Vol. 57, No. 5INFECTION AND IMMUNITY, May 1989, p. 1380-13830019-9567/89/051380-04$02.00/0Copyright X 1989, American Society for Microbiology

Campylobacter-Wolinella Group Organisms Are the Only OralBacteria That Form Arylsulfatase-Active Colonies on a

Synthetic Indicator MediumC. WYSS

Department of Oral Microbiology and General Immunology, Dental Institute of the University of Zurich,Plattenstrasse 11, CH-8028 Zurich, Switzerland

Received 14 November 1988/Accepted 24 January 1989

Most oral bacteria tested formed colonies on a chemically defined medium with a chromogenic arylsulfatasesubstrate. Arylsulfatase activity was, however, restricted to Campylobacter-Wolinella group organisms,including Wolinella recta, a possible periodontopathogen. W. recta was the only arylsulfatase-active speciesagainst which consistently high levels of antibody were detected in human sera.

In all forms of periodontitis oral bacteria are believed to beinvolved in pathogenesis. However, despite extensive stud-ies, so far no specific pathogen has been identified among thelarge number of bacterial species studied (11). It is possible,therefore, that disease is the result of the action of any oneof a number of species, each able to produce compoundsleading directly or indirectly to the degradation of periodon-tal structures. With this in mind it appeared useful to screensubgingival plaque populations not only for specific bacterialspecies but also directly for potential virulence factors.Arylsulfatases are among the group of compounds whichmight be directly involved in tissue breakdown. Arylsulfa-tases have been demonstrated with numerous different assaysystems in a wide range of bacteria (reviewed in reference 2),but so far no studies relating to oral bacteria have appeared.The arylsulfatase activity demonstrable in crevicular fluid ofinflamed gingiva has been explained by the release from hostcells of lysosomal enzyme (7, 8).The direct labeling of bacterial colonies as a result of

bacterial enzyme activity during development on agar mediadepends on a specific, stable, and nontoxic substrate incor-porated into the growth medium being converted to a non-diffusible, visible, and nontoxic product. Blood agar, themost commonly used medium for primary, nonselectiveisolation of oral bacteria, appeared unpromising for thispurpose, owing to its intense color and high content ofenzymes. In contrast, the synthetic culture media currentlyin development in our laboratory for in vitro models ofmixed populations of oral bacteria are transparent andalmost colorless and support a wide range of facultative andobligate anaerobic oral bacteria. However, like all presentlyavailable culture media, they are to some degree selective,since some species, including Treponema species, are notrecovered. The applicability of this approach has beendemonstrated with the use of the chromogenic beta-galac-tosidase substrate 5-bromo-4-chloro-3-indolyl-,-D-galac-topyranoside to directly differentiate Haemophilus aphro-philus from Actinobacillus actinomycetemcomitans inperiodontal pocket samples selectively growing on bacitra-cin-vancomycin-supplemented synthetic medium (17).

In the present study, a chromogenic arylsulfatase sub-strate was incorporated into a synthetic agar medium todirectly label colonies containing this enzyme. All primaryoral isolates with arylsulfatase activity and obtained in thisway proved to be Campylobacter-Wolinella group organ-

isms. Results with numerous reference strains supported theconclusion that arylsulfatase activity in oral microorganismsmay be restricted to this taxonomic group.

MATERIALS AND METHODS

Bacteria. The origin and maintainance of most laboratorystrains used in this study were as described elsewhere (6,15). All strains preceded by "Be" were kindly provided byS. Edwardson, University of Lund, Malmo, Sweden; allstrains preceded by "OMZ" were from our laboratory.Other sources are indicated in the text. Primary subgingivalplaque samples were obtained by the paper point techniqueand plated with a spiral dilutor in accordance with theroutine procedures of our laboratory (5).

Synthetic indicator medium. The chromogenic arylsulfa-tase substrate 5-bromo-4-chloro-3-indolyl sulfate (X-Sulf;Sigma Chemical Co.) was incorporated (100 mg/liter) into anonselective synthetic medium. This medium consisted oftissue culture medium ZW (16) with the following additions(milligrams per liter): NaHCO3 (300), guanine (10), adenine(10), uracil (10), cytosine (10), thiamine pyrophosphate (5),hemin (0.5), menadione (0.25), ascorbic acid (100), hydro-cortisone (10), N-(2-acetamido)-2-aminoethanesulfonic acidbuffer (3,644), p-aminobenzoic acid (10), spermine hydro-chloride (5), (NH4)2SO4 (500), insulin (1), transferrin (5),sodium fumarate (500), sodium formiate (1,500), potassiumnitrate (250), acetic acid (17), propionic acid (6), n-butyricacid (4), n-valeric acid (1), isovaleric acid (1), isobutyric acid(1), and 2-methylbutyric acid (1). To partially compensatefor the osmotic effects of these additions we reduced theconcentration of KCI to 1,255 mg/liter. This medium (finalpH, 6.9) has previously been used without fumarate, formi-ate, and nitrate to define growth factors for Treponemavincentii (1). Doubly concentrated medium without ascorbicacid, lipids, hemin, menadione, X-Sulf, and NaHCO3 wasstored at 4°C in closed bottles. Plates were prepared bymixing prewarmed medium with stock solutions of themissing ingredients and an equal volume of a precooled 3%solution of Noble agar (Difco Laboratories) and were storedat 4°C in plastic bags in air for up to 14 days. No specialprecautions for the exclusion of oxygen were taken until theinoculated plates were incubated anaerobically (GasPaksystem; BBL Microbiology Systems) at 36°C. Most oralfacultative and obligate anaerobes, with the exception ofTreponema and Bacteroides spp., were capable of colony

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formation on this agar medium within 5 days of incubation(unpublished data; see below).

Gel electrophoresis and Western blotting (immunoblotting).Gel electrophoresis and Western blotting were performed asdescribed elsewhere (17).

Arylsulfatase activity. In addition to the synthetic indicatormedium a more sensitive assay with the fluorogenic sub-strate 4-methylumbelliferyl sulfate (MUS; Sigma) was used.Colonies were transferred to 0.1 ml of a solution ofMUS (0.5mg/ml in saline), and after 5 to 60 min (at room temperaturein air) fluorescence was detected under long-wave UV light.

RESULTS AND DISCUSSION

When aliquots of subgingival plaque samples were platedon the synthetic indicator medium, the green arylsulfatase-active colonies became visible after 3 to 5 days of anaerobicincubation. Color intensity increased on prolonged incuba-tion and even further after 5 to 10 min of exposure to air.Single green colonies were readily detectable even on a lawnof unreactive colonies and were purified by dilution streak-ing on X-Sulf indicator agar.

Surprisingly, with one exception, all arylsulfatase-activecolonies detected in more than 20 subgingival plaque sam-ples on primary plates consisted of gram-negative, highlymotile rods reminiscent of Wolinella and Campylobacterspecies. To test this possibility, we grew a number ofreference strains on the synthetic indicator medium.

Arylsulfatase activity was readily detectable (green colo-nies) in Wolinella recta (Dl3a-g, P13a-g, HG563, HG567,FDC267R, FDC303, and FDC371), W. curva (Bal3a-g andFDC285), Wolinella sp. (H9a-f), Campylobacter sputorumsubsp. bubulus (NCTC 10355), C. concisus (FDC569 andFDC484), and Bacteroides gracilis (FDC1084).No arylsulfatase activity towards the chromogenic sub-

strate X-Sulf was detectable in colonies of W. succinogenes(FDC602W), Eikenella corrodens (OMZ342; obtained fromJ. Carlsson, University of Umea, Umea, Sweden), Seleno-monas sputigena (0MZ317, D19B-28, and 1304), Capnocy-tophaga ochracea (BCS1; obtained from F. A. Gusberti,University of Bern, Bern, Switzerland), Actinobacillus acti-nomycetemcomitans (Y4, ATCC 25923, ATCC 25924, JP2,OMZ346A, 650, and 652), H. aphrophilus (NCTC 55906,HK310, HK315, and OMZ384A), H. paraphrophilus(HK159), Actinomyces viscosus (Nyl, T14V, and Be64), A.israelii (Be26), A. naeslundii (Be84), A. odontolyticus(Be3l), Streptococcus mutans (OMZ176 and NCTC 10494),S. sanguis (OMZ9), S. mitior (OMZ89), S. milleri (58012;obtained from Unilever Research Laboratories), Propioni-bacterium acnes (Be6O), Bifidobacterium eriksonii (Be67),Arachnia propionica (Be25), Peptostreptococcus anaero-bius (VPI 4330-1), Veillonella parvula (ATCC 17745 andOMZ193), Eubacterium alactolyticum (Be48), Fusobacter-ium nucleatum (OMZ274 and FDC364), and Lactobacillusacidophilus (OMZ4).To substantiate the restricted distribution of arylsulfatase

activity in oral bacteria, we used a more sensitive assay withthe fluorogenic substrate MUS. MUS incorporated into agarplates does, however, not allow precise localization ofenzyme-containing colonies, because the fluorescent reac-tion product is diffusible. With this test the above-describedresults were confirmed. In addition, the screening wasextended to bacteria which grow only when the syntheticmedium contains further supplements, including, as the onlynondefined ingredient, 5% fetal calf serum (1). No MUS-degrading arylsulfatase activity was detectable in B. inter-

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FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis of extracts of 8 reference strains and 11 new isolates of oralbacteria with arylsulfatase activity. (A) Acrylamide (7.5%) gelstained with Coomassie brilliant blue. (B) Western blot of a similargel immunolabeled with human serum (1:50) and goat anti-humanimmunoglobulin serum coupled to alkaline phosphatase (1:1,000;Sigma). Lanes: 1, Wolinella sp. H9a-f; 2 to 4, W. recta FDC303,FDC371, and D13a-g, respectively; 5 to 9, OMZ448, OMZ449,OMZ450, OMZ451, and OMZ453, respectively; 10, W. curva Bal3a-g; 11 and 12, OMZ455 and OMZ456, respectively; 13, C. sputorumsubsp. bubulus NCTC 10355; 14, C. concisus FDC484; 15 to 18,OMZ454, OMZ457, OMZ452, and OMZ458, respectively; and 19, B.gracilis FDC1084. Molecular mass markers (kilodaltons) are on theright. The arrow in panel B indicates a group of prominent W.recta-specific antigens (lanes 2 to 8).

medius (OMZ248), B. gingivalis (W83 and OMZ409), B.forsythus (FDC331, ATCC 43037, and OMZ408), T. vincentii(LA1), T. denticola (CD1), and T. pectinovorum (ATCC33768).

Since only a few metabolic characters appear to be usefulin the characterization of Campylobacter-Wolinella grouporganisms (12), we decided to compare the protein andantigen profiles of some of the fresh clinical isolates and anumber of reference strains with arylsulfatase activity afterseparation on sodium dodecyl sulfate-polyacrylamide gels.

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Figure 1A shows the protein patterns of 8 reference strainsand 11 fresh clinical isolates, all with arylsulfatase activity.Isolate OMZ458 (lane 18) was the only clinical isolatedetected on the arylsulfatase indicator agar which wasnonmotile. It did show protein pattern similarity to nonmo-tile, arylsulfatase-active B. gracilis (FDC1084, lane 19) as

well as to the motile isolate OMZ452 (lane 17) and thus couldnot be definitively classified. The motile clinical isolatesOMZ452, OMZ457, and OMZ455 (lanes 17, 16, and 11,respectively) also were not identical to any of the referencestrains, although similarities were apparent. IsolatedOMZ453 (lane 9) appeared sufficiently similar to referencestrain Bal3a-g (lane 10) to suggest that it was identical to W.curva. Similarly, isolate OMZ454 (lane 15) could be classi-fied as C. concisus (reference strain FDC484, lane 14), andisolates OMZ448, OMZ449, OMZ450, OMZ451, andOMZ453 (lanes 5 to 9, respectively) could be classified as W.recta (reference strains FDC303, FDC371, and D13a-g, lanes2 to 4, respectively). Isolate OMZ456 (lane 12) might berelated to C. sputorum subsp. bubulus (reference strainNCTC 10355, lane 13). Reference strain H9a-f (lane 1),classified as Wolinella sp. (15), could not be grouped withany of the reference strains identified to the species level,despite considerable similarities.The evidence from immunolabeled Western blots was

consistent with the taxonomic grouping of the new isolatessuggested from their protein patterns. Figure 1B shows aWestern blot of a gel (as in Fig. 1A) immunolabeled with theserum of a healthy control person (dental hygienist) with no

history of gingivitis or periodontitis. Most striking in theimmunolabeling pattern with this and the 11 other sera tested(from persons with different oral health, including localizedjuvenile and severe adult periodontitis) was the prominentreaction of a group of antigens in the molecular mass range

of 20 kilodaltons (arrow in Fig. 1B) which appeared to becharacteristic of W. recta (lanes 2 to 8). These antigenicbands could correspond to the W. recta-specific yellowbands on silver-stained electropherograms (12).

In addition, the immunolabeling results revealed differ-ences between strains which were not apparent from theprotein pattern comparisons. The same plaque sample oftenyielded different arylsulfatase-active isolates (Fig. 1). Iso-late pairs OMZ448-OMZ449, OMZ451-OMZ452, OMZ453-OMZ454, and OMZ455-OMZ456 were obtained from oneplaque sample each. In particular, three different W. rectastrains were obtained from one patient: OMZ448 andOMZ449 (lanes 5 and 6), isolated from one plaque sample,and OMZ450 (lane 7), isolated from a different site (which, inaddition, contained a W. recta strain indistinguishable fromOMZ449).

In the present study a new method was used to screensubgingival plaque populations for colony-forming bacteriawith arylsulfatase activity. This enzyme activity was de-tected with the chromogenic substrate X-Sulf or the fluoro-genic substrate MUS only in motile W. recta, W. curva, C.concisus, and C. sputorum subsp. bubulus and nonmotile B.gracilis as well as a number of primary isolates which, bymorphological, protein composition, and antigen patterncriteria, were determined to be closely related to the Wo-linella-Campylobacter group. With arylsulfatase activity as acriterion W. succinogenes would be placed into a separategroup, in accordance with the recent genealogical resultsbased on 16S rRNA sequencing which placed this species faroutside the more closely related species C. concisus, C.sputorum (subsp. bubulus?), W. recta, W. curva, and B.gracilis (9, 10). Discrepant grouping would result for E.

corrodens, which here proved to be arylsulfatase negativebut was placed in the Wolinella-Campylobacter group in acomparative study which did, however, not include nucleicacid sequence comparisons (12). Whatever the genetic rela-tionships among these bacteria, arylsulfatase activity mayprove to be a new useful criterion for characterizing thisrather inconspicuous group of organisms. According to theprotein and antigen patterns shown in Fig. 1, this group maybe more heterogeneous than so far recognized. Whetherdifferences within this group also include differences inarylsulfatases remains to be investigated. Detailed informa-tion, including data on substrate specificity and specificactivity, will also be required to evaluate the potentialcontribution of microbial enzymes to arylsulfatase activitydetected in crevicular fluid (7, 8).The present results with a synthetic medium for both the

primary isolation and the further propagation of oral Wo-linella and Campylobacter species show that these bacteriaare not quite as fastidious as previous studies have suggested(4). However, no attempt has been made to define theminimal requirements and optimal growth conditions for thissmall segment of the oral flora. Instead, current efforts areaimed at defining a milieu compatible for the whole range oforganisms forming dental plaque.A number of studies have implicated W. recta as a

periodontopathogen (3, 13), and arylsulfatase might be oneof its virulence factors. However, this enzyme activity wasalso detected in related oral species not found to be associ-ated with periodontitis. A major difference between W. rectaand the other arylsulfatase-active oral species studied hereproved to be the level of specific antibodies in human sera.All 12 sera analyzed, including one from a person with nosigns of gingivitis or periodontitis (Fig. 1B), contained prom-inerit antibody activity against W. recta. This may have beenthe result of regular exposure to enteric W. recta (14). Suchantibodies might contribute to pathogenesis once W. rectahas colonized "ectopically" the gingival crevice.

ACKNOWLEDGMENT

I thank B. Guggenheim for support and comments on the manu-script.

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2-desoxy-a-D-glucopyranosyl-1-phosphat in Kuhmilch alsWachstumsfaktor fur Treponema vincentii. Helv. Chim. Acta71:818-821.

2. Dodgson, K. S., G. F. White, and J. W. Fitzgerald. 1982.Sulfatases of microbial origin. CRC Press, Inc., Cleveland,Ohio.

3. Dzink, J. L., A. C. R. Tanner, A. D. Haffajee, and S. S.Socransky. 1985. Gram negative species associated with activedestructive periodontal lesions. J. Clin. Periodontol. 12:648-659.

4. Gillespie, J., and S. C. Holt. 1987. Growth studies of Wolinellarecta, a gram-negative periodontopathogen. Oral Microbiol.Immunol. 2:105-111.

5. Gmur, R. 1988. Applicability of monoclonal antibodies to quan-titatively monitor subgingival plaque for specific bacteria. OralMicrobiol. Immunol. 3:187-191.

6. Gmur, R., and C. Wyss. 1985. Monoclonal antibodies to char-acterize the antigenic heterogeneity of Bacteroides intermedius,p. 91-119. In A. J. L. Macario and E. Conway de Macario (ed.),Monoclonal antibodies against bacteria, vol. I. Academic Press,Inc., New York.

7. Lamster, I. B., R. I. Vogel, L. J. Hartley, C. A. DeGeorge, andJ. M. Gordon. 1985. Lactate dehydrogenase, ,-glucuronidaseand arylsulfatase activity in gingival crevicular fluid associated

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10. Romaniuk, P. J., B. Zoltowska, T. J. Trust, D. J. Lane, G. J.Olsen, N. R. Pace, and D. A. Stahl. 1987. Campylobacter pylori,the spiral bacterium associated with human gastritis, is not a

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Campylobacter concisus, Bacteroides gracilis, and Eikenellacorrodens by polyacrylamide gel electrophoresis. J. Clin. Mi-crobiol. 24:562-565.

13. Tanner, A. C. R., J. L. Dzink, J. L. Ebersole, and S. S.Socransky. 1987. Wolinella recta, Bacteroides gracilis, andEikenella corrodens from periodontal lesions. J. PeriodontalRes. 22:327-330.

14. Van Dyke, T. E., V. R. Dowell, Jr., S. Offenbacher, W. Snyder,and T. Hersh. 1986. Potential role of microorganisms isolatedfrom periodontal lesions in the pathogenesis of inflammatorybowel disease. Infect. Immun. 53:671-677.

15. Werner-Felmayer, G., B. Guggenheim, and R. Gmur. 1988.Production and characterization of monoclonal antibodiesagainst Bacteroidesforsythus and Wolinella recta. J. Dent. Res.67:548-553.

16. Wyss, C. 1982. Ecdysterone, insulin and fly extract needed forthe proliferation of normal Drosophila cells in defined medium.Exp. Cell Res. 139:297-307.

17. Wyss, C. 1989. Selected low-cohesion variants of Actinobacillusactinomycetemcomitans and Haemophilus aphrophilus lackdistinct antigens recognized by human antibodies. Arch. Micro-biol. 151:131-136.

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