Calcification of Selected Strains of Streptococcus

Transcription

1 JOURNAL OF BACTEROLOGY, OCt. 1974, p Copyrght ( 1974 Amercan Socety for Mcrobology Vol. 120, No. 1 Prnted n U.S.A. Calcfcaton of Selected Strans of Streptococcus mutans and Streptococcus sangus JOSEPH L. STRECKFUSS, WLLARD N. SMTH, LEE R. BROWN, AND MARON M. CAMPBELL The Unversty of Texas Dental Branch, Dental Scence nsttute, Houston, Texas Receved for publcaton 3 July 1974 Nne strans of carogenc Streptococcus mutans and two strans of Streptococcus sangus were tested for ther ablty to form hydroxyapatte. The cells were examned by X-ray dffracton and electron mcroscopy for apatte crystals after growth n a synthetc calcfcaton medum. Each of the test solates, except for one stran of S. sangus, produced ntracellular mneral. Two strans of S. mutans formed both ntra- and extracellular crystals. There was no apparent relatonshp between calcfablty and serotype. Calcum phosphate smlar to hydroxyapatte, Ca10(PO4)6(OH)2, s the major mneral of vertebrate hard tssues. t s also dentfed as a consttuent of the mneralzed depost of calcfed oral mcroorgansms whch develop n dental calculus at the tooth gngval nterface (3). The present study was undertaken to determne the calcfcaton potental of selected oral streptococc whch are known to colonze on tooth surfaces and have been mplcated n dental cares (18). Calcfcaton from pure cultures was frst reported for Bacteronerna matruchot. Ths flamentous member of the oral mcroflora of man and some lower anmals forms ntracellular apatte (4, 19). A recent survey of 14 oral solates obtaned from humans and marmosets showed that four enterc speces also form apatte. Calcfcaton on a pure culture bass, therefore, s not unque to B. natruchot, but nether s t unversal among oral mcroorgansms. One of the noncalcfyng solates n the survey was dentfed as Streptococcus salvarus (8). Subsequently a streptococcal varant obtaned from B. matruchot calcfed, formng ntra- and extracellular deposts (6). These fndngs ndcated that streptococc of recognzed lneage should be studed, especally those assocated wth oral dsease. Nne strans of carogenc S. mutans representatve of the establshed serotypes (1) and two strans of S. sangus were examned for calcfcaton. stran desgnaton and serotypes are presented n Table 1. Meda. Each organsm was grown at 37 C n 250 ml of the synthetc medum of Ennever, Vogel, and Streckfuss (10) for 14 days. The ph of the medum was determned before and after ncubaton. The cultures were harvested by centrfugaton at 27,000 x g for 5 mn at 4 C and washed three tmes wth deonzed water. The concentrated cells of each solate were then frozen at -15 C and dvded nto two equal parts. One porton was ar-dred at 45 C and rado-frequency ashed for 2 to 4 h at 55 C for X-ray dffracton. The remanng porton of each sample was processed for electron mcroscopy as prevously reported (10). TABLE 1. Calcfcaton of streptococcal strans Stran Serotype Hydroxy- Orgn apatte S. mutans OMZ-61 a +a Rat (16) AHT-12 + b Human (24) FA-1 b + Rat (12) GS5 + Human (15) PK1 c + Human (15) S3 +a Human (14) SL-1 d + Human (11) Human (13) LM7c E + Human (15) S. sangus ATCC Type Human Type MATERALS AND METHODS S. salvarus 125C Human (8) Strans. Nne strans of S. mutans ncludng serotypes a, b, c, d, and E, and two strans of S. sangus, ntracellular. a ntra- and extracellular. Types and, were examned for calcfcaton. S. c S. mutans wth antgen reactng wth Lance feld salvarus 125C served as a noncalcfyng control. The group E (2, 18). 502

2 VOL. 120, 1974 CALCFCATON OF S. MUTANS AND S. SANGUS 503 RESULTS Growth. The ntal ph of the medum was 7.2 and after ncubaton was 6.9. S. mutans OMZ-61, AHT-12, GS5, PK1, S3, SL-1, and 6715 produced rapd growth reachng a maxmum after 30 h of ncubaton. The remanng solates of S. mutans (FA-1 and LM7), S. sangus Types and H, and S. salvarus 125 C grew sparsely. The slow-growng solates were subsequently noculated nto trplcate volumes of medum to obtan a suffcent mass of cells for X-ray dffracton. X-ray dffracton. An X-ray dffracton pattern of dental calculus s presented for reference n Fg. 1A. The test solates produced patterns for apatte except S. mutans FA-1 and S. sangus Type (Fg. 1G and H). Fgure H also represents the noncalcfable control, S. salvarus 125C. Patterns representatve of the serotypes of S. mutans and S. sangus Type are llustrated n Fg. 1B through F. The dffuse bands n the dffracton patterns ndcate the apatte crystals were small. Wth S. mutans strans GS5 and LM7, (Fg. 1C and E), the pattern was fant because the concentraton of apatte n the ashed materal was just at the pont of X-ray resoluton. Apatte wth S. mutans stran FA-1 was not detectable by X-ray dffracton (Fg. 1G) but crystals typcal of apatte were found by electron mcroscopy (Fg. 2B). Electron mcroscopy. Characterstc needlelke ntracellular crystals of apatte represented by one solate of each serotype of S. mutans and for S. sangus Type are llustrated n Fg. 2A through G. Fgures 2H and show the lack of crystals respectvely for the noncalcfyng S. sangus Type and S. salvarus 125C. Extracellular crystallnty was noted n S. mutans OMZ-61 (Fg. 2A) and S3 (Fg. 2D). Calcfcaton n S. mutans stran GS5 (Fg. 2C) and SL-1 (Fg. 2E) appeared to be assocated wth the cell membrane (arrows) by the darker electron dense areas at the perphery of the cells. DSCUSSON A varety of genera of oral mcroorgansms are known to calcfy when mplanted n the rat pertoneum (17). The defned cultural system utlzed n ths study, however, provded a means to observe relatve dfferences n calcfablty wth pure cultures. Bologcal apatte was readly detectable n the rapd-growng solates, whereas the slower developng strans produced weakly detectable mneral or were noncalcfable. These observatons and the presence of extracellular crystallnty ndcate the need to relate crystal nucleaton to the growth rate of the streptococc. Once crystal nucleaton occurs, crystal growth s favored n the medum. Apatte was detectable after 5 days of ncubaton and from 12 to 14 days wth the rapd- and slow-growng strans, respectvely. Under these condtons, t s estmated that about 5% crystallnty s detectable n ashed cell resdues by X-ray dffracton. The level of apatte detecton rased the queston of whether the mass of cells ashed n the noncalcfable strans was adequate for detecton of low levels of crystallnty. S. sangus Type and S. salvarus 125C have not produced crystallnty that was detectable by X-ray dffracton or electron mcroscopy. These solates, therefore, were repeatedly examned to nsure that the mass of cells ashed was n excess when compared wth that of the calcfable forms. No apatte was detectable by X-ray dffracton n the rat stran FA-1, but crystals characterstc of apatte were found by electron mcroscopy. Ths probably occurred because mneral could not be suffcently concentrated n the ashed cell resdue. The varable capacty of solates from the same speces to calcfy was demonstrated when S. mutans stran FA-1 was compared wth another rat stran, OMZ-61. Ths solate n contrast wth FA-1 produced rapd growth and extracellular deposts of apatte. A smlar observaton was made wth human strans when LM7 was compared wth S3. t was evdent that calcfablty was varable wth solates obtaned from dfferent and from the same speces, and there was no apparent relatonshp of serotype to calcfablty. There were no ultrastructural features that provded a bass for the predcton of calcfcaton. Each calcfable solate formed ntracellular deposts. n S. mutans, GS5 and SL-1, some of the foc appeared orented at the membrane. n addton, S3 produced extracellular crystals radatng from the cell. Ths feature may result from frequent breaks along the cell wall where nucleatng stes of the membrane are exposed to the medum. The cell-free apatte of OMZ-61 probably represents crystals released from lysed cells. Nucleaton of apatte s thought to begn wthn the cell snce extracellular foc were consstently found n the presence of ntracellular deposts, as prevously reported (6). The ntracellular deposts, some of whch appeared assocated wth cell membranes, suggest that there may be a common nucleatng moety n the streptococc. These observatons are also n accord wth the proposal that there s

3 504 A.-.T STRECKFUSS ET AL. L J. BACTrEROL. B.t C 0 E G H FG. 1. Comparatve X-ray dffracton patterns. (A) Dental calculus reference standard for apatte; (B) OMZ-61; (C) GS5; (D) SL-1; (E) LM7; (F) S. sangus Type ; () FA-l; (H) noncalcfable S. sangus Type and S. salvarus 125C. a common nucleator n phosphatc calcfcatons (4). The mechansm of mcrobal calcfcaton has been studed usng B. matruchot (4, 19, 5) and n ths organsm, the phospholpd fracton nucleates apatte (20, 23). Phospholpd s a major component of bacteral membranes and the reducton of these structures n calcfable cells wth a synthetc lathyrogen produces noncalcfable forms (21). Lpd extracts from vertebrate bone (9), dentn (J. Vogel, personal

4 .1, W ~~~B. :1 JWW. as 7 r2.b ' 4l,.,..f X..:.". S %. Fh.. 1"..1 Downloaded from 'V N 4 #4' S, 9.4 F A `4 0 '.-AA16 N.- 'wr.1 f,v on August 21, 2018 by guest.a. N- P G W 'N' FG. 2. Electron mcrographs of solates. (A) OMZ-61, ntra- and extracellular crystals; (B) FA-1, ntracellular crystals; (C) GS5. membrane assocated (arrow) and ntracellular crystals: (D) S3, crystals ntracellular and radatng from the cell; (E) SL-1, membrane assocated (arrow) and ntracellular crystals; (F)LM7,ntracellular crystals; (G) S. sangus Type, ntracellular crystals; (H) noncalcfable S. sangus Type ; () noncalcfable S. salvarus 125C; marker bar: 500 nm. 505

5 506 STRECKFUSS ET AL. J. BACTEROL. communcaton), and dental calculus (7) have also nucleated apatte. A recent report, however, questons the proposal of whether there s a unversal calcfcaton factor and suggests nstead that calcfcaton s a matter of common mechansm (22). The potental of the streptococc to produce hydroxyapatte s relevant to expandng studes n mcrobal calcfcaton. The most sgnfcant observaton, however, was that organsms known to contrbute to the destructon of tooth mneral can themselves form the same mneral n vtro when condtons are favorable. ACKNOWLEDGMENTS We thank J. Ennever for hs assstance wth X-ray dffracton, Frank E. Summers for assstance n electron mcroscopy, and Leonard Graves for techncal assstance n the cultvaton of the organsms. Ths nvestgaton was supported by a Publc Health Servce grant DE from the Natonal nsttute of Dental Research. LTERATURE CTED 1. Bratthall, D Demonstraton of fve serologcal groups of streptococcal strans resemblng Streptococcus mutans. Odontol. Revy 21: Bratthall, D mmunofluorescent dentfcaton of Streptococcus mutans. Odontol. Revy 23: Cron, P., G. J. Van Campen, and. Lnstrom Human dental calculus. norganc and crystallographc composton. Arch. Oral Bol. 12: Ennever, J ntracellular calcfcaton by oral flamentous mcroorgansms. J. Perodontol. 31: Ennever, J., and H. Creamer Mcrobologc calcfcaton: bone mneral and bactera. Calcf. Tss. Res. 1: Ennever, J., J. L. Streckfuss, and. Takazoe Calcfcaton of bacllary and streptococcal varants of Bacteronema matruchot. J. Dent. Res. 52: Ennever, J., J. J. Vogel, and L. A. Benson Lpd and calculus matrx calcfcaton n utro. J. Dent. Res. 52: Ennever, J., J. J. Vogel, and L. R. Brown Survey of mcroorgansms for calcfcaton n a synthetc medum. J. Dent. Res. 51: Ennever, J., J. J. Vogel, and B. M. Levy Lpd and bone matrx calcfcaton n vtro. Proc. Soc. Exp. Med. 145: Ennever, J., J. Vogel, and J. L. Streckfuss Synthetc medum for calcfcaton of Bacteronema matruchot. J. Dent. Res. 50: Ftzgerald, R. J., and H. V. Jordan Polysaccharde producng bactera and cares. p n R. S. Harrs (ed.), Art and scence of dental cares research. Academc Press nc., New York. 12. Ftzgerald, R. J., H. V. Jordan, and H. R. Stanley Expermental cares and gngval pathologc changes n the gnotobotc rat. J. Dent. Res. 39: Ftzgerald, R. J., P. H. Keyes, T. H. Stout, and D. M. Spnell The effects of dextranase preparaton on plaque and cares n hamsters, a prelmnary report. J. Amer. Dent. Ass. 76: Gbbons, R. J Metabolsm of ntracellular polysaccharde n Streptococcus mts and ts relaton to nducble enzyme formaton. J. Bacterol. 87: Gbbons, R. J., K. S. Berman, P. Knoettner, and B. Kapsmals Dental cares and alveolar bone loss n gnotobotc rats nfected wth capsule formng streptococc of human orgn. Arch. Oral Bol. 11: Guggenhem, B., K. G. Kong, and H. R. Muhlemann Modfcatons of the oral bacteral flora and ther nfluence on dental cares n the rat.. The effects of noculatng 4 labelled strans of streptococc. Helv. Odontol. Acta 9: Rzzo, A. A., D. B. Scott, and H. A. Bladen Calcfcaton of oral bactera. Ann. N. Y. Acad. Sc. 109: Streptococcus mutans and dental cares, rd Ann. Meet., Amer. Soc. Mcrobol. DHEW Pub. No. (NH) Takazoe, Study on the ntracellular calcfcaton of oral aerobc leptotrcha. Shkwa Gakuho 61: Takazoe,., J. J. Vogel, and J. Ennever Calcum hydroxyapatte nucleaton by lpd extract of Bacteronema matruchot. J. Dent. Res. 49: Takazoe,., J. J. Vogel, and F. E. Summers Ultrastructural changes n Bacteronema matruchot nduced by osteolathyrogen. Tex. Rep. Bol. Med. 28: Tryggre, L., and S. A. Knut Calcfcaton of oral bactera: an ultrastructural study of the two strans of Bacteronema matruchot. Scand. J. Dent. Res. 82: Vogel, J. J., and J. Ennever The role of lpoproten n the ntracellular hydroxyapatte formaton n Bacteronema matruchot. Cln. Orthop. 78: Znner, D., J. Jablon, A. Aran, and M. Saslaw Expermental cares nduced n anmals by streptococc of human orgn. Proc. Soc. Exp. Bol. 118: