii I-S-1000.
Stock cultures were maintained by monthly transferin fluid thioglycollate mediumt supplemented withmeat extract (20% v/v) and excess CaCO3. Fresh hu-man plaque isolates of streptococci were identifiedmorphologically on mitis salivarius agar and theiridentities were confirmed biochemically. The "rough"and "smooth" notation for S. salivarius characterizestheir colonial morphologies on mitis salivarius agar.Prior to experiments, cultures were adapted to growthin the complex medium of Jordan et al.7 supplementedwith 50 mg/liter of Na2CO3 and 5% sucrose.
For plaque growth on enamel, culture tubes of thecomplex medium were inoculated with 0.2 ml of 24-hrculture of the adapted strain. 6 Sterile, wire-mountedenamel specimens were then suspended in the culturetubes and incubated microaerophilically at 37°C. Theenamel specimens were transferred daffy to uninocu-lated medium, and after 2 days the enamel specimenswere observed visually for surface bacterial growthand processed for electron microscopy.
Electron Micro~scopic Preparation
The specimen.,~ to be processed for electron micros-copy were remowed from the growth medium and fixedwith a 2.5% gluteraldehyde in 390 mOsM phosphatebuffer (pH 7.4), and postfixed in 1% osmium tetroxidein veronal buffer (pH 7.3). s They were then washed inthe phosphate buffer and placed in an acidic gel con-taining 0.1 N HC1 and 15% gelatin (BBL) for 3.5 hr demineralize sliglhtly the enamel surface.~ After dehy-dration in acetone and embedment in epoxy medium,9
the resin was polymerized at 70°C.The specimens were prepared for sectioning as de-
scribed previously, 6 so that only the acid-softenedenamel and the organic films on the surface of theenamel remained. Thin sections were prepared with aReichert ultramicrotome using a diamond knife. Sil-
T Difco, Detroit, Michigan.
ver-gold-colored sections were stained with aqueousuranyl acetate followed by lead citrate ~° and examinedat 90 kV with a Zeiss EM 10 electron microscope.
Results
Adherent bacterial masses on enamel were evidentwith all S. mutans, S. sanguis and Actinomyces cul-tures tested. Only one of the nine S. salivarius strainstested failed to form plaque on enamel, whereas bothof the enterococcus strains failed to form plaque.
Ultrastructurally, enamel incubated with a non-plaque-forming microorganism, S. faecalis, showedneither microorganisms nor films on the enamel sur-face. The organisms that produced visible deposits onenamel exhibited bacteria surrounded by an extracel-lular electron-dense matrix (Figs. 1-4). Electron-lucent"holes" (0.02-0.30/zm) were noted in all strains ob-served, consistent with the presence of polyphosphategranules. ~
Bacterial attachment to enamel appeared to be me-diated by a thin, 0.01-0.10 pm, electron-dense filmwhich covered the enamel surface. This film appearedcontiguous with the surfaces of Cells in close associa-tion with the enamel.
No differences could be noted among the plaque-forming streptococci, either in their morphology or inthe matrix between these bacteria and the enamel.The Actinornyces strains, however, differed from thecocci in that these organisms were pleomorphic, vary-ing from coccal to bacillary in form (Fig. 4).
Discussion
Many of the current concepts concerning bacterialattachment to teeth have been derived from studies ofmarine bacterial sorption to glass surfaces. The at-tachment of these organisms has been reported toinvolve an initial reversible phase and a time-depen-dent irreversible phase.
In the first phase the bacteria are not firmly at-tached to the surface and can be desorbed readily.Later these organisms have been noted to producepolymeric fibrils which may cause an irreversible at-tachment to the surface. ~e Two stage attachment hasalso been noted for in vitro S. rnutans plaque forma-tion. For example, Clark and Gibbons~ demonstratedinitial reversible adsorption of S. rnutans to enamel.When these organisms were allowed to produce extra-cellular polymers, the attachment of bacteria to en-amel became irreversible, as demonstrated by de-creased ability to desorb the cells.
Glucan was suggested as the polymer mediatingfirm attachment of these organisms to enamel.~a Glu-can production by S. rnutans has also been implicatedas essential for this organism to form large bacterialaccumulations.2. ~4-~
PELLICLE AND POLYPHOSPATE OF ENAMEL-ADHERENT ORGANISMS2 Tinanoff and Tanzer
Fig. 1. Transmission electronmicrographs of the enamel-bac-terial interface after incubation ofStreptococcus sanguis UCHC-FG with enamel for 2 days incomplex medium containing 5%sucrose. Apparent bacteriallyderived extracellular material (ar-rows) is present on the enamelsurface at low magnification.Note electron-lucent holes incells. X9.200. Inset shows onecell approximating the enamelwith extracellular material appar-ently mediating attachment.X22.500.
Fig. 2. Electron micrograph ofthe enamel-bacterial interfaceafter incubation of S. salivariusUCHC-FL (rough) with enamel for2 days in complex medium con-taining 5% sucrose. Extracellularmaterial on the enamel is evidentat low magnification. Also noteenamel prism structures andelectron-lucent holes in cells.x5,040. Inset shows a highermagnification of one cell approx-imating the enamel, x 22,400.
In a previous report, we noted sucrose dependencyfor plaque formation only by Bratthall serotypes a, b,and d of S. mutans. Serotypes c and e were noted toform plaque on enamel in glucose-containing as wellas in sucrose-containing media, although they adheredless tenaciously under the former condition. Addition-ally, a bacterially produced "pellicle" on the enamelappeared electron microscopically to mediate attach-ment of S. mutans to the enamel.
Since this extracellular material was formed in glu-
cose-containing cultures as well as in sucrose-contain-ing cultures, it was believed not to be glucan. Chemicalanalysis confirmed that no glucan was present in theglucose-grown plaques. Furthermore, only bacterialproducts of adherent cells were believed to have pro-duced this "pellicle" since: (1) polishing of the enamelwas noted to remove organic films; (2) enamel was notexposed to saliva to form a "salivary pellicle"; (3)sterile bacterial growth medium incubated with en-amel did not precipitate any surface layer on the
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Fig. 3. Electron micrograph ofthe enamel-bacterial interfaceafter incubation of S. mutansNCTC 10449 with enamel for 2days in complex medium con-taining 5% sucrose. At low mag-nification extracellular materialcan again be observed on theenamel surface and electron-lu-cent holes are apparent in cells(white arrows). X9.200. Highmagnification shows the interac-tion of one cell with the enamel,x 22,400.
Fig. 4. Electron micrograph ofthe enamel-bacterial interfaceafter incubation of Actinomycesviscosus M-100 with enamel for2 days in complex medium con-taining 5% sucrose. A "bacterialpellicle" apparently mediatescell attachment to enamel. A striaof Retzius forming surface peri-kymata can also be noted as partof the enamel structure. Lowmagnification, x 19,000. Highermagnification shows one cell ap-proximating the enamel with ex-tracellular material apparentlymediating attachment, x 35,000.
enamel; and (4) nonadherent cells failed to form suchsurface film.6
In the present study a similar pellicle-like structurewas evident at the enamel-plaque interface for allplaque-forming organisms studied. This bacterially de-rived, presumptive polymer covered the enamel andapparently mediated bacterial attachment to this sur-face. Since the production of "bacterial pellicle" wasnoted to be common to the adherent organisms stud-ied, the ability of bacteria to produce this extracellular
polymer may be a critical determinant of initial stagesof bacterial attachment to enamel. Further studies arenecessary to explore the chemical nature of this struc-ture.
Traditionally, the pellicle found at the enamel-bac-terial interface has been thought to be derived fromsalivary glycoprotein components.17"19 Such pellicleshave been observed in previous studies upon incuba-tion of enamel with saliva for a brief period of time.6
Since the enamel in this study was not exposed to
PELLICLE AND POLYPHOSPATE OF ENAMEL-ADHERENT ORGANISMSTinanoff and Tanzer
saliva, salivary pellicle was not found essential forattachment of S. mutans, S. sanguis, S. salivarius, orActinomyces sp.
Others have also demonstrated that glycoprotein-rich (hog gastric) mucin is not essential for attachmentof S. mutans to nichrome surfaces.2° Salivary films,however, have been shown to enhance adsorption ofcertain plaque-forming microorganisms to enamel andto inhibit the adsorption of others.~3’ 21.22 Hence, theacquired salivary pellicle may have a role in determin-ing the relative proportion of different bacteria whichadsorb to enamel, but it does not appear requisite forthe attachment of plaque-forming bacteria to enamel.
.Bacterial attachment and subsequent formation ofmicrobial masses on enamel are indeed essential forcaries of smooth tooth surfaces.23 S. sangius report-edly adheres to enamel more readily than S. mutans,while S. salivarius has a weak affinity for enamel.24
Additionally, relatively large percentages of A. viscoosus have also been noted in the early dental plaque ofsome individuals. ~ However, S. sanguis has not beenshown to be associated with human caries, 25 while S.mutans has been shown to have a strongcorrelation, ~-e7 and there is only limited evidence thatcaries may be induced by A. viscosus~s’ ~9 and S. sali-varius. ~° Hence, other traits ~ appear to be necessaryfor Caries beside the capability of bacteria to adhereto the teeth.
The electron-lucent "holes" noted incidentally inthe bacteria were a common finding in all of theadherent strains. These holes probably represent elec-tron-dense bacterial polyphosphate granules whichhave been volatilized by the electron beam,a2’ ’~’~ whichis typical of polyphosphate. Polyphosphates have beenidentified in a variety of microorganisms,~1 includingS. mutans.34 This highly anionic phosphate is believedformed in cells when nutritional conditions are notfavorable to growth.~1
It should not be concluded, however, that the poly-phosphates apparent in the adherent cells are relatedto adhesion because nonadherent cells were not stud-ied ultrastructurally. The presence of polyphosphatesin the adherent cells does indicate, however, that thesecells are not growing optimally. Other data directlysupport this conclusion.35
Conclusion
Strains of S. sanguis, S. mutans, Actinomyces, andS. salivarius were noted to attach to enamel in vitro.Ultrastructurally, the bacterial attachment to enamelis mediated by an electron-dense film which coveredthe enamel surface. The formation of this "bacterialpellicle" at the enamel-plaque interface may be nec-essary for stable bonding of bacteria to this surface.
Electron-lucent "holes" in some of the bacteria werenoted in all-the strains observed. These holes are
believed to represent electron-dense bacterial poly-phosphate granules which have been volatilized by theelectron beam.
Acknowledgment
The technical assistance of F. N. Woodiel is gratefully acknowl-edged.
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Dr. N. Tinanoff is Assistant Professorin the Department of Pediatric Dentistryat the University of Connecticut HealthCenter. Requests for reprints shouldbe addressed to Dr. N. Tinanoff, De-partment of Pediatric Dentistry, Univer-sity of Connecticut Health Center, Far-mington, Connecticut 06032.
Dr. J. M. Tanzer is Professor in theOral Diagnosis Department also at theUniversity of Connecticut Health Cen-ter, Farmington.
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