t Faculty of Graduate Studies and Research

M.Sc.

Comparative Growth and Locomotion of

Anaerobie Oral Spirochetes

by

Antonia Klitorinos

A thesis submitted to the Faculty of Graduate Studies and Research, McGi:' University, in partial fulfillment of the requirements for the degree of Master of Science.

Department of Microbiology and Immunology McGill University Montreal, Quebec, Canada

© March 1991 1

1 / - 1 ABSTRACT

A positive relationship has been established between the number of oral spirochetes in diseased periodontal pockets

and the severit~ of periodontal disease. Elucidation of the role that spirochetes play in periodontal disease has been hampered by the difficulty of growing these organisms. Growth studies were carried out in or,jer to obtain a better

und~rstanding of the nutritional requirements of three specÎes of oral spirochetes. Growth was followed by cell counts and turbidity measurements. Long-chain fatty acids were shown to support the growth of ail treponemes studied with the

exception of Treponema ~anskjj. Short-chain fatty acids and rabbit serum were found to be essential for growth. Glucuronic acid was shown to stimulate growth.

Locomotion of oral spirochetes has been suggested to ba an invasive factor for tissue penetration. Video time-Iapse microscopy using darkfield optics was employed to study the locomotory behaviour of spirochetes in media of different densities. Optimal migration of spirochetes was found to be viscosity-dependent. Treponema denticola ATCC 35404 and T. dentjcola ATCC 35405 exhibited the greatest mean speeds at 0.2 - 0.3 % (wt.lvol.) Noble agar. Optimal motility for T. vjncentjj ATCC 35580 and 1. socranskjj ss. socranskii ATCC 35536 was achieved at 0.35 - 0.40 % (wt./vol.) Noble agar.

1

i '" .., 1 RESUME

Il existe une association positive entre les nombres de spirochètes buccaux trouvés dans les poches des gencives et la sévérite de la périodontite. Le rôle joué par ces microbes dans le développement de la périodcntite n'a pas été élucidé, surtout à cause de la grande difficulté dans la culture de ces organismes. En effet, des études entreprises au sujet des besoins nutritifs nécessaires à la culture des spirochètes ont établi une meilleure connaissance de trois espèces de spirochètes buccaux. La croissance fut suivie en comptant le nombre de bacteries viables en fonction de la transmltance à l'aide de spectrophotomètre. Les acides gras à longue chaîne aidèient la croissance tous les spirochètes, à l'exception de T. socranskii. tandis que les acides gras à courte chal'ne et le sérum de lapin se sont avérés absolument nécessaires. L'acide glucuronique stimula la croissance.

Il fut suggéré que la virulence dépend directement de la mobilité des spirochètes buccaux. La microscopie avec illumination sur fond noir, combiné avec le vidéo-accéléré a été utilisé dans If étude du comportement migratoire des spirochètes dans des milieux de culture de différentes consistances. La migration optimale des spirochètes dépend directement de la densité des milieux. Les I. Win.1jcola ATCC 35404 et ATCC 35405 affichaient la plus grande vitesse

moyenne avec 0.2 - 0.3 % (w/v) d'agar Nob!e. La mobilité optimale pour T. vincentii et T. ~ocranskil fut établie à des concentrations de 0.35 - 0.40 % (w/v) d'agar Noble. t

11 ACKNOWLEDGEMENTS

These investigations were carried out in the Lyman Duff Medical Sciences Laboratory and S+rathcona Anatomy and Dentistry Laboratory, McGili University, Montreal.

First and foremost, 1 would like to express my heartfelt gratitude to my parents and my brother for their support and encouragement.

1 would like ta thank Dr. E.C.S Chan for his supervision and guidance throughout the course of my study.

Finally, 1 would like to thank Dr. P. Noble for his technical assistance in the mati lity study and Dr. J. Dealy for the use of the Wells-Brookfield Micro-Viscometer and his expert advice in rheology.

1

iii 1 TABLE OF CONTENTS

Page ABSTRACT ...... i RESUME ...... ii ACKNOWLEDGEMENTS ...... iii LIST OF TABLES ...... vi LIST OF FIGURES ...... vii .NTRODUCTION ...... 1

LlTERATURE REVIEW:

1. HISTORY ...... 3

II. GENERAL CHARACTERiSTICS OF SPIROCHETES ...... 3 i. SlimeLayer ...... 5 ii. OuterSheath ...... 5 iii.Axial Fibril...... 6 iv.ProtoplasmicCylinder ...... 7

III. TAXONOMY OF SPIROCHETES ...... 7 i. Borrelja ...... 8 ii. Leptospjra ...... 9 iiLSpjrochaeta ...... 10 iv.Crjstjspjra ...... 1 0 v. Trepooema ...... 11

IV. CLASSIFICATION OF ORAL SPIROCHETES ...... 11

V. THE ISOLATION AND MAINTENANCE OF ORAL SPIROCHETES ...... 12

VI. LOCOMOTION OF SPIROCHETES ...... 14

! ------~~ --~~ ~

1 VII. PERIODONTAL DISEASE...... 16 i. Clinical Characteristics of Periodontal Disease ...... 16 iL Positive Numerical Correlation between Oral Spirochetes and Penodontal Disease ...... 18 iii. Evidence of Spirochetal Invasion of the GingivaITissue ...... 20

VIII. PUTATIVE PERIODONTOPATHIC MECHANISMS ...... 22 1. Toxic Metabolic End Products and Enzymes Released by Oral Spirochetes may Contribute to Periodontal Disease ...... ,...... 22 iL Inhibition of Cell Proliferation ...... 24

IX. CELLULAR AND HUMORAL IMMUNITY ...... 25

X. A METHOD FOR THE DETECTION OF ORAL BACTERIA ...... 26

MATE RIALS AND METHODS...... 27

RESULTS:

1. GROWTH STUDIES ...... 50 Il. EFFECT OF AGAR DENSITY ON GROWTH ...... 64 III. COLLAGEN BREAKDOWN TEST ...... 68 IV. ISOLATION OF COLONIES ...... 68 V. CHEMOT AXIS ...... 69 i. Hard agar plug technique ...... 69 ii. Weil plate technique ...... 72 iii. Test-tube method ...... 73 iv. Bacteria in plug method ...... 73 v. Chemical in pond method ...... 74 VI. MOTILITY EXPERIMENTS ...... n

DiSCUSSiON ...... 84 , BiBLIOGRAPHY ...... 95

./ r1. 1 LIST OF TABLES

Table Page

1. Morphological characteristics of four oral spirochetes...... 28

2. Comparative growth of oral anaerobic spirochetes ln

growth media (expressed in % values as calculated from cell counts) ...... 63

3. Comparative growth of oral anaerobic spirochetes in growth media (expressed in % values as calculated from 0.0 readings) ...... 65

4. Chemotacti(; behavior of I.dentjcola ATCe 35404 and A TCe 35405 to a variety of test-chemicals by the hard agar plug ter.:hn,:que ...... 70

5. Average locomotory speeds of spirochetes in NOS medium of different Noble agar concentrations ...... 78

6. Range of persistence of spirochetes in NOS medium of different Noble agar concentrations ...... 81

1 LIST OF FIGURES

Figure "age

1 . Schematic diagram of a spiror.ht:tte cell and its cross-section ...... 4

2. Schematic representation depicting how a spirochete cell swims (Bergls Model) ...... ' ...... 15

3. Schematic drawing of the periodontîum ...... 17

4. Schematic representation of a Wells-Brookfield Microviscometer ...... 47

5. Mathematical relationship of the viscosity calculation for non-Newtonian materials ...... 49

6. Growtll curves of three Treponema species in a variety of test media. (a)-(c) : 1. dentjcola ATCC 35404 ...... 51 (d)-(f) : 1. den1icola ATCC 35405 ...... 54 (g)-(i) : 1. vincentii ATCC 35580 ...... 57 (j)-(k) : T. socranskii ss. socranskii ...... 60

7. The affect of agar density on the growth of oral spirochetes. (a) T.denticola ATCC 35405 ...... 66 (b) I.dentjcola ATCC 35404 ...... 67

vi: r

1 8. Chemotaxis concentration response curves

to L-cysteine h~'drochlonde (a) T. dentlcola ArCC 35405 ...... 75 (b) 1. denticola ATCC 35404...... 76

9. Relationship of the viscosity of NOS medium wlth varying concentrations of Noble agar wlth mcreasmg shear rates...... '"'''''''' 82

1

vi.: 1. INTRODUCTION

Oral splrochetes have been Impllcated as etiological agents of pcnodontal dlsease. A positive quant.i:ative relatlonshlp between the pereentage of oral splroehetes and the seve rit y of penodontal dlsease has been observed by a number of Investlgators (Llstgarten and Levln, 1981 , Armitage, et al., 1982 , and Listgarten, 1976). Recently, the tirst quantitative eVldence of a positive correlation was reported (Slmonson, et al., 1988). A twu-to-three fold mcrease in 1.. dentlcola was

observed ln patients at severe diseased sites compared to healthy gingival suie!. However, the aSSOciation between ot'al splrochetes and periodontal dlsease is not simply a numencal one. Spiroehetal invasion of the gmglval tissue has been observed in periodontitis (Mlkx, et al, 1984).

Dlfficultles have been encountered in cultlvating spirochetes because very Iittie Information is known about their nutrition. Consequently, growth studies were carned out in a

vanety of test media ln order to gain a better understandlng rnto their nutntlonal and growth requirements.

The appearance of oral spirochetes in the sulcular

epithelium impllcates them ln rnitlating the pathogenlcity of periodontal dlsease (Mlkx, 1990) Th,,": motility of splrochetes ln viscous envlronments may be consldered an invasive factor for gingival tissue penetration. The abillty of spirochetes to locomote through hlghly VISCOUS envlronments may confer on 1 them an ecological advantage enabling them to negotiate J mucosal surfaces and inter- and mtra-cellular spaces Spirochetes like LeQ1Q,s QI ra and Ba r reli.a. have been shawn to remain motile ln environments that render other flagellated prokaryotes immobile Reports on the locomotory behavlour of oral splrochetes are sca"ce As a result, the locomotory betlaviour of three species of oral ,:;plrochetes was rnvestlgated usrng video time-Iapse microscopy under darkfleld optlCS

Chemotaxis may be one of the many mechan.sms by whlch bacteria are attracted to or repelled trom mucosal surfaces such as the gingival crevlce (Allweiss, et al, 1977) The chemotactlc

behaviour of bacteria may also play an Important role ln thelr invasiveness. Consequently, the chemotactlc charactenstlcs of two strams of Trepooema dentjcola to a vanety of test­ chdmicals were investigated

1 LlTERATURE REVIEW

1 • HISTORY

Oral spirochetes were first observed microscopically in periodontal disease by Antonie Van Leeuwenhoek in 1674. He described these organisms obtained from the teeth of an old man as "living animalcules aswimming more nimbly than any 1 had ever seen up to this time. The biggest sort bent their body into curves in going forward" (Holt,1978). It was not until 1950, with the discovery of the electron microscope, that the complex structure of these organisms could be fully realized.

Il. GENERAL CHARACTERISTICS OF SPIROCHETES

Spirochetes are Gram-negative, chemoheterotrophic, helically-shaped microorganisms that have a unique body structure and a distinctive motility. Spirochetes divide by transverse binary fission. Ail spirochetes have common morphological characteristics. The outermost layer of the spirochete cell is called the slime layer or S-Iayer (Schmid, 1989). This layer is followed by the outer sheath or outer <;ell envelope which surrounds the protoplasmic cylinder. Wound around the protoplasmic cylinder are endocellular flagella known as axial fibrils, endoflagella, or axial filaments 1 (Figure 1.). The number of axial fibrils per cell varies from 3 .E.ig.. 1. (A). Schematic diagram of a spirochete cell in longitudinal profile with one axial fibril. (B). Cross- section of a spirochete with six axial fibrils. The mucoid layer , protoplasmic cylinder and outer sheath are also depicted . 1 A

...... ,'......

B

uter sheath

~t--- Protoplasml c cyl1nder

.,

4 1 one to several hundred depending on the type of spirochete.

i. Slime layer

The slime layer also refarred to as the mucoid layer is the outermost layer of the spirochete cell. The slime layer is composed of carbohydrates and lipoproteins and may contribute to the virulence of spirochetes. The slime layer can be removed from the rest of the spirochete cell with Triton X-100 (0.1 0/0) treatment.

ii. Outer sheath

The outer sheath which contributes to the morphological integrity of the spirochete cell is composed of a three-Iayered membrane. It is composed of two electron dense layers of 5 to 8 nm separated by an electron transparent layer of similar dimensions (Holt, 1978 ; Canale-Parola, 1978). It is similar to the outer membrane of Gram-negative bacteria and consists of protein, lipid and carbohydrate. Electron micrographs of the outer sheath reveal a pinstriped motif or transverse stnatlons. The outer sheath is antigenic and immunogenically specifie for spirochetes and may eontain lipopolysaccharides. But no endotQxin activity has been reported for Listerja and Borrelja • (Holt, 1978 ; Strugnell, et al., 1990). The outer sheath can be solubilized into its polygonal subunits with sodium dodecyl sulphate (SOS), myxobacter AL-1 protease, or the detergent 1 Teepol. Reaggregation can be aceomplished with dlvalent and .1 trivalent cations and EDTA (Holt, 1978).

iii. Axial fibril

Axial fibrils are located between thl3 outer sheath and the protoplasmic cylinder. Eaeh axial fibril is attached subterminally to each cell pole extending toward the center of the cell and overlapping. Axial fibrils are morphologically and chemieally similar to other proearyotic flagella except that they are completely endocellular organelles. They are

composed almost entirely of protein. TV'.~ polypeptides of 38 and 35 kDa have reeently been purified from the flagellum of Treponema dentjcola (Cockavne, et al. , 1989).

The axial fibril has a diameter of 15 to 22 nm depending on the spiroehete. It is composed of two components: a shaft and an insertion apparatus. The shaft of the axial fibril is covered with a sheath. The insertion apparatus consists of a terminal knob composed of a proximal hook and insertion dises. The proximal hook is eontinuous with the insertion dises by means of a narrow neck and bends into the protoplasmie cylinder. Axial fibrils are susceptible to hydrogen bond­ breaking compounds, protein denaturants and heating at 60° C (Holt, 1978). Axial fibrils can be removed with Triton X-100 (O.1 %) (Strugnell, et al.,1990).

1 ~ 1 IV. Protoplasmic cylinder

The protoplasmic cylinder consists of a cytoplasmic membrane covered by a cell wall giving rise to a cell wall­ cytoplasmic membrane complex (Holt, 1978). The cytoplasrr'lc membrane is similar to that of Gram-positive and Gram-negative bacteria (Holt, 1978). The cell wall-cytoplasmic membrane

complex contains peptidoglycan which is chemically slmll~r to that of Gram- negative bacteria. Treatment with lysozyme or penicillin leads to the loss of the helical shape of the calI. Hence, the peptidoglycan maintains the helical shape of the ce Il (Canale-Parola. 1978).

III. TAXONOMY OF SPIROCHETES

Spirochetes belong to the order Spirochaetales which is comprised of two families : Spirochaetaceae and Leptospiraceae. Four genera are members of the Spirochaeteceae family Spirochaeta , Cristispira , Borrelia • and Treponema. The family

Leptospiraceae consists of the Leptospjra genu~ (Holt , 1978 ; Canale-Parola, 1978). Differences between the genera are based on characteristics like disease manifestations, animal or vector tropism, serology, morphology under electron microscopy, and DNA homology (Schmid, 1989).

Spirochetes are ubiquitous in nature. They are present in a variety of habitats which include: aquatic environments, the colon of mammals, gingival crevices of humans, the rumen of

7 l cows and sheep as weil as the gut of termites (Canale-Parola, 1978). Sa me spirochetes are pathogenic causing diseases like lyme borreliosis, relapsing fever, leptospirosis, venereal syphilis, yaws and pinta (Schmid, 1989).

i. Borrelia

Borreliae are fastidious, strictly anaerobic or microaerophilic organisms. Borrelja is zoonotic in nature, that is, it has an animal reservoir and is able to infect humans via

direct or indirect contact ~ith the animal reservoir (Schmid , 1989). Ce Ils can be grown at 33° C in BSK medium (Barbour­ Stoenner-Kelly medium ; Kelly, 1971 ; Barbour, 1984 ; and Stoenner, 1974). Growth is detected after three weeks of incubation. Cells possess 4 to 30 irregular coils which are widely spaced in their helical structure. CeUs range from 10 to 30 )lm in length and 0.2 to 0.7 Ilm in width. Cell poles are pointed or tapered. The guanine to cytosine mole ratio ranges from 28 to 30.5 percent.

Borrelja buccaljs is the largest oral spirochete. It is associated with acute necrotic ulcerative gingivitis (ANUG). Cells are 10 ta 30)lm long and possess 10 axial fibrils. Other members of Borrelia include B. recu rrentis and Il. burgdorteri. Borrelia recurrentjs is 10 to 20)lm long and has 12 axial fibrils. It is the etiological agent of relapsing fever. Borrelja burgdorferj is 20 to 30 Ilm long and has 7 ta 11 axial fibnls. It was first isolated by Willy Burgdorferi in 1 1982 trom Ixodes dammjni ticks. r i

l E}Q rre 1ja bu rgdo rfe ri is the causative agent of Lyme disease or Lyme borreliosis which is presently the most common tick-borne disease in the world (Johnson, 1989). Lyme disease is a multisystemic illness that begins wlth a skin rash called erythema chronicum migrans (ECM) at the site of the tick bite. The secondary and tertiary manifestations of the disease involve the heart, nervous system and the joints. Borrelia burgdorferi is transmitted by hard-bodied tick vectors that belong to the Ixodes rjcjnus complex as weil as by hematophagous arthropods (Steere, 1989; Burgdorfen, 1984 ; Anderson, 1989). The most commonly used antibiotics are tetracyclin, erythromycin and ampicillin (Johnson , 1989).

ii. Leptospjra

Leptospjra cells are 0.1 !lm in diameter and 6 to 20 !lm in length possessing tight regular coiling. A single axial fibril is attached at each cell pole extending toward the

center of the oeil. The axial fibrils do not overlap as IS the case in other spirochetes (Holt, 1978). Bromley and Charron

(1979) have reported that the axial fibrils are involved ln the motility of.L.. i nte rrog an s. In translating cells, the anterior end is spiral-shaped, and the posterior end is hook-shaped. In non-translating cells both ends are either hooked or spiral­ shaped (Goldstein and Charron, 1988). Cells are aerobic and grow in bovine serum albumin-Tween 80 medium. Growth can be detected after two to six weeks of incubation (Johnson, J 1989). The guanine to cytosine mole ratio of their DNA ranges from 36 to 39 percent.

Members of this genus are serologically heterologous and contains both saprophytic and pathogenic species. The natural reservoir of the se cells are in the nephritic tubules of rodents as weil as wild and domestic animais. Humans become infected and acquire leptospirosis by direct contact with contaminated urine or indirect contact with waters contaminated with leptospiral urine (Van der Hoeder, 1958). The antibiotics most commonly used against leptospirosis are penicillin and tetracyclin (Johnson, 1989).

iii. Spjrochael.l

Cells are free living in aquatic environments. They are obligate or facultative anaerobes. Cells have a diameter

of 0.2 to 0.75 ~m and a cell length of 5 to 500 !lm. The cell poles tend to be straight with rounded ends. Two axial fibrils are present, one arising from each cell end. The guanine to cytosine mole ratio ranges from 50 to 66 percent (Holt, 1978 Canale-Parola, 1978).

iVe Crjstjspjra

Cells are present in the digestive tract fluid of marine and fresh water molluscs. They range from 0.3 to 3.0 !lm in diameter and 30 to 150 !lm in length. Several hundred axial fibrils are present at each pole. They have never been isolated and their oxygen requirements are 1 unknown.

10 v. Treponema

Treponemes are Gram-negative, strictly anaerobie or microaerophill ie cells; ail are fastldious in culture. They are 0.5 to 0.9 J.lm in diameter and 5 to 20).lm in length. The number of axial fibrils varies fram one to several depending on the species and strain. The cell poles are rounded or tapered. The guanine to cytosine mole ratio of thelr DNA ranges from 37 to 46 percent (Holt, 1978).

80th pathogens and nonpathogens exist within this

genus. The nonpathogens have b~en identifled as part of the normal flora of the oral cavity, genital and intestinal tracts. The pathogenic treponemes are associated with dlseases which include : venereal syphilis (L. pail id u m), yaws (L. ~enue), pinta (1. carateum) and endemic syphilis (1. pallidum endemicum) (Smibert, 1981).

IV. CLASSIFICATION OF ORAL SPIROCHETES

Listgarten and Socransky (1965) differentiated the oral spi rochetes on the basis of the diameter of the protoplasmic cylinder and the number of axial flbrils. Organisms were subscribed into "smalt", "intermediate" and "large" oral spirochetes on the basis of those two cnteria. The small-size oral spirochetes include those that have an 1 endoflagella configuration of "1-2-1" and "2-4-2". The IIJ

diameter of their protoplasmic cylinder ranges tram 100 to 250 nm. Intermediate-size oral spirochetes possess 3 to 20 axial tibrils at each cell pole. The dlameter of the protoplasmic cylinder ranges from 200 to 500 nm. The number of axial fibrils in large-size oral spirochetes ranges from 12 to more than 20. The diameter of the protoplasmic cylinder is approximately 500 nm.

Oral spirochetes that have been reportedly isolated in pure culture include: I.. dentjcoia, 1.. macrode ntjum, ,L. scoliodontum, 1. orale and T. vincentii. Spirochetes of the species 1. dentjcola have been divided into two serovarieties (Cheng, et al., 1985). These treponemes are intermediate-size spirochetes possessing 6 to 10 axial fibrils.

v. THE ISOLATION AND MAINTENANCE OF ORAL SPIROCHETES

Oral spirochetes could be isolated trom other microorganisms in the subgingival plaque by three methods: the membrane filter technique, the weil plate technique and by antibiotics. In the membrane tilter technique, a membrane with a pore size of 0.2 ta 0.45 Jlm is placed on the surface of the agar medium. Other contaminating bacteria are retained on top of the filter because of the smalt pore size (Smibert, 1981). In the weil plate technique, three ta five millimeter deep wells are made into the agar medium and

1... 1 1 the subgingival plaque sam pie is then placed withm the wells. Spirochetes are able to grow and locomote through the agar away from the weil formmg a subsurface growth (Canale-Parola, 1973). This IS due to the abliity of spirochetes to locomote thro:.Jgh VISCOUS milieu whereas other bacteria are restrained wlthin the weil (Canale­ Parola, 1978). Antibiotlcs su ch as polymyxln B, nalidixlc aCld and rifampin can be used to facilitate the Isolation of spirochetes (Flehn, 1984). Leschine and Canale-Parola (1980) also reported the use of rifampin as a selective agent for the isolation of oral spirochetes. Cheng and Chan (1983) reported the isolation of Intermediate-slze sp!rochetes fram periodontal pockets with a combinatlon of the membrane tilter technique and nfampin incorporated mto the agar medium as an effective methad.

Spirochetes could be maintained by subculturing, Iyophilization, freezing in glycerol medium and low tempe rature storage. Oral treponemes isolated could be maintained by monthly transfers mto tresh new oral spirochete medium (NOS) wlth 0 7% Noble agar. Freezlng

0 with glyceral in medium kept at _70 C and Iyophillzatlon of cells in brain heart infusion, fresh serum and glucose have also been successfully tested for long-term maintenance o (Cheng, et al., 1983). Stonng agar stab cultures at 5 Chas also been reported (Canale-Parala, 1973).

l l VI. LOCOMOTION OF SPIROCHETES

Axial fibrils play a role in the motility of spirochetes (Bromley and Charon, 1979 ; Goldstein and Charon, 1988). AXial fibrils Isolated from nonmotile mutants of .L.. !illlli were shown to be stralght and had lost the hook-and­ spiral-shaped ends. On the other hand, axial fibrils of wildtype cells were cOlled and exhibited hook-and-spiral-shaped ends. Revertants to motility regained the hook-and-spiral shaped ends and coiled axial fibrils. Hence, axial fibrils of .6.. illini play a role in locomotion.

Spirochetes exhitJit three types of locomotory behavior: translational motility, rotation about the longitudinal axis and flexing (Canale-Parola, 1978). Insight into the mechanism of motility is still in the early stages. Berg, et al.

(1976) proposed a model for the moti~:ty of splrochetes (Figure 2.). Berg's model proposes that axial fibrils rotate and cause the rotation of the protoplasmic cylinder. Assuming the outer sheath is free to move , the outer sheath rotates in a direction opposite to that of the axial fibril and the protoplasmic cylmder. A change in direction of rotation of

the aXial fibrils results ln a change in direction of the protoplasmic cylmder. As a result, spirochete cells are able

to propel themselves III a screw-like manner like a corkscrew through a cork rotating about their longitudinal axis.

1 Ela.. 2. Schematic representation of a !-lpirochete ce;!. (a), protoplasmic cylinder. (b), axial fibril:3. (c), outer sheath. The rotation of the axial fibrils causes the rotation of the protoplasmic cylinder. The outer sheath rotates in a direction opposite to that of the protoplasmic cylinder.

l 1

f "

1 1 VII. PERIODONTAL DISEASE

i. Clinical Characteristics of Periodontal Disease

Periodontal disease or , as it is eommonly known, pyorrhea, is the major cause of tooth loss in adults (Carranza, 1979). This is a progressive disease, which starts with gingivitis, in which the gingiva become inflamed, swollen and tender. Left uncheeked, the inflammation progresses and the gingivae begin to reeede from the teeth due to degeneration of eollagen fibres and the slaekening of the transseptal and eireumdental bands (Figure 3.). This results in the formation of sulcular poekets which harbor bacteria and pus. Periodontal fibers anehoring the teeth in their soekets weaken and gradually the alveolar bone supporting the teeth is destroyed leading to tooth loss, this is referred to as periodontitis.

Periodontal disease is elassified on the basis of radiographie evidenee of generalized alveolar bone loss, the presence of inflamed gingival tissue, probing depth measurements, as weil as, bleeding upon probing (Lai, 1986). For example, individuals with moderate periodontitis exhibit moderate gingival inflammation, bleeding upon gentle probing, poeket depths of 4 to 6 mm, and early to moderate radiographie evidenee of alveolar bone loss. Individuals with advanced periodontitis exhibit severe gingival inflammation, bleeding upon probing, multiple sites with poeket depths of > 1 6 mm, radiographie evidenee of severe alveolar bone loss 1, 1

flg,. 3. SchematÎc drawing of the periodontium . l

J

17 "1 l and pus discharge (Simonson, 1988). The most destructive form of the disease is chronic adult periodontitis. A relatively common gingival disease, present predominately in young adults, is acute necrotizing ulcerative gingivitis (Vincent's infection). This disease is characterized by "tender and painful gingiva, bleeding on pressure and marginal gingival ulceration and necrosis" (Wintrobe, et al., 1974).

iL Positive Numerical Correlation Between Oral Spirochetes and Periodontal Disease

Oral spirochetes have been implicated as etiological agents of periodontal disease. The healthy gingival crevice is predominanted by Gram-positive microorganisms such as StreptQCOCCUS and Actinomyces (Slots, 1979). Anaerobic spirochetes account for less than 2% of the microscopic flora at healthy gingival sites. The proportion of oral spirochetes increases to 25 to 58% in periodontitis (Listgarten and Levin, 1978). In acute necrotizing ulcerative gingivitis spirochetes comprise about 30% of the microscopie count (Loesche, et al., 1982).

Recurrent investigations have iIIustrated a clearcut difference in the microbial flora of healthy and periodontally diseased sites. A positive quantitative relationship between the percentage of oral spirochetes and the severity of periodontal disease has been observed (Listgarten and Levin, 1 1981 ; Armitage et al., 1982 ; and Listgaten, 1976). The 1 percentage of spirochetes was positively correlated with gingival index, plaque index, gingival exudate measurements, gingival bleeding tendency, connective tissue attachment loss and pocket depth (Armitage, et al., 1982). Chan, et al.(1981) and Lai (1986) also reported a significant Increase in

spirochete COU,lts in patients with periodontitis than ln periodontally healthy patients using fluorescence microscopy. The proportion of spirochetes have been shown to be good predictors of the susceptibility of subjects to periodontal disease (Listgarten and Levin, 1981).

Microscopie stlJdies have revealed that it is small and intermediate-size spirochetes which are found in increased proportions in gingivitis and periodontitis patients (Listgarten and Hellden, 1978). Westergaard and Fiehn (1987) have also reported small-size spirochetes with two endoflagella as the most frequently observed spirochetes in advanced marginal periodontitis .

Recently, Simonson, et al., (1988) reported the first quantitative evidence of a positive relationship between a specifie spirochete strain and periodontal disease. They observed a two- to three-fold increase in T. microdentium from patients at severe disease sites (pocket depth > 6 mm) compared to healthy gingival sites. As a consequence, this microorganism has been suggested to be one of the etiological agents of periodontitis (Moore, et al., 1983).

The association between bacteria and periodontal ,1 disease is not restricted to spirochetes Armitage, et al.

l' 1 (1982) observed a higher percentage of motile bacteria of ail cell types, other than spirochetes in clinically diseased sites. Furthermore, some forms of periodontal disease are associated with weil defined microbiota. Black-pigmented a. gjngjvalis is one species which has also been implicated in chronic adult periodontltis (Loesche, 1985). Hernophjlus (Actjnobacjllus) actjnomycetemcomjtans has been associated with juvenile periodontitis which occurs in young patients (Zarnbon, 1985).

iil. Evidence of Spirochetal Invasion of the Gingival Tissue

The relationship between oral spirochetes and periodontal disease is not simply a numerical one. Spirochetal invasion of the gingival tissue has been observed in periodontitis by a number of investigators. Mikx, et al. (1984) were able to induce artificial periodontitis in beagle dogs by ligature placement resulting in an increase in b 1a c k -P i 9 men te d a. g j n g i y i t i s, li. a sac cha roi Ytic us, B... interrnedjus and spirochetes.

Small and intermediate-size spirochetes were observed to be located in histological sections in "the deeper layers of the subgingival plaque at the tooth side and in the plaque

area facing the sulcular spithelium ft. Spirochetes were also found to be present in destroyed connective tissue and edernatous and necrotic epithelium. Others have also reported the presence of oral spirochetes in the apical 1 surface of the subgingival plaque in direct contact with 20 r l the sulcular epithelium in periodontitis (Newman, 1976).

Recently, Mikx, et al. (1990) were able te induce necrotizing ulcerative gingititis (NUG) in beagle dogs by supplying plaque debris trom NUG carriers to experimental dogs. Coccoidal, red-shaped bacteria and intermediate-size spirochetes were ebserved intercellularly and intracellularly in sulcular gingival epithelium. Interestingly eneugh, the detection of these microerganisms in the epithelial tissue preceded the appearance of the symptoms of NUG. This suggests that oral spirochetes may have a role te play in initiating ulcerative gingivitis.

The appearance of oral spirochetes in the connective tissue (Mikx, et al., 1984) implicates them in the invasion, possibly intra or interepithelially, and their subsequent migration through the basement membrane. Splrochetes may attach to epithelial cells and cause damage through the secretion of enzymes as has been shown by the keratinolytic activity exhibited by 1. dentjcola and 11. gjngjvaljs (Mikx, et al., 1987). Microulcerations in the epithelium could also allow the passage of spirochetes to the base ment membrane (Grenier, 1990). Attachment to the basement membrane mediated by adhesin molecules on the surface of spirochetes a.nd the subsequent degradation of this barrier and access into the connective tissue may play an important role in pathogenic1ty. Dawson, et al. (1990) reported the binding of ail strains of L. dentjcola ,a,b,c,d,e and e' , to human fibronectin in the basement membrane. J .------,

c VIII. PUTATIVE PERIODONTOPATHIC MECHANISMS

i. Toxic Metabolic End Products and Enzymes Released by Oral Spirochetes May Contribute to Periodontal Disease

Subging ival bacteria play a major role in the pathogenieity of periodontal disease althoug h the exact etiology is not known. Oral baeteria may interaet directly, eausing damage to epithelial tissue and inflammation by the secretion of enzymes or toxie metabolic end products ; or indireetly through their antigenic constituents by inducing host-mediated tissue in jury (Listgarten, 1987).

Oral anaerobic spiroehetes actively secrete phosphatidylcholine-hydrolyzing phospholipase C in the gingival erevicular fluid in periodontally diseased sites but not in healthy sites (Siboo, et al., 1989). The release of phospholipase C may contribute to periodontal disease. Phospolipase C is known to hydrolyze membrane phospholipid leading to the release of arachidonic acid metabolites sueh as prostaglandins and leueotrienes. These metabolites may contribute to the Inflammation and alveolar bone loss prevalent in periodontal lesions (Offenbacher, et al., 1986).

A trypsinlike (Ohta, et al., 1986) and a chymotrypsinlike (Uitto, et al., 1988) enzyme have been reeently purified from

, ) 1.. denticola and may be associated with penodontal pathology. The trypsinlike enzyme has been shown to break down arginine and Iysine-containing peptides which are present in subgingival plaque and saliva (Ohta, et al., 1986). The chymotrypsinlike enzyme isolated trom 1. denticola ATCC 35405 is capable vÎ hydrolyzing transferin, fibrinogen, gelatin, immunoglobulins G and A, laminin, fibronectin, type IV collagen and a 1-antitrypsin (Grenier, 1990 ; Uitto, 1988). The ability of the chymotrypsinlike enzyme to degrade basement membrane components may play a role in the dissolution and subsequent invasion of the basement membrane. It is the ability to degrade serum and tissue proteins that may en able the spirachete to ellade host defense mechanisms.

IL-1 has also been implicated in contributing to periodontal disease. IL-1-like activity is present in the crevicular fluid of patients with chronic inflammatory periodontal disease. The release of neutral metalloproteinase, from IL-1 induced chondrocytes, has been shown to degrade the proteoglycan matrix of cartillage and induce calcium release fram bone resulting in bone resorption. IL-1 also stimulates the secretion of collagenase and prostaglandins from macrophages or fibroblasts (Kabashima, et aL, 1990).

Bacteria can also contribute to periodontal dlsease through the production of metabolic end products. Short­ chained fatty acids, namely, propionic, butyric, isobutyric, and isovaleric acids have been detected in gingival tluid from 1 periodontal pockets. These substances have been shown to penetrate the oral mucosa (Siegal, 1977) and their concentration is associated with the severity of periodontal disease (Botta, et aL, 1985). Short-chain fatty acids have been shown to inhibit degranulation of polymorphonuclear leucocyte killing capacity thus affecting host defense mechanisms against bacteria (Tonetti, et aL, 1987). These substances have also been shown to inhibit gingival fibroblast prollf'3ration \I~' .... !!1ger, e t a.,1 1981) . Other bacterial metabolic end products such as ammonia, indole, hydrogen sultide aria polyamines may also potentiate periodontal diseas,EI (Listgarten, 1987).

ii. Inhibition of Cell Proliferation

Other periodontopathic mechanisms have been investigated. These include: the inhibition of fibroblast proliferation in the lamina propria by 1. dentjcola leading to the loss of collagen in the connective tissue (Boehringer, et aL, 1984), the suppression of lymphocyte response by spirochetes (Shenker, et al., 1984) and the inhibition of neutrophil granulocyte function (Taichman, et aL, 1982). The degeneration or loss of collagen fibres in the periodontium may be responsible for the loss of anchorage and loosening of the teeth.

Ali these etiological factors and pathological 1 mechanisms may be interacting together in the periodontium 1 to produce disease (Newman, 1985).

IX. CELLULAR AND HUMORAL IMMUNITY AND PERIODONTAL DISEASE

Cellular and humoral immunity in periodontal disease is evident as it is for most inflammatory dlseases. Polymorphonuclear leucocytes and antibodles are the focus of widespread investigation in periodontal disease. Polymorphonuclear leucocytes are the predominant cell type in the gingival crevice, cornprising up to 47% of the tissue cells, that ma)' play a role in the control of spirochetes. Polymorphonuclear leucocytes empty their Iysosomal contents upon bacterial or antigen-antibody stimulation. The Iysosomal contents have been shown to kill : Streptococcu..s. mutans , Bacterojdes gjngjyaljs, Actjnomyces vjscosus and \leillonella

alcalescens (Wilton, 1982). Phagoc~·tosis by macrophages may also contribute to the control of spirochetes.

Antibody-producing plasma cells and T lymphocytes are present in periodontal lesions. T lymphocytes can be stimulated by bacterial antigen or antigen-antibody complexes to produce lymphokines like macrophage inhibitory factor and osteoclast activating factor. These factors inhibit macrophage migration to the site of tissue in jury and cause alveolar bone resorption (Burnette, et al., 1976). 1

2J l Elevated levels of IgG and IgA to T. vincentii and T. dentjcola are present in adult periodontitis compared to healthy controls (Lai, et aL, 1986 ; Jacob, 1982). A 53 kOa surface antigen on I. dentjcùla may act as an immunogen (Umemoto, et aL, 19l3g). High levels of antibody may bind to

molecules on th~ surface of baeteria and induee the formation of antigen-antibody complexes. These immune complexes '.. an also activate the complement system via the Classical pathway and subsequently induce the release of inflammatory mediators (bradykinin, histamine etc.) leading to the infiltration of more polymorphonuclear cells to the site of injury inevitably causing more tissue damage.

x. A METHOD FOR THE DETECTION OF ORAL BACTfRIA

Besides darkfield microscopy, another method has reeently been developed to deteet oral bacteria in the subgingival plaque called benzoyl-OL-arginine-naphthylamide test or the SANA test. Oral microbes like T. denticola , Il. gjngjvalis, .6.. forsythus and others produce an enzyme that breaks down SANA (Loesche, et aL, 1990). A quick diagnosis of oral anaerobic infection provided by the SANA test may aid the periodontist to manage the disease. 1 MATERIALS AND METHODS

1. BACTERIA

The four oral treponemes used in ail the studies were: Treponema denticola ATCC 354G4, ATCC 35405 were isolated from human subgingival plaque and mamtained in our laboratory (Cheng and Chan, 1983); Treponema vincentij 35580 and Treponema socranskii subspecles socranskii ATCC 35536 were purchased fram the American Type Culture Collection (Rockville, Md.) and maintained in our laboratory as weil (Table 1).

Il. pH MEASUREMENTS

Ali pH measurements were recorded with a Beckman zeramatic pH meter.

III. DEMINERALIZATION AND DISTILLATION OF WATER

Single-distilled water was demineralized by passmg it through a demineralizer (Corning, Model LD-2) and double­ 1 distilled with a water distillation apparatus (Cornmg Madel

• 7 Table1. Morpho log ical Characteristics of Oral Spirochetes

I.dentjcola I. dentjcola 1. vjncentjj I.socranskjj Characteristics ATCC 35405 ATCC 35404 ATCC 35580 ss.socranskjj

Cel! length (J.1m) 7.7 4±O. 94 9. 02±2. 59 ? 5. 45±O. 07

Cel! diameter (j.I.I1\) O.20±O.02 O.22±0.03 0.2 to 0.25 O.15±0.10

Wavelength (~m) 1. 23±0 .15 1.14±O. 09 ? O. 71±0. 01

Amplitude (~m) O. 50±0. 05 O. 49±O. 08 ? 0.3 O±O. 05

No.of Axial fibrils 10 6 4 to 6 2

1 , ,-

1 AG-2).

IV. STANDARD CUlTIVATION CONDITIONS

The oral treponemes employed throughout these studies were grown anaerobically in "new oral spirochete" (NOS) medium devised by Canale-Parola (1980). They were maintained in this basal medium by subculturing monthly.

V. NUTRITIONAl STUDIES

i. Chemical Ingredients

The four spirochetes were grown anaerobically at 350 C in an atmosphere of 85% N2 ' 100/0 H2 and 5% C02 in an anaerobic glove box (Coy Laboratory Products, Ann Arbor, Mich.) in a TY3BS broth medium supplemented with 5% rabbit serum (Ohta, et al., 1986 ; Suzuki and Watanabe, 1989). TYGBS medium had the following composition (in grams pel' liter of distilled water) : trypticase, 10.0; yeast extract, 10.0 and brain heart infusion broth in place of veal heart infusion broth, 5.0. Ali ingredients were purchased from BBl (Becton Dickinson and Company, Cockeysville, M.D.) ; gelatin (Difco laboratories, Detroit, Mich.), 10.0. The salt solution consisted of (grams par liter dist. water) : (NH4)2S04 ' 0.5; K2HP04 ,1.13; KH2P04, 0.9; 1 NaCI, 1.0, ail obtained trom Fisher Scientific (Chemical Manufacturing Division, Fair Lawn, New Jersey); and MgS04.7H20 (BOH, The British Orug House, Toronto) 0.1. The pH of the medium was adjusted to 7.2 with 4N KOH (BDH). The medium was then dispensed into 10ml screw capped test­ tubes and autoclaved at 121 0 C for 20 minutes. It was then allowed ta cool at room temperature before the addition of 5% normal rabbit serum (Flow Laboratories, McLean, Virginia) which was filter sterilized with a Millipore membrane with a pore size of 0.45 flm (Millipore Corporation, Bedford, MA.).

Four consecutive transfers of each of the spirochetes in TYGBS medium was performed to eliminate any carry over from the original inoculum (NOS medium with 0.3% Noble agar). One ml of each of the spirochetal suspensions from the fourth subculture was transferred to 99 ml of the following media: TYGBS broth medium ; complete fluid NOS medium ; fluid NOS medium devoid of volatile fatty acids ; fluid NOS medium with L- .c.-phosphatidylcholine or lecithin (Sigma Chemical Company, St. Louis, MO.) of which 0.33g was dissolved in 10 ml alcohol and 0.2 ml was used per 100 ml medium, in place of volatile fatty acids; fluid NOS medium with polyoxyethylene sorbitan monoleate or Tween 80 (Nutritional Biochemical Corporation, Cleveland, Ohio) of which 2 ml were suspended in 100 ml distilled water and 0.2 ml was used per 100 ml medium in place of volatile fatty acids ; fluid NOS medium with Tween 80 in the absence of normal rabbit serum; and fluid NOS medium with 0- glucuronic acid (Sigma) at 0.2g per 100 ml medium in place 1 of dextrose. l The constituents of the complete NOS medium are (in grams per 100ml of distilled water): brain heart infusion

broth, 1.25; trypticase, 1.0; and yeast ex~ract, 0.25, ail purchased from BBl ; sodium thioglycollate, 0.05 ; l-asparagine, 0.025 and dextrose, 0.2 were purchased from Fisher Scientific ; l-cysteine hydrochloride (BDH), 0.1; Noble agar (Difco), 0.3 for maintenance and 0.7 for isolation of spirochetes. The medium 0 was autoclaved at 121 C for 20 minutes before the addition of the following supplements (mil 100ml medium) : 0.20/0 (wt.lvol.) thiamine pyrophosphate (Nutritional Biochemical Corporation), 0.3 ml; 100/0 sodium bicarbonate (Fisher Scientific), 2.0 ml ; normal rabbit serum (Flow laboratories), 2.0 ml; and a mixture of volatile fatty acids, 0.2 ml. The mixture is made from 0.5 ml of each of the following volatile fatty acids dissolved in 100 ml 0.1 N KOH: isobutyric acid (Fisher Scientific) ; D,l - 2-methylbutyric acid, isovaleric and valerie acids (Eastman Kodak Company, Rochester, N.Y.).

Ali the above media were placed in the anaerobic glove box for a period Of 24 hours before inoculation for a purity check.

ii. pH Reading

The pH of ail the media were recorded before autoclaving and at the end of the incubation period.

1 ,

( iii. Growth Measurements

Two techniques were used to follow the growth of the spirochetes in the different media on a daily basis: optical density readings and direct cell counting.

a. Optical Density

Growth was investigated by pipetting, under anaerobic conditions, 5 ml from each of the cell cultures growing in the different growth media to spectrophotometer tubes. The cell cultures in the speotrophotometer tubes were vortexed and the turbidity measurements were then reoorded with a Gilford Starsar Il SpectrolJhotometer (Gilford Instruments Laboratory Ino., Oberlin, Ohio, U.S.A) at 620 nm.

b. Direct Cell Count

The number of spiroohete oells per ml of growth medium was counted using a Petroff-Hausser counting chamber (C.A Hausser and Son, Philadelphia, U.S.A) under darkfield illumination with a final magnification of SOOX. Very dense suspensions were diluted with 0.85% NaCI solution (Fisher Scientific). A drop of the bacterial sample was then placed onto the ruled area of a clean Petroff-Hausser counting chamber. A glass coverslip was then placed carefully over the drop so as to avoid air bubbles. The counting chamber a was then allowed to stand for 10 minutes to permit the

"/ bacteria to settle into the same focal plane as much as possible.

The counting chamber is ruled into squares that are 1/400 mm 2 in area ; the glass coverslip rests 1ISO mm above the chamber, 50 that the volume over a square is 1/20,000 mm 3 or 1/20,000,000 ml. If, for example, an average of 5 cells are present in each ruled square then there are 5 cells x 20,000,000 ml- 1 or 108 bacteria/ml of medium.

VI. COLLAGEN HYDROLYSIS

A collagen gel matrix at a concentration of 1.5 mg/ml was constructed by gently mixing 0.5 ml of Minimal Essential Medium without glutamine (Flow Laboratory Products, McLean, Virginia, U.S.A) with 4 ml of collagen (rat tait ; Sigma). The pH was adjusted to 7.2 by the addition of sodium bicarbonate. Subsequently, 2 ml of 10X Minimal Essential Medium was incorporated to the collagen solution, thoroughly mixed with a pipette and placed into sterile precipitin o tubes. The precipitin tubes were then incubated at 37 C for polymerization of the gel to commence.

Upon polymerization, the precipitin tubes were incubated in the anaerobic glove box for a period of 24 hours for a purity check. One-tenth ml of midlogaritnmic phase cells of T. denticola ATCC 35404 and ATCC 35405 were 1 placed on the surface of the collagen gel matrrx and r------~

incubated anaerobically. The breakdown of collagen was visually examined after three days incubation.

VII EFFECT OF AGAR DENSITY ON GROWTH

The effect of the density of the medium on growth of I. dentjcola A TCC 35404 and ATCC 35405 was determined by growing the cells in NOS medium with varying concentrations of Noble agar (Difco).

Four-day old cultures were centrifuged at 4,OOOxg for 15 minutes at 25 0 C (Beckmann J2-21 centrifuge). The medium was decanted and the cells were suspended in complete NOS media with : 0.3 , 0.4 , 0.5 , 0.7 , 1.0 , 1.5 , 2.0 , o and 2.5 % (wt.lvol.) of Noble agar '

VIII. ISOLATION OF COLONIES

Isolated colonies of 1. denticola ATCC 35404 and ATCC r

1

1 ! 35405 were obtained in NOS agar medium.

i. Streak and Spread Plate Technique

A 0.7°/0 and a 1°/0 NOS agar medium were prepared and autoclaved at 121° C. The media were cooled to 45°_50° C and 15 ml of each NOS agar medium was poured into 100 x 15 mm Petri plates and allowed to gel for 20 minutes. In the first set of plates containing the 0.7°/0 NOS agar medium , 0.1 ml of the spirochete suspension was pipetted onto the surface and spread using a Pasteur pipette that was constructed into a 'hockey-stick' by flaming. In the second set of plates containing 0.7°/0 NOS agar medium, as weil as plates containing 1% NOS agar medium, a loopful of the bacterial suspension was streaked across the plates using tht;) quadrant method of streaking for the isolation of colonies. Ali plates were incubated anaerobically for two weeks in the glove box at 35°C.

ii. Pour Plate Method

A one-tenth ml bacterial suspension was incorporated into 15 ml of 0.7 0/0 molten NOS agar medium kept in a 45° C water bath container in the anaerobic glove box. The spirochetal cells in the NOS agar medium were vortexed to obtain a thorough distribution of cells in the agar and poured into 100 x 15 mm Petri plates. The agar medium containing the spirochetal cells was then allowed to J solidify.

Ali plates were wrapped in aluminum foil 24 hours following anaerobic incubation to prevent any moisture loss and drying of the agar.

IX. CHEMOTAXIS ASSAYS

i. Test Chemicals

Ali the test chemicals employed in the chemotaxis assays were filter-sterilized with a 0.45 Jlm pore size Millipore filter. The test chemicals used were: sodium acetate, succinic acid, sodium citrate, sodium thioglycolate, isobutyric acid, NaHC03, NaCl, K2HP04, dextrose, and sodium salicylate, ail purchased from Fisher Scientific; sodium pyruvate , MgS04.7H20 , L-cysteine hydrochloride and N- acetylglucosamine were purchased fram BDH; L-methionine, L-fucose, taurine, D+xylose, D+raffinose, lactose, indole, and anthranilic acid obtained from Nutritional Biochemical Corporation ; L-aspartic acid, L-serine, L-proline, and L­ threonine were from Sigma ; collagen was from leN Pharmaceuticals (Cleveland, Ohio) ; and valerie acid, DL-2 dimethylbutyric acid and isovaleric acid were from Eastman Kodak.

1 l ii. Hard Agar Plug Technique

Hard agar plugs containing test chemicals were prepared according to Hugdahl, et aL, (1980) and Tso and Adler (1974) with minor modifications. A 4% Bacto-agar solution was made by dissolving 2 9 of Bacto-agar (Difco) in 50 ml PBS (10mM, pH 7.0). The agar solution was autoclaved and kept in a 70° C water bath. The test chemicals were made by dissolving the individual test chemicals in PBS at two times the required concentration with the exception of collagen which was dissolved in O.SM acetic acid. The pH of the test chemicals was then adjusted to 7.0 with 4N NaOH (BDH) and 1N HCI. The hard agar plugs containing the test chemicals were prepared by adding 8 ml of the testchemical to 8 ml of the 4% Bacto-agar solution. Hard agar plugs containing PBS were also prepared as controls. The solution was then vortexed for 10 seconds and poured into

100 x 15 mm Petri plates ~Fisher Scientific).

The plates containing the hard agar plugs were allowed to solidify at room temperature and refrigerated overnight. Hard agar plugs ware made by using the mouth of a sterile test tube with an inner diameter of 10 mm to cut the agar into circular plugs.

Five-day old cultures of I. denticola ATCC 35404 and ATCC 35405 were counted using a Petroff-Hausser counting chamber. The cells were centrifuged at 4,000 x 9 for 15 minutes at 25° C. Motility of the ce"s was not hampered by centrifugation as observed by darkfield microscopy. The 1 me,jium was decanted and the bacterial ce" concentration was )7 l adjusted to 1x1 09 cells/ml by the addition of 0.4% Noble agar in PBS (10mM, pH 7.0) tempered to 42°C. Approximately 12 ta 15 ml of the bacterial suspension in soft-agar was poured into 100 x 15 mm Petri plates. Hard agar plL.:gs containing the test chemicals were gently placed in a concentric manner into each Petri plate with a sterile toothpick, control plugs were placed at the center of each Petri plate. The plates were then incubated anaerobically and examined for positive or negative chemotaxis at 24 and 48 hours post-incubation by holding the plates against a direct source of light. The distance from the edge of the chemotactic zone to the other edge of the chemotactic zone was measured using a ruler.

iii. Weil Plate Method

Sterilized stainless steel cylinders with an inner diameter of 6 mm and a height of 10 mm were placed with forceps, dipped into 95% alcohol and flamed, in 60x15 mm sm ail Petri plates (Fisher Scientific). In the first group of plates, three cylinders were placed in a linear array equidistant from eachother within the plate. In the second and third group of Petri plates, one stainless steel cylinder was placed in the center of each Petri plate. Then 8 ml of 0.4% Noble agar in PBS (10mM, pH 7.0) kept in a 50° C water bath was pipetted carefully into both groups of plates without disrupting the cylinders. In the third group of plates, 4 ml of 0.8% Noble agar in PBS was added to 4 ml of the test chemical that was dissolved in PBS at two times the required concentration, vortexed and pipetted carefully into small Petri plates. The agar solutions in ail three groups were allowed tc.l solidify at

3 1 room temperature.

Upon solidification of the agar, the stainless steel cylinders were individually removed with sterile forceps trom each Petri plate. As a result, 6 mm size wells were created that could hold the test chemical solution and splrochete suspension.

ln the fi.st group of plates, 0.1 ml of the test chemical in PBS was added to the left weil and 0.1 ml of the control (PBS) was added to the right weil. The plates were anaerobically incubated for twenty-four hours to allow the diffusion of the test-chemica Is.

Four-dt:y old cultures of T. denticola ATCC 35404 and ATCC 35405 were counted using a Petroff-Hausser counting 0 chamber and centrifuged at 4,000 x 9 for 15 minutes at 25 C. The medium was decanted and the cell concentration adjüsted to 10 8 cells Iml with PBS. One-tenth ml of the spirochetal suspension was then pipetted into the wells of ail the plates including those that had been anaerobically incubated for 24 o hours. Ali the plates were then incubated anaerobically at 35 C and examined daily for positive or negativ'3 chemotaxis.

iv. Test-tube Method

The test-tube method for negative chemotaxis was prepared according to Tso and Adler (1974) wlth slight modifications. A 4% Bacto-agar solution in PBS was prepared o ,1 and autoclaved at 121 C. Then 2 ml of the Bacto-agar solution •

was pipetted into 2 ml of the test chemical in 10 mi screw­ capped test-tubes. The Bacto-agar test chemical solution was allowed to solidify at room temperature. The test chemicals employed in this method were those that were found to be repellents for 1. denticola ATCC 35404 and ATCC 35405 by the hard agar plug technique.

The cell concentration of four-day old cell cultures, grown in NOS medium, was determined using a Petroff-Hausser counting chamber. The cells were centrifuged at 4,000 x 9 for 15 minutes at 25° C and the medium was decanted. The cell concentration was adjusted to 108 cells Iml with 0.4% Noble agar in PBS kept in a 42° C water bath or with chemotaxis medium containing phosphate buffer (pH7.0, 10-2 M) and disodium salt ethylene diaminetetracetate (EDTA ; 10-4M). Subsequently, 4 ml of the bacterial suspension in Noble agar was placed into half of the screw-capped test-tubes containing the test chemical and 4 ml of the bacterial suspension in chemotaxis medium was added to the other half of the test- tubes. Ali the test-tubes were allowed ta stand for 10 minutes before they were incubated in the anaerobic glove box at 35°C. The test-tubes were analyzed visually at various times for a period of 10 days for the detection of clearing zones above the Bacto-agar test chemical substratum.

v. Bacteria in Plug Method

Sterilized stainless steel cylinders with an inner 1 diameter of 6 mm and a height of 10 mm were placed 1 ! 1 equidistant from eachother in 60 x 15 mm Petri plates. A 0.8% Noble agar solution in PBS was prepared and autoclaved. The agar solution, kept in a t1SoC water bath, was thoroughly mlxed with an equal volume of tne test chemical at twice the reqUired

concentration. A 0.02 ml volume of the test chemical ln the agar suspension was pipetted into each of the empty stainless steel cylinders except those at the center of each 60x15 mm plate. The test chemical agar suspensions were allowed to solidify at room temperature for 30 minutes.

Four-day old cultules of T. denticola. ATCC 35404 and ATCC 35405 were centrifuged at 4,000 x 9 for 15 minutes at 25 ° C. Th e supernatant was d ecante d an d t h e ce Il s were suspended in 0.4% Noble agar in PBS and pipetted into the central stainless steel cylinder of ail the plates. Upon solidification of the agar, ail the stainless steel cylinders were lifted gently with sterile forceps thus creating plugs containing bacteria surrounded by plugs containining test-chemicals. l'hen, 8 ml of a 0.3% Noble agar in PBS solution, kept in a 42°C water bath, was pipetted around the plugs. Ail the plates were allowed to solidify at room temperature before they were incubated anaerobically in the glove box. The plates were examined bath visually and with an inverted microscope to detect ::lny movement of spirochete cells toward the chemicc:.1 plugs.

vi. Chemical in Pond Method

The chemical in pond method was performed by Tso and Adler (1974) for the chemotaxis of .E.. k.QJl. The technique itself was described by : Mesibov and Adler in 1972, Adler in 1973 and Sment and Konisky in 1989 and further modified in our studies.

A 1 ml solution of sterile PBS (pH7.0, 10mM) was added to sterile 5 ml test-tubes. A 1 ml solution of 0.3% Noble agar in PBS, kept in a 420 C water bath, was also added to another set of sterile 5 ml test-tllbes. Sterile capillary tubed (Kimax, nO.34502 tram Kimble Products, Owens-Illinois, Toledo Ohio, U.S.A) with a size of 0.8-1.10 x100 mm were sealed at one end by holding one end directly into the flame of a bunsen burner

with forceps. Each ca~;lIary tube was then passed over the flame and immersed with open end into the 5 ml test-tubes containing 1 ml PBS or 1 ml 0.3% Noble agar in PBS. As the capillary tubes cooled, liquid was drawn in 1 cm. The capillary tubes were allowed to stand immersed in the solutions for 15 minutes.

Early logarithmi~ phase spirochete cells, namely, 1.. dentjcola ATCC 35404 and ATCC 35405 were again harvested by centrifugation at 4,000 x 9 for 15 minutes. The cells were suspended in 1 ml of the test chemical. The test chemical of choice was L-cysteine hydrochloride because it was shawn ta be a repellent by the hard agar plug technique. 10-5 , 10-4 , 10-3, 10-2, 10-1, and 0.5 M of L-cysteine hydrochloride was prepared in PBS. lhe pH was adjusted to 7.0 with 1N KOH and 1N Hel and filter-sterilized. T"e 1 ml bacterial suspension was pipetted into sterile 5 ml test-tubes. The capillary tubes containing either PBS or 0.3% Noble agar in PBS were then plunged, with 1 forceps, open end down into the 5 ml test-tubes containing the

'" • bacterial suspension and test-chemical. As a control, a capillary tube was immersed into a bacterial suspension of PBS. Ali the test-tubes were anaerobically incubated for 18 hours.

Post-18 hour incubation, the sealed ends of the capillary tubes were broken off gently and rinsed with 1 ml sterile PBS. The number of bacteria entering the capillary was determined using a Petroff-Hausser counting chamber.

X. MOTI LlTY TRACKING

i. Viscous Mil ieu

NOS agar media containing 0 ,0.1 , 0.15 . 0.2 , 0.25 , 0.3 ,

0.35 , 0.4 , 0.5 , and 0.6 % (wt.lvol.) of Noble agar were prepared at twice the required concentration and autoclaved at 121 ° C for 20 minutes. The agar solutions were kept in a 42°C water bath.

ii. Cell Cultures

The following logarithmic phase bacteria were used: 1. dentjcola ATCC 35404 (four-day old culture), six axial fibrils; I. denticola ATCC 35405 (four-day old culture), ten axial fibrils ; I. vjnce.nlli ATCC 35580 (seven-day old culture), four to six axial fibrils; T. §ocranskii ss. socranskji ATCC 35536 (ten-day old culture), two axial fibrils (Cheng and Chan, 1983).

----- iii. Microscopie Method

One ml of each of the cell cultures grown in NOS medium, kept in the anaerobic glove box, was pipetted into each 1 ml NOS agar media concentrations in 5 ml test-tubes resulting in final agar concentrations of 0, 0.1 , 0.15 , 0.2 ,

0.25, 0.3, 0.35 , 0.4 , 0.5 , and 0.6 %. Each cell-agar suspension was vortexed for ten seconds and allowed to equilibrate at room temper3ture for 5 to 10 minutes. One drop (approx. 0.05 ml) of

each cell-agar sus~ension was then placed onto a clean glass slide (Fisher Scientific microscope slide 3" x 1" x 1 mm thick) and carefully covered with a coverslip (Fisher Scientific) to prevent the formation of any air bubbles between the glass and the coverslip. The four corners of the coverslip were then sealed with non-toxic varnish and allowed to dry for a few minutes. The four sides of the coverslip were subsequently sealed as weil with varnish resulting in anaerobiosis. The cells were allowed to settle for 5 minutes before video-taping was commenced. Treponema denticola ATCC 35405 and T. socranskii ss. socransk;; were further incubated for 2 hours on the slide at that NOS agar concentration where the maximum speed was attained to determine wh'3ther their speed decreased with time of incubation. Furthermore, NOS agar concentrations where these two treponemes exhibited the lowest speeds were diluted with fluid NOS medium to determine whether motility could be restored.

iv. Motility Recording

1 Translation motility of the spirochetes was video-taped

,.. , fI r1

1 at a magnification of 400X using a GYYR time-Iapse cassette video recorder and a television monitor connected to a Panasonic videocamera that was mounted on a microscope fltted for darkfield illumination. Different fields were viewed and recorded for each of the four treponemes in each viscosity milieu. Recordings were conducted at an approximate room o temperature of 26 C.

Measurements of time and distance travelled by each spirochete were made by playing back the video recording. The motility tracks of each spirochete cell were reproduced on paper by the method of Pietrantonio, et aL, (1988). The video image of the cell tracks on the monitor was reflected with the help of an illumination lamp (120V) and traced onto paper on a

horizontal surface from a 50 % reflection mirror.

v. Speed Calculation

The speed expressed in micrometers per minute was calculated using the following formula described by Vaituzis and Doetsch (1969): V=( LxE )/T ; L (the actual pathlength traversed by the spirochete in millimeters) x E (micrometer tJquivalent per millimeter as calculated from the enlargement) / T (time of exposure in minutes). The actual pathlength travers6d by the spirochete was determined by subtracting from the total pathlength or trajectory of the organism the cell-Iength of the spirochete (head to tail). For example, if 25 mm on the

photograph is equal to 10 ~m, i.e. 0.4 J..lm/mm at a final magnification of 400X and the actual pathlength traversed by the bacteria is 60 mm for a 3-minute exposure time then the speed of the spirochete is 60 mm x 0.4 J.1m/mm + 3 minutes = 8 J..lm/minute.

vi. Persistence Measurements

Persistence, direct distance/aetual pathlength travelled, of the spirochete cell movement was determined from the projected image from the monitor to paper using a 50 % refleetion mirror.

vii. Viscosity Measurements

A Wells-Brookfield eone/plate microviscometer was used to obtain rheologieal data of our motility medium (Figure 4). The viscometer is made up of a eone-shaped spindle, whieh is an almost fiat dise, a graduated adjusting ring assembly, a sample cup and a rotating torque meter.

The viscometer was supported on a vertical bar with a stand. The following calibration steps were taken for each viseometer reading of our motility media. The spindle was initially screwed onto the upper coupling of the viseometer avoiding cross-threading. The viscorneter dial zero was brought to a center point on the vision glass by starting and stopping the motor. The sample eup was then slipped on around the spindle and supported by the arma The viseometer was started at 12 rpm. The separation between the cone-shaped spindle and the plate (cup) was calibrated by sl.rewing the mierometer adjusting ring by small increments. The arm that supported f the spindle was then swung aside, the eup removed and 1.0 ml r

.E.iQ...4. Schematic representation of a Wells-Brookfield Microviscometer. The viscometer consists of a graduated adjusting ring, a cone-shaped spindle and a sample cup. l

edjustlng rlng

cone-shtlped splndle

stlmple plete/cup

47

~~------r

l of the motility rnedium was inserted into the cup. The cup was remounted around the the spindle and relocked wlth the arm. The speed of rotation was selected and the motor was turned on. The torque dial meter was allowed to rotate five times before the clutch was engaged and the motor turned off and the torque reading measured. This procedure was undertaken for ail our NOS agar samples employed in our motility studies (0, 0.1 , 0.15 , 0.2 . 0.25 ,0.3 , 0.35 , 0.4 , 0.5 , and 0.6 % [wt.lvol,] Noble agar) at the following speeds : 60 , 30 , 12 , 6 , 3, 1.5, 0.6 , and 0.3 rpm.

To determine whether the Wells-Brookfield Viscometer was working properly or not the viscosity of a known substance, namely, n-butyl alcohol (Fisher Scientific) was measured at o 20 C by the sa me method as stated above.

The torque meter senses the resistance the sample medium imposes on the cone spindle rotating on the plate which is a function of the shear stress of the sample. The viscosity was calculated trom the known geometric constants of the cone, the shear stress of the sample, rate of rotation and the stress which is related io the torque (Figure 5).

1

J 1

fUl. 5. Mathematical relationships of the viscosity calculation for non-Newtonian materials. Absolute viscosity calculation for non-Newtonian solutions

Shear stress (dynes/cm2 ) = T 2/3 1tr3

Shear rate (seconds·1) = 21tN/60 sind

Viscosity (eps) = Shear stress x 100 Shear rate r=spindle radius=2.409 cm N=instrument speed (rpm) o d=cone angle = 1 .565 T =% scale reading x full scale torque (dyne-cm.)

4 : RESULTS

1. Growth Studies

Initial growth of the four oral treponemes was established in TYGBS medium supplemented with 5% normal rabbit serum following transfer from complete NOS medium with 0.3% Noble agar. A period of three to four weeks was required to establish growth in TYGBS medium. Growth appeared milky­ white in appearance and cells tended to settle at the bottom of the test-tubes during the early incubation period. After a period of three weeks growth appeared hazy and diffuse.

Nutrition and growth of our four anaerobic treponemes in the different media was followed by optical density readings and direct cell counting. The growth curves for ail the four treponemes are shown in Figures 6a to 6k. The average growth yield at stationary growth in complete NOS medium for : I.dentjcola ATCC 35404 was 1.2x109 cells/ml; I.dent;cola ATCC 35405 was1.5x109 cells/ml; T. socranskii ss. socranskii was 2x108 cells/ml and I..yjncentjj ATCC 35580 was 1.7x108 cells/ml. Tregonema denticola ATCC 35404 and ATCC 35405 took approximately one to one-hait week to reach maximum growth whereas T .vincentii and 1. socranskii took approximately two to four weeks to reach maximum growth, respectively.

Table 2 depicts the growth of the four oral treponemes expressed as percent growth in the following media: complete NOS medium (control) ; fluid NOS medium devoid of volatile fatty

r \ 1

.EiQ..6a. Growth curves of T. denticola ATCC 35404 in : complete

NOS medium 1 NOS devoid of volatile fatty acids, NOS with lecithin in place of volatile fatty acids and TYGBS medium. Fig. 6a. Treponeme denticola AlCC 35404

10~------~

-E 9 -c -:s o -u -8 8 complete Q o TYGBS ..J • lecithin o no volatile fatty acid

o 24 48 72 96 120 144 168 192 216 240 264

lime (hours)

5] Eig,. 6b. Growth curves of I. dentjcola ATCC 35404 in : complete NOS medium , NOS with tween 80 in place of volatile fatty acids in the presence of normal rabbit serum. Fig. 6b. lreponemil dentjcola AlCC 35404

10------~

-E 9

-c -~ o -() 8 ..() œ o ...1 7 El complete -0-- tween 80 transfer 1 --0-- tween 80 transfer 2

6~~~----~~~~~~----~~--~,-~~ o 24487296120144168192216240264288

Tlme (hours)

"

5? t

Elg,. 6c. Growth curves of I. dentjcola AlCC 35404 in : complete NOS medium , and NOS with glucuronic acid in place of glucose. Fig. 6c. Treponema dentjcola ATCC 35404

10,------~

- 9 -E C -::1 0 8 -u -CD U Q 0 ..J 7 • glucuronate El complete

6 0 24 48 72 96 120 144 108 192

Time (hours)

~~~--~- ---- 1

Elg.. 6d. Growth curves of 1. dentjcola ATCC 35405 in : complete NOS medium , NOS devoid of volatile fatty acids , NOS with lecithin in place of volatile fatty acids and TYGBS medium. Fig. 6d. Treponema dentic.2la AlCC 35405 10------

-E - 9 C -::1 o U

-CI) -u 8 ID complete œ o TYGBS ..J 6 • no volatile fatty acid • lecithin 7 0 24 48 72 96 120 144 168 192 216 240 264 288

lime (hours) fig,. 6e. Growth curves of T. denticola ATCC 35405 in: complete NOS medium , NOS with tween 80 in place of volatile fatty acids in the presence of normal rabbit serum and NOS with tween 80 in place of volatile fatty acids devoid of normal rabbit serum. Fig. 6e. Treponema denticola ATCC 35405

10~------~

- 9 -E c El complete -:::1 0 8 --()- u tween 80 transfer 1 --0- tween 80 transfer 2 -CD u a no rabbit serum œ 0 7 ..J

6~~r-~~~-r~~~~~~~-'~-r-r~~~~ o 24 48 72 96 120 144 168 192 216 240 264 288

Time (hours)

1

c:: ,- r f

W. 6f. Growth curves of 1. dentjcola ATCC 35405 in: complete

NOS medium 1 and NOS with glucuronic acid in place of glucose. Fig. 6f. Treponema denticola ATCC 35405

10------~

-E 9

-c -:::s 0 u 8

-CD -u en 0 El complete .... 7 • glucuronate

6~~~_r~~~~~~~~~_r~T_~~~~ o 24 48 72 96 120 144 168 192 216 240 264 288

Tlme (hours)

J Elg,. 69· Growth curves of T. vincen.lli ATCC 35580 in : complete NOS medium , NOS with lecithin in place of volatile fatty acids, NOS devoid of volatile fatty acids , NOS with tween 80 in the presence of normal rabbit serum and TYGBS medium. Fig. 6g. Treponema vincentii ATCC 35580

8.0,------~

7.5 -E 7.0 -c -::a 0 u 6.5 -ca. -u iii complete Q 6.0 -6 TYGBS 0 ...J • lecithin 5.5 0 no volatile fatty acids --0- tween 80 transfer 1 5.0 0 48 96 144 192 240 288 336 384 Tlme (hours)

1 1

Eig,. 6h. Growth curves of T.vjncentii ATCC 35580 in complete

NOS medium 1 and NOS with glucuronic acid in place of glucose. .------•

(

Fig. 6h. Treponema vincentii ATCC 35580

9,------~

- -E C -::J o U - -CI) u a o -J complete • glucuronate

6~--~~-r __~~-r~~_,~~~~~~ o 48 96 144 192 240 288 336 384

Time (hours)

5 ' ri'

.Elg,. 6i. Growth curves of T. vincentii ATCC 35580 in : complete NOS medium and NOS with tween 80 in place of volatile fatty acids in the presence of normal rabbit serum. l

Fig. 6i. Treponem, ~incentii ATCC 35580

9~------

-E - 8 C -:J 0 U

-CD u 7 C) 0 ..J El complete --0- tween 80 transfer 2

6~---T----__-- __-- __--~ __-- __---T--~ o 100 200 300 400

Time (hours)

'). r

.El.g.. 6j. Growth curves of 1.. socran;qkii ss. socranskii in: complete NOS medium , NOS with lecithin in place of volatile fatty acids , NOS devoid of volatile fatty acids and TYGBS medium. ..

Fig. 6j. Treponemp socranskii SS. socranskii

9~------~

-E 8

-c: -:::::a 0 u 7 - -CI» U C El complete 0 -1 6 ta TYGBS • lecithin • no volatile fatty acids 5 0 200 400 600 800

Time (hours)

1

~~~~~~~~ -~--- r 1

1

ElQ.. 6k. Growth curves of T. socranskii ss. socranskii ln : complete NOS medium, NOS with tween 80 in place of volatile fatty acids in the presence of normal rabbit serum. ..

Fig. 6k. Treponeml socranskii ss. socranskii

9~------~

-E 8 -(1) c -~ o u - -G» U 7 oœ ..J complete --

o 200 400 600 800

Time (hours) •

acids ; fluid NOS medium with lecithin in place of volatile fatty acids; fluid NOS medium with Tween 80 in place of volatile fatty acids; fluid NOS medium with Tween 80 devoid of normal rabbit serum ; fluid NOS medium wlth D-glucuromc acid m place of glucose and TYGBS medium.

As can be seen in Table 2, T .denticola ATCC 35404 and 35405 grew moderately in : TYGBS medium with 61.0±2.0 % and

39.5±1.5 % average growth, respectivE1ly ; fluid NOS medium with lecithin with 65.0±7.5 % and 56.0±9.5% average growth, respectively; and fluid NOS medium devoid of volatile fatty acids with 57.0±1.0% and 40.0±16.0oI0 average growth, respectively. Our findings also indicate that the growth yleld in fluid NOS with Tween 80 was very good wlth 84.0±2.0% and 89.5±2.0% average growth, respectively. Growth in NOS medium

with Tween 80 was followed for a second transfer resultmg 10 a slight decrease in growth to 67.5±6.5°10 and 49.0±7 5°/0, respectively. However, NOS medium with Tween 80 devold of rabbit serum could not support the growth of T .denticola ATCC 35405 (Iess than 1 % growth was detected). Optimal growth was attained in fluid NOS medium with glucuronlc acid with 105.5±1 0.5% and 104.7±19.0%, respectively. Glucuronlc acid seemed to support the growth of these spirochetes as weil as in the basal medium (NOS).

Growth of 1. vjncentli ATCC 35580 in fluid NOS medium with lecithin and fluid NOS medium devoid of volatile fatty acids was moderate to low in growth with 48.0±5.0% and 31.0±2.00/o, respectively. Excellent growth was achieved in TYGBS medium 1 with 8B.5±1.5% growth. Optimal growth was attained ln fluid

.- )

------Ta b 1e 2. Comparative Growth of Oral Anaerobie Spirochetes in Growth Media (expressed in % values ±S.D. from cell eounts)

SPIROCHETES

FlUld MedIa 1.. denticola :1:. dent icola :ra yincentii 1:. socranskii ATCC 35404 ATCC 35405 ATCC 35580 sS.socranskii

Complete NOS 100 100 100 100

NOS WI th no volatlle fatty 57.0±1.0 40.0±16.0 31. O±2. 0 16.8±2.1 acids

NOS WI th L- ot-phosphatldyl- 65. 0±7 .5 56. 0±9 .5 48. 0±5. 0 11. 0±1.1 choline

NOS wi th polyoxyethylene sorbltan monoleate Transfer 1 84. O±2 . 0 89. 5±2. 0 111. 5±2. 0 31. O±13. 6 Transfer 2 67. 5±6. 5 49. 0±7 . 5 89. 6±16. 5 6.3±0.8 no rabblt serum ND O. 62±0 . 05 ND ND

NOS WI th glucuronic aCId 105.5±10.5 104.7±19.0 130.7±17.0 137.5±15.0

TYGBS 61.0±2.0 39.5±l.5 88. 5±l. 5 16.7±4.3 NOS medium with Tween 80 with 111.5±2.0 % growth resulting

only in a slight decrease to 89.6±16.5 % growth ln a

consecutive transfer in this medium. Growth was enhanced ln

fluid NOS medium with glucuronic acid with 130. 7±17 0 0/0 growth. None of the media tested cou Id support or influence the growth of 1. socranskjj ss. socraoskii. The average % growth for this species raoged from 6.3 to 31.0 % growth. I. socransk!j reached stationary phase in about four weeks with very low growth yields compared to the other treponemes.

Table 3, depicts the average 0/0 growth from optical density readings of I.dentjcola ATCC 35404, ATCC 35405 and I.vjncentjl ATCC 35580. As can be seen °/0 growth trom optical density readings was correlated to % growth trom cell counts Optical density measurements for T. socra nski! were very difflcult ta read because the cells are so thin and therefore data are not available for comparison.

The final pH of ail the culture media, except for ~. socranskji. at the end of the incubation period were alkal109, ranging in pH values from 7.1 to 7.5. This suggests that glucose and glucuronic acid were not used as carbohydrate fermentative sources. Culture media of T. socranskii ranged in pH values of 6.0 to 6.9.

Il. EFFECT OF AGAR DENSITV ON GROWTH

Growth curves of 1.. dentjcola ATCC 35404 and ATCC

35405 in complete ~OS medium with varying concentrations of 1 Noble agar are depicted in Figures 7a and 7b. The concentrations r------

( I.I.b...l.Jl 3. Comparative Growth of Oral Anaerobie Spiroehetes in Growth Media (expressed in % values ± S.D. from optical density readings at 620 nm)

Spiroehetes

Fluid Medium 1:. dent l CQÙ I,. denticola T. vincentii ATCC 35404 ATCC 35405 ATCC 35580

Complete NOS 100 100 100

NOS wHh no volatile fatty aClds 54. 3±0. 3 29. 5±2 .0 30.3±O.7

NOS wHh L-a­ phosphatldylchollne 60.8±4.2 33 .1±8. 5 37 .1±6.l

NOS wHh polyoxyethylene sorbHan rnonoleate Transfer 1 97 . 7±1. 7 87. 4±3 . 3 101.7±1. 7 Transfer 2 68.6±2.7 33. 9±6. 9 94. 0±14. 0

NOS with glucuromc aCld 96. 8±1. 5 93. O±l. 0 127. 2±11. 2

TYGBS 64.3±8.8 42.6±O.9 80.8±8.4

( f..Ul. 7a. The effect of agar density on the growth of 1. dentjcola ATCC 35405. \

(a) lreponema denticola AlCC 35405

120

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Percent Noble Agar Concentration Eig,. 7b. The effect of agar density on the growth of 1. dentjcola ATCC 35404. (b) Treponema dentjcola ATCC 35404

120

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Percent Noble Agar Concentration

67 of Noble agar incorporated into the medium were 0.3, 0 4, 0.5, 0.7, 1.0, 1.5, 2.0, and 2.5 % (wt.lvol,). Complete NOS medium with 0.3% Noble agar was chosen as the control since this is the agar density employed for the maintenance of oral splrochetes. Growth of both strains of treponemes , in NOS medium, was best at lower concentrations of Noble agar and decreased gradually with higher concentrations of Noble agar. No contammation of these culture media in the spectrophotometric tubes was evident under darkfield microscopy.

III. COLLAGEN BREAKOOWN TEST

No collagen breakdown was observed to occur, iD..ld1rQ., by I. dentjcola ATCC 35404 and ATCC 35405 upon visual examination for the presence of liquefaction of the collagen gel matrices 24 to 72 hours following anaerobic incubation.

IV. ISOLATION OF COLONIES

Isolated colonies of I. dentjcola ATCC 35404 and ATCC 35405 were obtained using the streak plate technique. White. small to medium size colonies with entire edges were observed on the subsurface of the streaked plates contaming complete NOS medium with 0.7% and 1.0% Noble agar two weeks followll1g incubation. The presence of spirochetes was confirmed by picking samples near the edge of many colonies and prepanng wet mounts from individual colonies. Hellcally-shaped spirochetes were observed under darkfield mlcroscopy Ail the colonies on the agar plates appeared morphologlcally slmilar, no contaminants were present. 1 Unexpectedly, no isolated colonies were obtained on the subsurface of the agar in the pour plate technique. This is contrary to the finding of Cheng and Chan (1982) where subsurface growth was detected after 10 days' incubation in rifampin-supplemented molten agar (0.7%).

v. CHEMOTAXIS

i. Hard Agar Plug Technique

A number of carbohydrates, inorganic ions, organic acids, fatty acids, as weil as constituents of the oral cavity and connective tissue, were tested by the hard agar plug technique procedure for their effect on the chemotactic behavior of 1. dentic.wa ATCC 35404 and ATCC 35405 (Table 4). The results were generally similar for both treponemes. For chemoattractants such as NaHC03, K2HP04, dextrose, L­ aspartic acid, lactose and saliva the zones of bacterial accumulation ranged fram 1.4 cm to 2.5 cm. Surrounding the zones of bacterial accumulation around the plugs were faint clear zones or rings. The zone of accumulat!on for chemoattractants like L-fucose, sodium pyruvate, and sodium salicylate was simply a dense or diffuse ring of bacteria around the plug. Othflr chemoattractants like N- acetylglucosamine exhibited a diffuse large zone of bacterial accumulation ranging from 3.8 t.-,l 6.0 cm in 1 diameter. 6; ------~ -~------~ 1

Table 4. Chemotactic response of 1.. denticola ATCC 35404 and ATCC 35405 to a variety of test chemicals.

CHEMOTACTIC RESPONSE OF I·denticola T. denticola ATCC 35404 ATCC 35405 Chemical tested Canen. tested (M) 24hr 48hr 24hr 48hr

Organic acids sodium aC'etate 0.01, 0.1 ° 0 ° 0 sodium pyruvate 0.1, 1.0 ° 0 +,0 - , - succinic acid 0.01,0.1 ° 0 -,0 -,0 sodium citrate 0.01,0.1 0,- 0,- 0 0 anthranilic acid 0.01 ° 0 ND ND

Inorganic ions NaHC03 0.01,0.1 0 0 0,+ NaCl 0.0085,0.085 ° 0 K2 HP04 0.01,0.1 0,+° +,+ +,0° +,0° MgSo4· 7H20 0.01,0.1 ° 0 ° ° PBS 0.01(pH7) ,0.01(pH9) ° ° ° ° Amino acids and analo\lues L-glutamic acid 0.01 ° 0 ND ND L-methionine 0.01 ° ° ° ° L-fucose 0.01,0.1 ° +,0 ° ° L-aspartlc acid 0.01, 0.1 ° 0,+ ----, , L-serine 0.01,0.1 ° ° ° ° L-proline 0.01,0.1 ° 0 ° ° L-threonine 0.01 ° ° ° ° ( L-cysteine hydroch:orlde 0.01

70 cont'd T. denticola T.llimticola ATCC 35404 ATCC 35405 Chemical tested Conc.tested(M) 24hr 48hr 24h1' 48h1' taurine 0.01,0.1 o o o o indole 0.01,0.1 o o -,0 -,0

Carbohydrates and analogues N-acetylglucosarnine 0.01,0.05 0 +,0 +,0 +,0 dextrose 0.01,0.06 0 a 0,+ 0,+ D+xylose 0.065, 0.1, 0.13 0,0,+ 0,0,+ +,O,ND +,O,ND D+raffinose 0.01,0.1 0 0 o 0 b lactose 0.01,0.1 0 0 +,0 +,0

Fatty acids isobutyric acid 0.001,0.01 a o o a valeric acid 0.001 o o o a isovaleric acid 0.001 o o o a DL-2-dimethylbutyric 0.001 o o o o sodium thioglycollate 0.01 o o o a collagen 0.000005, 0.00005 -,0 -,0 -,0 -,0

saliva ? ? ± ±

sodium salicylate 0.01 o o + +

0, no response +, chemoattractant chemorepellent

7 ln negative chemotaxis, faint clear zones were observed around plugs containing sodium citrate, sodium pyruvate, L­ aspartic ac!d, indolR, succinic acid, and collagen ranging from 1.3 cm to 3.3 cm in diameter. The zone of repulsion or clearance around the L-cysteine hydrochloride plug ranged from 1.2 cm to 2.0 cm in diameter which was surrounded by a ring of bacteria driven away or repelled. Most of the chemicals tested elicited no response. The ring of bacterial accumulation initially observed at 24 hours around the sodium pyruvate plug may in fact exemplify the driving away of cells away from the plug at the earliest stages since a distinct zone of clearance or repulsion was observed at 48 hours ranging from 2.5 cm to 3.3 cm in diameter.

Positive and negative chemotactic results were reproducible most of the time for both organisms for the following plugs: L-cysteine hydrochloride, K2 HP 04, sodium salicylate, L-aspartic aCid, collagen, indole, and xylose. However, the size of the zone of accumulation or clearance varied somewhat.

ii. Weil Plate Method

ln this method, a suspension of bacteria was placed within a weil at the center of the Petri plate and the test­ chemical was either placed in a weil or contained within the soft agar throughout the Petri plate. A zone of bacterial diffusion migrating toward the test-chemical in the weil or

72 1 irto the soft agar incorporated with the test-chemical was not .Jpparent in ail the trials attempted. The adherence and the precipitation of the bacterial cells onto the plastic Petri dish was interfering. This method was repeated and modified to try and solve the problem of precipitation and adherence. This was unsuccessfully accomplished by obliterating wells that would serve to hold bacterial cells and instead cells were inoculated directly into the soft agar at the center of the plate. Unfortunately, when the cells were injected into the molten agar they overflowed and spi lied out onto the agar surface.

iii. Test-Tube Method

The test-tube method was used ta demonstrate negative chemotaxis. One would expect bacterial cells to migrate away from the Bacto··agar test-chemical substratum at the bottom of the test tube resulting in a clearing region above the agar. Unfortunately, no zones of clearance were observed for ail the test chemicals. In fact, the spirochetal cells tended to settle at the interface between the Bacto­ agar test chemical substratum and the chemotaxis medium or 0.4% Noble agar in which they were suspended.

iv. Bacteria ~n Plug Method

ln this method hard agar plugs were constructed with bacterial cells and test chemicals embedded in 0.4% agar. A 1 0.3% Noble agar solution was poured around the plugs. Following one week of anaerobic incubation the plates WfJre visually examined for the migration of cells away from the plug toward the adjacent plug containing the test chemical. No movement of cells was observed with the naked eye. Furthermore, the handling of the Petri plates containing the 0.3% Noble agar solution was very difficult because the agar tended to break or dislodge.

V. Chemical in Pond Method

The chemical in pond methoo' was used as a method of detecting negative chemotaxis. A repellent, L-cysteine hydrochloride , as determined by the ,t,ard agar plug technique, was present in a pond of b:l.cteria, and none was put into the capillary. The bacteria enter the capilla\fy for refuge from the repel/ent and the number of bacteria in the capillary was determined. As can be seen in the chemotaxis concentration response curves (Figure Ba and 8b) the threshold value for repulsion, i.e., the lowest concentration of a chemical in the pond giving a response bigger than background (Mesibov and Adler, 1972) for T. denticola ATCC 35404 and ATCC 35405 was 10-4 M of L-cysteine hydrochloride. As the concentration of the repellents in the pond was increased the number of spirochete cells entering the capil/ary was also increased. The number of cells entering the capillary filled with 0.3% Noble agar compared to PBS was greater for 1. dent;cola ATCC 35405. This was not observed with 1. denticola ATCC 35404.

At higher concentrations of 0.5 M there was a decline in the number of spirochete cells entering the capillary. The J peak concentration, i.e., the concentration of a chemical in Elg.. Sa. Chemotaxis concentration response curves of T. denticola ATCC 35405 to L-cysteine hydrochloride as performed by the capillary in pond method. (a). Chemotaxis concentration response curve 8.2

8.0 --0- P8S • 0.3 % Noble agar 7.8 -E

-c 7.6 -::::J 0 Co) 7.4 -CD Co) en 7.2 0 ..J 7.0

6.8 0 Molarity of I-cysteine hydrochloride

75 1

f.lJ;L. 8b. Chemotaxis concentration response curves of 1.. dentjcola ATCC 35404 to L-cysteine hydrochloride as performed by the capillary in pond method. (

(b). Chemotaxis concentration response curve 7.6

7.4 -E -c 7.2 ~ 0 u

Q) u 7.0 en 0 ..J 6.8 -0- PBS

- ....~ 0.3 % Noble agar 6.6 +-_.---r--...------.--...... ,..------I o 10- 5 10- 4 10- 3 10 - 1

Molarlty of I-cysteine hydrochloride

( • the capillary which gives the greatest negative ct.emotactic (esponse for both 1. dentjcola ATCC 35404 and ATCC 15405 was 10-1 M L-cysteine hydrochlonde. It was at thls concentration of repellent where the cells were mast repelled.

When tl1e pond of bacteria contained no chemorepellent, a relatively small number of spirochete cells entered the capillary. In fact, the number of cells entenng the caplliary ln the control (PBS) were less than the threshold. On average 7 8 x 106 cells Iml 1ntered the capillary by random sWlmmmg ln the control situation.

VI. MOTILITY EXPERIMENTS

Video time-Iapse microscopy using darkfield optlCS at

a magnification of 400X was employed ~") monitor the motility of spirochetes in media of dlfferent densltles or Vi3cosities. The motility tracks of the distinctive movement of the individual bacteria were drawn and the speeds and persistence measurements were calculated tram motility tracks.

Table 5 depicts the mean speeds of T. dentlcola ATCC 35404, ATCC 35405, I. vjncentji ATCC 35580 and 1. socranskjl ss. socranskii. The mean speeds of the splrochetes vaned with the differe'lt agar densities used. As can be seen, the greatest rnean speed for l. denticola ATCC 35404 was

achieved between 0.25 to 0.30 % Noble agar (5 1±3.2 ~m/mln.

, 1 Table 5. Average Locomotory Speeds of Spirochetes in NOS Medium of Different Noble Agar Concentrations Caiculated from Motility Tracks

Average speed (J,.lm/min. ± S.D.)

% Noble T.denticola T.denticola T.yincentii T.socrafiskii agar conc. ATCC 35404 ATCC 35405 ATCC 35580 ss.socranskii

(w. Iv.)

0 1. O±O. 5 2.9±0.4 9.9±6.2 5. 7±1. 8 0.10 3. 6±1. 3 6.6±6.7 14.2±6.9 8.4±4.5 0.15 3.6±1.5 10. 5±7 .1 14.2±8.0 10.5±5.7 0.20 1. 8±l. 2 18.7±4.4 9.3±5.0 14.1±7.5 0.2'5 5.1±3.2 11.1±5.1 16.3±7.5 6.7±4.4 0.30 8. 7±2. 0 6.4±2.3 19.5±8.6 15.ü±5.9 0.35 3. 3±1. 8 3. 3±1. 8 41.9±14.9 33.4±13.2 0.40 5.2±2.2 7.3±2.3 32.8±14.2 21.3±6.1 0.50 3.3±2.9 6.7±2.2 11.3±6.2 11.2±5.5 0.60 2.8±2.2 4.5±2.3 3.S±3.3 3.0±2.3 and 8.7±2.0 }lm/min., respectively). The hlghest mean speeds for 1. dentjcola ATCC 35405 was exhiblted at 0 2 to 025 °/0

Noble agar (18.7±4.4 ~!m/min. and 11 1±5 1 pm/mm. respectively). Trepone..m.aS,Qcranskij and l vjncentjl moved most rapidly at agar concentrations of 0.35 to 0 40 0/0

(33.4±13.2 }lm/min. and 41.9±14.9 ~m/min., respectively). Hence, spirochetes were able to travel many times thelr cell

lengths per mir~ul~.

The maxi.~urn mean speeds varied between the I. deotjcola strains. Trepon.e.r:na denticola ATCC 35405 exhibited the greatest me an speed as cûmpared to L. dentjcola ATCC 35404. Furthermore, the concentration of agar at which the greatest mean speed was achieved also varied slightly between the 1. denticola straios. In general, the mean speeds a!so varied greatly between the three species. Trepooema vlOceotll being the faste st moving organism followed by T soc ra n s k Il. Treponema yjncentij exhibited a jerky, erratlc type of locomotory behaviour whereby statiooary periods were followed by abrupt and rapid translational movement.

ln the absence of agar, and at low agar densities minimal translational motility of ail the splrochetes was observed. At agar densities of 0.35% or hlgher mean speeds for the T. denticola.. strains was reduced. At hlgher agar

densities of 0.5 to 0.6 % (wt /vol.) the three spec:es of spirochetes were more or less Immobllized translatlonally within the agar. Other types of movement such as flexlng and undulation were also reduced at these agar densities. Ta test whether the motility apparatus was damaged at these agar 77

densities a 0.6 % Noble agar cell suspension of 1. ~çransk;; was diluted with 1 ml of complete fluid NOS medium. The motility was restored to 6.0±1.6 J.1m/min. At 0.35 % Noble agar, the mean speed of 1. d.a.ntjcola ATCC 35405 was 3.3±1.8 ~m/min. This agar solution was also diluted with 1 ml complete NOS medium and the motility was also restored to

9.6±1.7 ~m/min. Hence, the se agar densities at which low mean speeds were obtained did not damage the motility system in thtt spirochetes.

The persistence of each spirochete species in the different agar densities was also determined. The ranges of persistance of the spirochetes are depicted in Table 6. As can be seen there is a broad range of persistence among the population of cells. To determine whether the broad range of persistence obtained was due to the short period of tracl

The viscosities in centipoises (cP) of the agar densities employed were determined with a Wells-Brookfield Microviscometer. Thtt viscometer measures the viscosity by measuring the force required to rotate a spindle in the material tested. The viscosities in cP at a given shear rate are depicted in Figure 9. A decreasing viscosity with an increasing shear rate is observed with 0.2 , 0.25 , 0.3, 0.35, 0.4, and 0.5 % Noble agar. If the viscosity changes with a change in shear rate then the material is sa id be non-Newtonian. The viscosity of

~~~ --~~--~------Table 6. Range of Persistence of Spirochetes in NOS Medium of Different Noble Agar Concentrations Calculated fro m Motility Tracks

RANGE OF PERSISTENCE

% Noble T.denticola î.denticolg T. yincentii 1:. socranskii agar conc. ATCC 35404 ATCC 35405 ATCC 35580 ss.socraDskii

(w. Iv.)

0 0.52-0.93 0.66-1.00 0.24-1.00 0.68-0.97 0.10 o .l4-0 .96 0.42-0.93 0.45-0.93 0.46-0.98 0.15 0.31-0.87 0.76-1.00 0.43-1.00 0.17-0.88 0.20 0.26-0.98 0.20-1. 00 0.53-0.98 0.83-0.98 0.25 0.09-0.85 0.70-0.95 0.37-1.00 0.43-0.93 0.30 0.25-0.97 0.31-0.95 0.30-1.00 0.52-0.97 0.35 0.35-0.90 0.38-1.00 0.22-0.99 0.38-0.99 0.40 0.31-0.97 0.29-1.00 0.61-0.95 0.45-0.94 0.50 0.39-0.92 0.37-0.90 0.28-0.80 0.58-0.96 0.60 o . 56-0.96 0.10-0.95 0.51-1.00 0.28-0.80

81 1

Ei.g,. 9. Relationship of the viscosity (cP) of NOS medium with varying concentrations of Noble agar with an increasing rate of shear.

, 800

700 El 0% 0.1% 600 • • 0.15% D. u 500 0 0.2% .. 0.25% ..>- 400 • en D 0.3% 0 u .-en 300 * 0.35% > A 0.4% 200 -0- 0.5%

100

0 0 20 40 60 80 100 120

Shear rate( per sec) n,Jn-Newtonian solutions depends on the rate of shear at which they are measured. The shear rate will depend on the speed at which the spindle rotates (Barnes, et al., 1989). At low agar densities viscosity measurements were very small and relatively constant at different shear rates indicating that the material was Newtonian in nature, i.e., the viscosity is independent of shear rate.

8) DISCUSSION

Difficulties have been encountered in growing oral anaerobic spirochetes because very little information is avaiiable on their nutrition al requirements as weil as their metabolism Th€H'efore, nutritional and growth studies were performed on three species of oral treponemes by both cell counting and optical density readings. Su ch information may be useful in interpreting the environmental conditions that allow spirochetes to thrive and proliferate in subgingival areas. Similar results were obtained by both techniques (Tables 2 and

3). Variations in % growth were observed between the three species of treponemes in the different test media.

The deletion of short-chain volatile fatty acids from complete NOS medium resulted in low to mediocre growth yields of ail the fou r treponemes tested namely, 16 .8±2.1 % of growth in complete NOS medium for T. socranskii to 57.0±1.0 % for I. dentjcola ATCC 35404. Hence, our findings indicate that short-chain fatty acids are essential for growth of our oral treponemes and it can also be concluded that these spirochetes are not able to synthesize them. Socransky, et al. (1964) found isobutyrate, a short-chain fatty acid present in our media, to be a growth factor for T. denticola.

The findings concerning volatile fatty acids as essential growth factors are controversial. The deletion of volatile fatty acids from complete NOS medium was shown not to affect the growth of nine oral small-size spirochetes containing ( one endoflagellum from eaeh ce" end (Fiehn, 1989). As a result, fatty acids may not be required for the growth of 1 :2:1 spirochetes. It can be eoncluded that these spirochetes are able to synthesize them. The deletion of short-chain fatty aeids from NOS medium were also not required for the growth of six oral strains of Treponema (on the first transfer in this medium) studied by Cheng and Chan (1983).

Short-chain fatty aeids like those present in NOS medium have been shown to be metabolie by products of oral bacteria like Bactero~ (Rotstein, et aL, 1985). The only fatty acid product released by Treponema has been shawn ta be acetic acid (Cheng and Chan, 1983). Short-chain fatty acids namely, propionie, butyrie, isobuty'ric, isovaleric, and succinie acid have been detected in gingival fluids from periodontal pockets (Tonetti, et al., 1987). Fatty acids have also been shown to penetrate the oral mucosa and inhibit gingival fibroblast and lymphocyte proliferation thereby impairing host defense mechanisms leading the way for bacteriéil invasion. As a result, short-chain fatty acids may have a raie ta play in periodontal disease (Singer and Buckner, 1981). We may postulate that the production of short-chain fatty acids by other oral bacteria may subsequently be used as growth factors by oral treponemes to grow and maintain their infectivity. Alternatively, oral treponemes are selected for because of the content of fatty acids in the diseased sulcular pocket.

Ta determine whether long-chain fatty acids affect the growth of oral treponemes, long-chain fatty acids nameiy, l-o<­ phosphatidylcholine (Iecithin) and polyoxyethylene sorbitan ------

monoeleate (tween 80) were substituted for volatile fatty acids. Tween 80 has been successfully used for the growth of Leptospira (Johnson and Harris, 1967). Tween 80 supported the growth of the two l. dentjcola strains and stimulated the growth T. vincentji. However, % growth of ail three spirochetes was decreased in a second transfer in Tween 80. Lecithin moderately supported the growth of the two l. dentjcola. strains but could not support the growth of l. vincentii. Both long-chain fatty acids were found to Inhibit the growth of l. socranskii. Fiehn (1989) also reported the inhibiton of growth of nine s;mall-sized spirochetes by long­ chain fatty acids. It may be that spirochetes that are inhibited by long-chain fatty acids lack the appropriate enzymatic activity ta degrade these fatty acids into shorter chain fatty acids that would be metabolized and assimilated. Other investigators have found long-chain fatty acids to be essential for the growth of 2:4:2 spirochetes (Fiehn and Westergaard, 1969).

Rabbit serum was found to be essential for the growth of l . dentjcola ATCC 35405. Suzuki and Loesche (1989) have shown rabbit serum to be essential for the growth of l. vjncentji. Besides being a source of long-chain fatty acids, rabbit serum also serves as a source of protein. Serum proteins like human ceruloplasmin, albumin, a-1-antitrypsin, a-1-acid glycoprotein, and a-2-macroglobulin have been shown to stimulate the growth of 1.. dentjcola. In fact, human ceruloplasmin can altogether substitute for rabbit serum. An increase in serum glycoproteins in the gingival exudate in severe periodontitis has been observed (ter Steeg and van Hoeven, 1990). The availability of these growth factors in. Y..im may simply be a selection factor accounting for the preponderance of

spirochetes in the gingival crevice. Alternativel~', these growth factors provide the oral spirochetes with the means for inciting disease.

Glucuronic acid stimulated the growth of ail three treponemes tested. ConsequentlY, it can be substituted for glucose in the NOS medium. Glucuronic acid has also been demonstrated to stimulate the growth of nine oral small-size spiroclietes strains (Fiehn, 1989). Chondroiton and hyaluronic acid, which are present in loose connective tissue and bone, are composed of glucuronic acid units (Burnett, et al., 1989). Hence, the presence of spirochetes in the gingival connective tissue may be due to the accessibility of nutrients like glucuronic acid.

lYGBS medium moderately supported the growth of I. dentjcola AlCC 35404 and 1. vjncentjj ATCC 35580. However, meager growth yields were obtained for I. denticola AlCC 35405 anc1 I. socranskjj. Suzuki and Loesche (1989) reported the growth of 1. denticola AlCC 35405 in TYH medium in the absence of rabbit serum (0.0 reading of 0.063) and a dose related' response to increasing concentrations of rabbit serum up to 5 0/0 (0.0 reading of 0.129). TYGBS medium lacks nutrients which have been shown to be essential for the growth of oral spirochetes, such as, sodium bicarbonate, glucose, and thiamine pyrophosphate (Cheng and Chan, 1983 ; and Austin and Smibert, 1982). The refore , this medium cannot substitute for complete r~os medium.

87 Neither glucose or glucuronic acid were used as fermentative sources as indicated by alkali pH values of 7.1 to 7.5 for 1. denticola strains and 1. yincentji . These findings are similar to those reported by Cheng and Chan (1983). Other carbohydrates substituted for glucose whieh have been shO·Nn not to be fermented inelude mannitol, galactose, celliobose. and maltose (Cheng and Chan, 1983). These spirochetes may utilize amino acids as carbohydrate sources as has been reported for other spiroehetes (Fiehn and Westergaard, 1986). However, slightly acidic pH values of 6.0 to 6.9 were detected for 1.. socranskii. Gas liquid chromatographie analysis would have to be undertaken to determine if fatty acids are produced as fermentative endproducts of carbohydrates by 1. socranskii.

Our findings indicate that both 1. denticola strains do not break down collagen in ri1r..Q.. Collagen fibres, present in the periodontal ligaments, anchor the tooth in the alveolar bone. Hence, collagen itself plays a structural raie. Oral spirochetes may use collageneous fibrils as a source of attachment for their colonization and invasiveness. However, recent investigations with T. denticola ATCC 33520 and strain e have revealed that there was no difference in the binding of these organisms to Type IV collagen and binding to BSA (control) with the exception of 1. dentjcola strain é (Dawson, 1990).

The effect of viscosity on the grawth of twa 1.. de nticola strains, ATCC 35404 and ATCC 35405, was studied. Complete NOS medium with varying concentrations of Noble agar was used. Concentrations of Noble agar incorporat&d into the medium were 0.3 , 0.4 , 0.5 1 0.7 , 1.0 1 1.5 , 2.0 , and 2.5 % (wt.lvol.) . As can be seen in Figures 7a and 7b growth was best at lower concentrations of Noble agar and decreased gradually and slightly with higher concentrations of Noble agar . These findings demonstrate the spirochetes' ability to th rive and fluorish in viscous environments such as the subgingival habitat.

Positive and negative chemotaxis, that is, the movement of organisms towards or away from chemicals was observed more than a century ago by Engelman 1881 and Pfeffer 1888 (TsC' and Adler, 1974). Chemotaxis may be one

of the sev~ral methods governing the interaction of spirochetes with gingival tissue. Allweiss, et al. (1977) microscopically observed chemotactic attraction and repulsion of a number of bacteria at mucosal surfaces sorne of which included ï.LbLi..a.cholera, Salmonella typhjmurjum and

Escherichia~. The chemotaxis by bacteria to intestinal mucosal surfaces may be as important as the chemotaxis by bacteria to gingival tissues in promoting or inhibiting their colonization and invasion into these tissues.

ln the hard agar plug chemotaxis assay performed, the spirochetes responded to the gradient of the test chemical created by the diffusion of the chemical from the hard agar plug into the spirochetal-soft-agar suspension. In the presence of an attractant, spirochetes accumulate around the plug containing the attractant. If the test-chemical is a repellent then the spirochetes are repelled by it and move away from the plug leaving a zone of clearance behind them. The clearing zone ,,. is surrounded by a ring of bacteria that were driven away. As can be seen in Table 4, vanations exist in the chemotactic response between the two strains of 1. dentjcola. N-acetylglucosamine, K2HP04 and D-xylose were observed to be attractants for bath L. denticola strams. L-cystetne hydrochloride and collagen were found ta be repellents for both T. denticola strains. The majority of chemlcals ellcited no response. Whether this phenomenon was due to the mablilty of the cells ta respond or the inadequacy of thls technique for testing for chemataxis of spirochetes is unknown. The fact that some positive and negative chemotactlc responses were observed supports the former hypothesis.

The capillary method or chemical in pond method have been used by a number of Investigators for the study of positive and negative chemotaxis, respectively. 1n the capillary method, a microcapillary containing an attractant IS Immersed in a suspension of cells. Cells which are attracted to the chemical enter the capillary. The chemotactic behavlour of Spjrochaeta aurantja M1 (Greenberg and Konisky, 1989) and Methanococcus voltae (Sment and Konisky, 1989) was studied by this method.

The chemical in pond method was used as a method for detecting the chemotactic response of two T. dentico la strains to L-cysteine hydrochloride which was found to be a repellent by the hard agar plug technique. Chemotactlc concentration response curves are depicted in Figures 8a and 8b The peak concentration for chemotaxis occurred at 10-1 M of L-cysteme hydrochloride for both strains. However, more 1.. denticola ATCC 35405 cells entered the capil!ary containing 0.3 % Noble agar th an the capillary containing PBS. This may be due to their capacity to th rive and locomote in viscous environments

The appearance of oral anaerobic spirochetes in sulcular epithelium implicates them in initiating the pathogenicity of periodontal disease (Mikx, et al., 1984). The motility of spirochetes in viscous envlronments may be considered an invasive factor for the penetration of the gingival tissue. The invasion of the gingival tissue by bacteria may explain the "cyclic course of the disease in which periods of quiescence is followed by phases of exace;bation" (Allenspach-Petrzilka and Guggenheim, 1983). For spirochetal colonization to be most efficient and the pathogenic mechanisms most destructive , cells would have to first bind to host periodontal tissue, invade and interact with sulcular epithelial cells. The binding of spirochetal cells to hast periodontal tissue has been illustrated to occur with the binding of 1. dent;cola to fibronectin (Dawson and Ellen, 1990). The invasion of the periodontium by spirochete cells depends on the ability of the cells to lacomote through viscous environments. In fact, studies on Vibrio cholera have shown that "bacterial interaction with mucosal surfaces is influenced by bacterial motility " (Allweiss, et al., 1977).

We compared the locomotory characteristics of three species of spirochetes using video time-Iapse microscof.,lY under darkfield illumination. Translational mati lity was found to be density dependent. We were able to show increasing locomotory speeds of three species of T re p 0 ne m a with increasing agar densities. Optimal locomotory speeds for the two T. denticola strains occurred at 0.2 to 0.3 % (wt./vol.) of Noble agar. As can bE} seen in Table 5 signlficant differences m speeds were exhibited by the two Treponema strains. Treponema dentjcola ATCC 35405 was the faster of the two strams This suggests a heterogeneity with respect tù their locomotory phenotype. Optimal locomotory speeds for 1. Y.in.centil and 1. socranskii were attained at 0.35 to 0.4 % (wt.lvol.) Noble agar.

These results indlcate (hat spirochetes are able to locomote through viscous environments. The abllity to locomote through viscous environments may confer ecological advantages such as the ability to swim in gingival crevicular fluid and the ability to penetrate sulcular epithelial linings and gingival connective tissue.

The ability of spirochetes, other than oral spirochetes, to locomote through viscous solutions has been demonstrated by other investigators as weil. Lyme disease spirochetes (Borrelia burgdorferi) were observed to exhibit greater locomotory speeds with increa 'inÇJ viscoelasticity. As the viscoelasticlty of the BSK medium increased from 1.3 to 205 cP the mean veloclty of the spirochetes also increased from 1.7 to 34.9 Ilm/sec and decreased when the viscoelasticity reached approxlmately 200 cP (Kims€y and Spielman, 1990). Kaiser and Doetsch, (1975) also reported an increased velocity of Leptospira interrogans. at viscosities exceeding 300 cP (0.35 % to 0.5 % w/v lonagar)

Spirochetes are in fact able to locomote in VISCOUS mlheu that render other prokaryotes immobile. Oral treponemes remain motile in viscosities up to 500-700 cP at a shear rate of

J

- ( 1.1 (j/sec. At an agar density of 0.6% (approx. 1420 cP at a shear rate of Î .16 Isec) treponeme cells were rendered immobile. Other prokaryotes, for example, Spjrjllium serpens, a polarly flagellated bacteria, has been shown to have an optimal speed at a viscosity of 2.5 cP of polyvinylpyrrolidone. At viscosities greater than 2.5 cP its velocity decreases. Bacjllus megaterj um. a peritrichously flagellated bacterium, exhibits its maximum velocity at a viscosity of 4.7 cP and again a rapid decrease in velocity is observed to occur at higher viscosities (Schneider and Doetsch, 1974). Average velocities of f.. ~ and f... aerog;nosa cells have also been shown to decrease with increasing viscosities beyond 2-5 cP. The inability of these bacteria and others to locomote through viscous environments may be hampered by the localization of their flagella. Unlike spirochetal flagella, which are completely endocellular organelles enclosed between the outer sheath and the protoplasmic cylinder, the flagella of other prokaryotes are located externally from the calI. Eubacterial flagella, as opposed to spirochetal flagella, are in direct contact with the external environment which may affect the efficiency of flagellar propulsion rendering them incapacitated in viscous milieu.

The broad range of persistence exemplified by oral spirochetes (Table 6) may be a reflection of the age of the ceHs. ln other words, spirochete cells may exhibit different locomotory behaviour at different stages of division. If this is the case, then the locomotory behaviour at the various stages of growth would have to be monitored. The broad range of persistence observed may also reflect a true heterogeneity, a true phenotype of the cells. This broad range of persistance observed is not uncommon, it was also observed by Pietrantonio, et al., 1988 for seven strains of I. dentjcola.

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