Oral Microbiology Immunology 2002: 17: 321–323 Copyright C Blackwell Munksgaard 2002Printed in Denmark. All rights reserved

ISSN 0902-0055

Short communication

J. Loubinoux,1,2Isolation of the provisionally C. Bisson-Boutelliez,3 N. Miller,3

A. E. Le Faou1

1Laboratoire de Bacteriologie-Virologie, UniteMixte de Recherche 7565 Centre National denamed Desulfovibriola Recherche Scientifique Faculte deMedecine, Vandoeuvre-les-Nancy, France,2Instituto de Tecnologia Quimica e Biologica,fairfieldensis from human Universidade Nova de Lisboa, Oeiras,Portugal, 3Departement de Parodontologie etd’Implantologie, Faculte de ChirurgieDentaire, Nancy, Franceperiodontal pockets

Loubinoux J, Bisson-Boutelliez C, Miller N, Le Faou AE. Isolation of theprovisionally named Desulfovibrio fairfieldensis from human periodontal pockets.Oral Microbiol Immunol 2002: 17: 321–323. C Blackwell Munksgaard, 2002.

Sulfate-reducing bacteria have recently been associated with periodontitis andproposed to play a role in the pathogenesis of this chronic inflammatory process.Eight isolates of sulfate-reducing bacteria belonging to the genus Desulfovibrio Key words: Desulfovibrio fairfieldensis;were obtained from the periodontal pockets of five out of seven patients presenting periodontitis; sulfate-reducing bacteriawith active periodontitis. A multiplex PCR was devised for their identification at

Julien Loubinoux, Service de Microbiologie,the species level. All isolates were identified as Desulfovibrio fairfieldensis, a recently Hopital Hotel Dieu, 1 Place du Parvis Notre-proposed new species. This finding reinforces the suggestion that Desulfovibrio Dame, 75181 Paris Cedex 04, Francefairfieldensis is a human bacterium that may present a pathogenic potential. Accepted for publication February 21, 2002

Periodontitis is a destructive disease ofthe supporting tissues of the teeth,namely the cementum, the periodontalligament, the alveolar bone and to alesser extent the gingiva. The etiologyof this disease is probably multifacto-rial. It involves the oral flora, but alsoenvironmental, genetic and immunefactors. The major symptom is the for-mation of periodontal pockets resultingfrom the loss of tissue attachment.These pockets harbor a complex bac-terial flora, with a gradient of anaerobicgram-negative bacteria, the concen-tration of which increases as the pocketgets deeper (4).

Sulfate-reducing bacteria represent aclass of anaerobic microorganisms thatcarry out dissimilatory sulfate reduc-tion. In this process, sulfate reductionpermits the dissimilative oxidation oforganic matter with the release of hy-drogen sulfide, a corrosive and cyto-toxic compound. Sulfate-reducing bac-

teria are commonly isolated from en-vironmental sources (17), but they arealso present in the digestive tracts(mouth and gut) of animals andhumans (2, 20, 22). As Desulfomonas pi-gra has been reclassified as Desulfovib-rio piger comb. nov. (11), human iso-lates of sulfate-reducing bacteria con-sist mainly of Desulfovibrio species (1,3, 23). So far, three species of Desulfovi-brio have been isolated from humanspecimens: D. desulfuricans, D. fairfiel-densis, and D. piger (10, 12). Recentfindings suggest that sulfate-reducingbacteria may have a role in human dis-eases. These bacteria have been impli-cated in inflammatory bowel diseases(3, 12, 16), and, more recently, have alsobeen associated with periodontitis (5,6). Desulfomicrobium orale and D. fair-fieldensis, a recently proposed new spe-cies (19), have been isolated from peri-odontal pockets (7). In order to eluci-date the roles of these bacteria in this

chronic inflammatory process, we haveundertaken a systematic search for sul-fate-reducing bacteria in the peri-odontal pockets of patients presentingwith periodontitis and attending theDepartment of Periodontology of theUniversity Hospital of Nancy (Lorraineregion, France).

Patients consulting for periodontaldisease, but otherwise healthy, were in-cluded in the study according to the fol-lowing criteria: periodontitis withpocket depths of at least 4mm and ab-sence of any periodontal treatment,either antibiotic therapy during the pre-ceding 4months or current medicationaffecting gingival inflammation. Theclinical status of patients was evaluatedon the basis of pocket depth, bleedingupon probing, gingival recession, toothmobility (Mühlemann’s index) (15),amount of supragingival plaque(plaque index) (18) and gingival in-flammation (gingival index) (9).

322 Loubinoux et al.

Supragingival plaque and saliva werewiped off before bacterial collection. Asubgingival plaque sample was col-lected from the periodontal pocketusing a sterile paper point. After 20s,the paper point was removed from thepocket and immediately transferredinto 1ml of sterile phosphate-bufferedsaline (PBS) buffer. After vortexing, the1-ml sample was inoculated through therubber cap of a ready-to-use liquid cul-ture medium for the detection of sul-fate-reducing bacteria (LabegeA Test-Kits, Compagnie Francaise de Geother-mie, Orleans, France). This mediumsupports the growth of most sulfate-re-ducing bacteria such as Desulfovibriospp., Desulfotomaculum spp., Desulfob-acter spp., and Desulfobulbus spp. Itwas chosen as it is more sensitive thanthe commonly used Postgate’s mediumfor clinical samples (12). All inoculatedmedia were incubated at 37æC for 1month. The presence of sulfate-reduc-ing bacteria was ascertained by the for-mation of a black precipitate (ferroussulfide). Sulfate-reducing bacteria werefurther isolated in Postgate’s solid me-dium E (17).

For identification at the species level,a multiplex PCR was performed. ThisPCR targets the 16S ribosomal RNAgene (16S rDNA) and was devised inorder to identify D. desulfuricans, D.fairfieldensis, and D. piger (12). Briefly,DNA extracts were obtained followingincubation from 500ml of culture me-dium (LabegeA Test-Kits), after centri-fugation and resuspension in Tris HCIEDTA (TE) buffer (10mM Tris HCl, 1mM EDTA, pH 8), by the standard

Table 1. Characteristics of patients with periodontal disease and clinical findings

Patient Sex Age Smoker Tooth SRB1 Pocket Bleeding upon Gingival Mobility Plaque Gingivaldepth (mm) probing recession (mm index index index

1 M 71 no 26 π 7 yes 5 2 3 32 M 37 yes 25 – 7 no 0 2 0 1

44 π 8 yes 1 1 2 13 M 64 no 25 – 8 yes 3 3 3 3

47 – 8 yes 3 2 1 148 – 7 yes 4 1 1 1

4 M 60 yes 13 – 5 no 0 0 3 123 – 10 no 0 3 3 2

5 M 48 yes 16 π 4 no 4 1 1 126 π 5 no 0 0 2 133 – 6 no 0 0 1 1

6 F 24 no 16 π 8 no 0 3 0 222 π 6 no 0 2 1 242 π 9 no 0 3 0 2

7 M 38 yes 37 π 8 no 0 3 1 245 – 6 yes 1 1 0 1

1SRB, isolation of sulfate-reducing bacteria from the periodontal pocket.

phenol-chloroform-isoamyl alcoholmethod. Each 50-ml PCR mixture con-tained 5 ml of DNA extract (approxi-mately 50 ng of DNA) and finalamounts of 0.4mM of each primer, 0.8mM of each deoxynucleoside triphos-phate (Boehringer Mannheim Biochem-icals, Mannheim, Germany), 20mMTris HCl buffer (pH8.4), 1.5mMMgCl2, and 1.5U of Taq DNA poly-merase (Gibco-BRL Life Technologies,Paisley, UK). The primers were 27K-F(5ƒ-CTG CCT TTG ATA CTG CTTAG-3ƒ), 27K-R (5ƒ-GGG CAC CCTCTC GTT TCG GAG A-3ƒ), Essex-F(5ƒ-CTA CGT TGT GCT AAT CAGCAG CGT AC-3ƒ), Fair-F (5ƒ-TGAATG AAC TTT TAG GGG AAAGAC-3ƒ), Pig-F (5ƒ-CTA GGG TGTTCT AAT CAT CAT CCT AC-3ƒ) andP687-R (5ƒ-GAT ATC TAC GGA TTTCAC TCC TAC ACC-3ƒ). All reactionswere carried out using the GeneAmpPCR System 2400 (Perkin-Elmer, Nor-walk, CT). An initial denaturation stepof 94æC for 4min was followed by 30cycles of denaturation (94æC, 1min),annealing (55æC, 1min) and extension(72æC, 2min), with a final extension at72æC for 5min. Negative (water insteadof DNA extract) and positive (D. fair-fieldensis DNA extract) controls wereincluded in each run. Amplified prod-ucts were resolved by electrophoresis in1.5% (w/v) agarose gels containing ethi-dium bromide (1.6mg/ml). A 100-bpDNA ladder was used as a size marker(Gibco-BRL Life Technologies). Theability of the multiplex PCR to identifycorrectly strains of Desulfovibrio wasascertained using four collection strains

(D. piger ATCC 29098T; D. desulfur-icans strain Essex 6 ATCC 29577T andstrain MB ATCC 27774; D. fairfield-ensis ATCC 700045) and 70 clinicalstrains. D. desulfuricans Essex 6 and D.desulfuricans MB were differentiated bythe multiplex PCR as the 16S rDNA se-quences of these strains exhibit a differ-ence of 3% (10). D. piger (primers Pig-F and P687-R), D. desulfuricans Essex 6(Essex-F and P687-R), D. desulfuricansMB (27K-F and 27K-R) and D. fairfi-eldensis (Fair-F and P687-R) were iden-tified by a 255-, 255-, 396- and 534-bpDNA band, respectively. D. piger andD. desulfuricans Essex 6 were furtherdifferentiated by separate PCR assaysusing respective specific primers.

Of the seven patients included in thestudy (six males and one female; meanage 49years, range 24–71years), fivewere found to harbor sulfate-reducingbacteria in eight periodontal pockets(Table 1). Using the LabegeA Test-Kits,growth of sulfate-reducing bacteria wasdetected from 2 to 14days after inocu-lation. All eight isolates were motile, as-accharolytic, gram-negative curvedrods, producing H2S from sulfate, andpositive for the desulfoviridin test.Thus, they possessed the phenotypicproperties of Desulfovibrio spp. (17, 21).They were all identified by PCR as D.fairfieldensis (Fig. 1). There was no cor-relation between the presence of D. fair-fieldensis and any of the clinical par-ameters used to evaluate the severity ofperiodontal lesions.

D. fairfieldensis has previously beenisolated from blood, abdominal ab-scesses, and a urine specimen (8, 10, 13,

Desulfovibrio fairfieldensis in periodontal pockets 323

Fig. 1. Multiplex PCR products obtainedwith four different strains of Desulfovibrio.Lanes 1 and 7: 100-bp DNA ladder (Gibco-BRL Life Technologies); lane 2: negativecontrol (water instead of DNA extract); lane3: D. piger (formerly Desulfomonas pigra)ATCC 29098T; lane 4: D. desulfuricans Essex6 ATCC 29577T; lane 5: D. desulfuricans MBATCC 27774; lane 6: D. fairfieldensis ATCC700045.

19). In these settings, it has been iso-lated mostly in pure culture or as a pre-dominant part of the flora, and maythus possess a higher pathogenic poten-tial than other species of sulfate-reduc-ing bacteria. More recently, D. fairfiel-densis was isolated from 10% (healthyindividuals) to 14% (individuals with in-flammatory bowel diseases) of humanfeces (12). Thus, D. fairfieldensis may bepresent in the human intestinal tract.Periodontitis is usually considered as apolymicrobial infection. The predomi-nant bacteria in periodontal pockets areanaerobic proteolytic and fermentatingbacteria (14). Their metabolic end-products (low-molecular-weight organiccompounds and hydrogen) can be util-ized as electron donors in the anaerobicrespiration of sulfates by sulfate-reduc-ing bacteria. Sulfates can be obtaineddirectly from alimentation or from thebacterial degradation of proteoglycansoriginating from connective tissues.Thus, sulfate-reducing bacteria mustrely on a complex microbiota to obtainthe substrates necessary for their owngrowth. This might explain the associ-ation of these bacteria with advancedcases of periodontitis (6). As a conse-quence, the accumulation of H2S inperiodontal pockets may play a role inthe destructive process. Our results sug-gest that D. fairfieldensis might be oneof the most prevalent species of sulfate-reducing bacteria in periodontalpockets, although Desulfomicrobium or-

ale has also been described (7). D. fair-fieldensis has been isolated only fromhumans and, thus, might be specific tothe human digestive tract. An extensivesurvey is in progress to determine if D.fairfieldensis is a common inhabitant ofperiodontal pockets, if it is found onlyin periodontal pockets in the oral cav-ity, if it constitutes a significant part ofthe periodontal flora, and if it is impli-cated in the onset or in the later stagesof the destructive process.

Acknowledgments

We are indebted to the late Dr Wee Tee(University of Melbourne, Australia)for kindly providing four strains of D.fairfieldensis, including strain ATCC700045. We thank D. Meng and A. M.Carpentier (Laboratoire de Bacteriolo-gie-Virologie, Unite Mixte de Recher-che 7565 Centre National de la Recher-che Scientifique) for their excellent tech-nical assistance.

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