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Oral Microbiology Immunology 2003: 18: 37–44 Copyright C Blackwell Munksgaard 2003Printed in Denmark. All rights reserved

ISSN 0902-0055

W. K. Leung1, J. Y. Y. Yau1, L. J. Jin1,Subgingival microbiota of renal A. W. K. Chan1, F. C. S. Chu1,C. S. P. Tsang1, T. M. Chan2

1Faculty of Dentistry, and 2Division ofNephrology, Department of Medicine, Facultytransplant recipientsof Medicine, The University of Hong Kong,Hong Kong SAR, China

Leung WK, Yau JYY, Jin LJ, Chan AWK, Chu FCS, Tsang CSP, Chan TM.Subgingival microbiota of renal transplant recipients.Oral Microbiol Immunol 2003: 18: 37–44. C Blackwell Munksgaard, 2003.

Renal transplant patients undergoing immunosuppressive therapy may experienceperiodontal side-effects such as gingival overgrowth. This study evaluated thesubgingival microbiota of renal transplant recipients with or without periodontaltissue destruction who may have concurrent gingival enlargement. Subgingivalpaper point samples taken from the deepest probing sites of 38 subjects (one perpatient) were examined using direct microscopy and culture techniques. Acomplex microflora comprising gram-positive and gram-negative cocci, rods andfilaments, fusiforms, curved rods and spirochetes was observed using microscopy.Yeasts were occasionally detected. Significantly higher proportions of gram-positive morphotypes, including gram-positive cocci, were observed in samplesfrom periodontally healthy patients. The predominant cultivable microflora fromanaerobic culture comprised several species of facultative and obligate anaerobes.Colonization of the subgingival sites by ‘foreign’ microbes that are normallydermal, intestinal or vaginal flora was detected in up to 50% of the samples. High Key words: anaerobic bacteria; gingivalmean proportions of lost or unidentified species were also occasionally noted. overgrowth; kidney transplantation;The results showed that the subgingival biofilm of renal transplant recipients with periodontal diseaseschronic periodontitis comprised mainly gram-negative rods and spirochetes.

W. Keung Leung, Faculty of Dentistry, TheBesides the usual predominant cultivable subgingival microbiota associated with University of Hong Kong, Room 3B39, 34periodontitis, the high prevalence of unidentified and ‘foreign’ microbes indicates Hospital Road, Hong Kong SAR, Chinathe possibility of subgingival microbial alteration in renal transplant patients. Accepted for publication July 30, 2002

Renal transplantation offers rehabili-tation of individuals diagnosed withend-stage renal failure (4). Renal re-placement therapy was introduced inHong Kong public hospitals 24yearsago (5). Immunosuppression therapyusing glucocorticoids, azathioprine,cyclosporin, tacrolimus, mycophenolatemofetil, anti-CD3 antibody or anti-lymphocyte globulin has effectively re-duced acute transplant rejection (7, 10,23). Cyclosporin A seems to interfere inthe early phase of T-cell activation.After complexing with cyclophilin,cyclosporin A inhibits the phosphatasecalcineurin which, in turn, blocks cyto-kine gene activation and hence exerts an

immunosuppression effect (35). Themajor drawbacks of cyclosporin A areopportunistic infections, predispositionto lymphoreticular and epidermal ma-lignancies, neuroectodermal problemsincluding gingival overgrowth and mes-enchymal disorders such as hyperten-sion (18). Tacrolimus, another immuno-suppression agent introduced in 1994,also inhibits calcineurin and preventsallograft rejection, but is associatedwith less gingival enlargement (3).

Gingival overgrowth is one of themost important complications for renaltransplant recipients (6, 33). Poorplaque control and pre-transplant and/or concurrent gingival inflammation are

associated with gingival overgrowth inthe renal transplant recipient (28).However, improved oral hygiene alonemay not be sufficient to prevent gingivalovergrowth (28, 29, 38).

The present report is part of a projectfocusing on the oral health status andtreatment needs of post-operative,stable renal transplant recipients inHong Kong. The prevalence of gingivalovergrowth in subjects in the presentstudy was high (53%) (6), comparableto values in reports from other parts ofthe world (12, 33). This study investi-gated the subgingival microflora of re-nal transplant recipients with or with-out periodontal tissue destruction who

38 Leung et al.

may or may not be affected by gingivalenlargement.

Materials and methodsSelection of subjects, study groups andsample sites

From a pool of 60 subjects who partici-pated in an oral health status survey(6), 45 subjects (19 women, age 21–68years) consented to the microbiologicalstudy. They were Chinese patients at-tending for regular review at the Neph-rology Clinic of the Department ofMedicine, Queen Mary Hospital, Fac-ulty of Medicine, The University ofHong Kong. All subjects had received arenal transplant at least 6months be-fore recruitment, were deemed to havestable renal functions and did not re-quire antibiotic prophylaxis for dentalprocedures. Dental histories were takento exclude individuals undergoing reg-ular dental care. Recruited subjects werenot under antimicrobial therapy cur-rently or during the preceding 4-weekperiod. The research protocol was ap-proved by The University of HongKong, Faculty of Dentistry EthicsCommittee, and informed consent wasobtained from participants.

A full-mouth manual periodontal ex-amination was carried out to identifythe subgingival sample site. Standardpanoramic and periapical radiographswere taken to assess the condition of thealveolar bone. The subjects were separ-ated into two groups: (i) subjects with-out radiographic signs of alveolar boneloss, i.e. without periodontal destruc-tion (group A), in whom gingival over-growth was either absent (subgroup A1)or present (subgroup A2); (ii) subjectswith radiographic signs of alveolarbone loss (10%) around at least onetooth per quadrant, i.e. with peri-odontal destruction (group B), inwhom gingival overgrowth was eitherabsent (subgroup B1) or present (sub-group B2).

Subgingival plaque samples were col-lected from the deepest probing sites(one per subject) from all recruits, in-cluding sites with and without gingivalovergrowth. For subjects who had shal-low probing depth measurements (3mm), a labial surface of one of the in-cisors or canines was chosen at random,for convenience, to be the sample site.The samples were used for microscopyand culture study. After sampling, theprobing pocket depths of the sampledsites were measured using an electronic

probe system (Florida ProbeA, FloridaProbe Corporaton, Gainesville, FL) toconfirm the clinical conditions of thesites sampled.

Microbiological sampling

Subgingival microbiological samplingwas performed as described previouslyusing four sterile endodontic paperpoints (medium absorbent paperpoints, DiaDent, Burnaby, Canada), in-serted two at a time to the depth of thesulcus/pocket with a sampling time of20s (16). All paper point samples wereplaced in a single coded bottle contain-ing 3ml of reduced transport fluid. Thebottle was then transported immedi-ately to the laboratory, where the speci-men was processed within 30–60 min byJYYY, who was blind to the clinicaldata and subject grouping.

Direct microscopy

The paper point samples in reducedtransport fluid were dispersed by vor-texing (Autovortex Mixer SA2, StuartScientific, London, UK) at maximumsetting for 15s. A total of 15 ml of thedispersed suspension was transferredand smeared onto a clean glass slide forgram-staining and examination. Thedifferential counts of various microbialmorphotypes were obtained as de-scribed previously using a light micro-scope (Nikon 104, Nikon Corporation,Tokyo, Japan) at ¿1000 magnification(16).

Anaerobic culture

The remainder of the dispersed mi-crobial suspension was serially dilutedand inoculated onto Columbia bloodagar base (Difco Laboratories, Detroit,MI) supplemented with 5% defibrillatedhorse blood, 5 mg/l hemin and 500 mg/lmenadione (CBABS) using a spiralplater (Spiral System, Cincinnati, OH).The plates were incubated for 5–7daysat 37æC in an anaerobic chamber (For-ma Scientific, Marietta, OH) under anatmosphere of 80% N2, 10% H2 and10% CO2. Anaerobiosis of the chamberwas monitored daily using disposableanaerobic indicator strips (BBL, BectonDickinson, MD). After incubation,plates with colonies that were well sep-arated and evenly dispersed were se-lected and a stainless steel template wasused to subdivide the agar surface intosectors with a defined area. The relative

proportions of all different colony mor-photypes were determined. Representa-tive colonies were subcultured as de-scribed by McNabb et al. (19) onCBABS plates to obtain pure cultures.The colony-forming units of these rep-resentative colonies and the dilutionfactor of the inoculum were recordedfor later quantification. All dispersion,plating and subculturing were per-formed on a laboratory bench.

Selective culture of aerobic and facultativeanaerobic gram-negative rods and yeasts

The dispersed microbial suspension wasinoculated by spiral plating onto dupli-cate MacConkey agar and Sabouraud’sdextrose agar (Oxoid, Hampshire, UK)plates and incubated for 18h at 37æC(14, 17). Cultures were examined andthe colony-forming units of representa-tive colonies and the dilution factor ofthe inoculum noted. The aerobic andfacultative anaerobic gram-negative rodand yeast colonies were subcultured ontheir respective culture media to obtainpure isolates.

Identification of isolates

Isolates were identified presumptivelyusing colony morphology, hemolysisand pigmentation, cell morphology,catalase and oxidase tests and the abil-ity to grow in air supplemented with10% CO2 (31). Additional tests for thefacultative anaerobic gram-positivecocci included colony morphology onmitis-salivarius agar (Difco Labora-tories) and characterization using API20 Strep and API Staph kits (AnalyticalProfile Index, BioMerieux, MarcyI’Etoile, France). Additional tests forthe facultative anaerobic gram-negativerods were glucose fermentation andmotility in a semi-solid medium (31).Facultative anaerobic gram-negativefusiforms were further characterized asper Koneman et al. (13). Obligate an-aerobes were characterized using therapid ANAII system (Innovative Diag-nostic Systems, Norcross, GA) and mo-tility in a semi-solid medium (31). Thecorresponding quantitative/percentageproportion data (colony-forming unitsper paper point/percentage proportionof the identified species per total countof the sample) of individual species iso-lated were then calculated.

Facultative anaerobic gran-negativerods isolates were further characterizedbiochemically using the API 20E kit.

Subgingival flora post-renal transplant 39

Table 1. Demographic data, post-transplant duration and immunosuppressant usage of subjects

Transplant donor

Living Immunosuppressant used

Years post- Not Geneti- Cyclo-n Age range transplant genetically cally sporin

Group (% female) (mean ∫ SD) (mean ∫ SD) Cadaveric related related A Tacrolimus None

A (subjects without periodontal destruction)Without gingival overgrowth (A1) 4 (50.0) 30–41 0.5–5.6 3 1 0 3 1 0

(35.4 ∫ 4.6) (2.5 ∫ 2.3)With gingival overgrowth (A2) 7 (42.9) 21–68 0.5–9.6 5 1 1 6 1 0

(38.3 ∫ 14.7) (3.7 ∫ 3.6)Total 11 (45.5) 21–68 0.5–9.6 8 2 1 9 2 0

(37.2 ∫ 11.8)a (3.3 ∫ 3.1)

B (subjects with periodontal destruction)Without gingival overgrowth (B1) 16 (43.8) 35–64 0.5–11.6 13 2 1 13 2 1

(44.6 ∫ 7.6) (5.2 π3.7)With gingival overgrowth (B2) 11 (36.4) 31–67 1.1–12.5 8 0 3 11 0 0

(46.3 ∫ 10.8) (5.0 ∫ 3.4)Total 27 (40.7) 31–67 0.5–12.5 21 2 4 24 2 1

(45.3 ∫ 8.9) (5.1 ∫ 3.5)aSignificant difference from subjects with periodontal destruction (P Ω 0.027, analysis of variance).

Table 2. Sampling sitesa

Group A Group B

Without gingival With gingival Total Without gingival With gingival TotalSample sites overgrowth (A1) overgrowth (A2) overgrowth (B1) overgrowth (B2)

Incisors/canines 4 2 6 4 6 10Premolars 0 2 2 1 1 2Molars 0 3 3 11 4 15Probing depthb (mm) 2.5 ∫ 0.5 6.2 ∫ 0.6 4.6 ∫ 2.0 5.3 ∫ 1.1 6.6 ∫ 1.9 6.0 ∫ 1.7aGroup A Ω subjects without periodontal destruction; Group B Ω subjects with periodontal destruction.bMeasured with the Florida Probe System.

Pure cultures of yeasts were identifiedbased on colony morphology, cell mor-phology, gram staining reaction, thegerm tube test (14) and the API 20CAUX and API ZYM tests.

Statistics

The demographic and microbiologicaldata of groups A and B were analyzed by 4.5 for Macintosh (SAS Insti-tute, Cary, NC). Differences betweengroups were tested by analysis of vari-ance (group A against group B) orFisher’s exact test, as appropriate. Thebacteriological data for the four sub-groups according to the presence/ab-sence of periodontal destruction and thepresence/absence of concurrent gingivalovergrowth, i.e. subgroups A1, A2, B1,and B2, were analyzed using Bonferroni’smultiple comparison. Groups or sub-groups were regarded as significantly dif-ferent from each other if P was 0.05.

ResultsClinical findings

Of the 45 individuals who consentedand were invited to participate in thestudy, 38 were eligible (Table 1). Fivesubjects were rejected because they wereeither under regular dental care or weretaking antibiotics. Two subjects, whosedeepest probing sites were associatedwith impacted mandibular third mo-lars, were excluded. No recruits had re-ceived more than emergency dentaltreatment during the preceding 24months (6). Relevant periodontal dataare shown in Table2. Moderate peri-odontal problems inclusive of gingivalovergrowth were diagnosed in 89% ofthe subjects. Sixty-four per cent (nΩ7)of group A subjects were affected bygingival overgrowth while 41% (nΩ11)of group B subjects also displayed gin-gival overgrowth with various levels ofperiodontal destruction.

Direct microscopy

As shown in Table3, a total of nine mi-crobial morphotypes were recognized.Gram-negative rods were observed inall samples while the prevalences ofgram-negative cocci, curved rods andspirochetes were more than 80%. Preva-lences of gram-positive cocci and fusi-forms were more than 60%. Gram-negative filaments were observed inabout 50% of the samples, while theprevalences of gram-positive rods ingroups A and B were 32 and 63%, re-spectively. The prevalence of gram-posi-tive coccal morphotypes as well as theproportions of total gram-positive spe-cies were significantly higher in group Athan in group B. The proportions ofother microorganisms were similar inthe two groups.

When groups A or B were furthersubdivided according to presence or ab-sence of gingival overgrowth, group A

40 Leung et al.

Table 3. Differential cell counts from gram-stained smearsa

Group A Group B Pb

Gram-positive microorganismsc

Cocci 0–50.5 (1.5, 21.1)d 0–27.5 (0, 3.2) 0.012Rods 0–10.5 (0, 2.2) 0–24 (0, 2.0) NSTotal 0–56.5 (1.5, 17.6) 0–51.5 (0.5, 5.2) 0.045Gram-negative microorganismsCocci 0–11.5 (4, 4.9) 0–17.5 (2.5, 4.7) NSRods 25–86.5 (53.5, 57.9) 22–88.5 (60.5, 58.6) NSFusiforms 0–7.0 (1, 1.9) 0–2.5 (1.5, 1.4) NSCurved rods 0–4.5 (1.5, 1.9) 0–60 (2.0, 2.4) NSFilaments 0–1.5 (0, 1.1) 0–26.5 (0.5, 1.6) NSSpirochetes 0–44 (11, 14.8) 0–66.5 (24, 26.1) NSTotal 43–100 (98.5, 82.4) 48.5–100 (99.5, 94.7) NSFungie

Yeast form 0 (0, 0) 0–20 (0, 0.1) NSaGroup A Ω subjects without periodontal destruction; Group B Ω subjects with periodontal destruction.bAnalysis of variance. NS Ω not significant.cNo gram-positive filaments observable.dData shown are percentage range, with median and mean in parentheses.eNo mycelial form observable.

subjects without any gingival over-growth (subgroup A1) were found tohave significantly greater percentageproportion of gram-positive cocci (33.3∫22.2%) than group A subjects withgingival overgrowth (subgroup A2; 5.3∫13.1%), and group B subjects without(subgroup B1; 11.2∫14.5%) or with(subgroup B2; 0.0∫0.0%) gingivalovergrowth (PΩ0.003, Bonferronimultiple comparison). Samples fromsubgroup A1 subjects were also foundto comprise a smaller proportion ofcurved rods (0.9∫0.8%) than thosefrom subgroup B1 subjects (13.7∫12.7%; subgroup A2 Ω22.1∫16.4%;subgroup B2 Ω3.3∫1.1%, PΩ0.029,Bonferroni multiple comparison). Over-all, samples from subgroup A1 hadgreater proportions of total gram-posi-tive species (37.6∫26.1% vs. subgroupA2 Ω6.3∫15.6%; subgroup B1 Ω13.8∫18.6% or subgroup B2 Ω0.5∫0.7%, PΩ0.008, Bonferroni multiple com-parison).

Anaerobic culture

Following purification, a total of 279isolates were obtained from the 38samples. The total numbers of isolatesobtained from the two groups weresimilar, as were the numbers of ident-ifiable species (group AΩ6.5; groupBΩ7.7). Fifty-six isolates, however,were lost or could not be identified.

Marked sample-to-sample variationswere observed in the two groups regard-ing the total quantity of predominantcultivable facultative and obligate an-

aerobic bacteria. The mean colony-forming units/paper point for groups Aand B were 2.4¿107 ∫3.9¿107 and 5.5¿107 ∫9.0¿107, respectively. No sig-nificant difference was detectable intotal quantity of microbes isolated ortheir subcategories between groups Aand B, because of the large data ranges.When data were transformed into per-centage proportions, differences in pro-portions could be measured (Fig.1). Upto 50% of the isolates in the groupswere facultative anaerobes, of which agreater proportion of gram-positivecocci were observed in group A. Ahigher proportion of total gram-posi-tive species and hence, conversely, alower proportion of total gram-negativespecies were observed in group A thanin group B (i.e., for gram-positive spe-cies, group A vs. B: 82.6∫20.1% vs.52.6∫27.2%, PΩ0.002; for gram-nega-tive species, group A vs. B: 19.1∫12.1% vs. 50.4∫37.3%, PΩ0.01). Thetotal proportions of gram-positive cocciand gram-positive facultative anaerobicspecies were higher in group A than ingroup B (47.3∫27.2% vs. 24.8∫21.3%,PΩ0.010 and 47.7∫26.2% vs. 28.5∫20.0%, PΩ0.019, respectively). Up to25% of the isolates were lost beforebeing tested (Fig.1). When the group Aand B data were subdivided with re-spect to the absence or presence of gin-gival overgrowth, a greater proportionof facultative anaerobic gram-positivecocci were found in samples from sub-group A1 subjects than in samples fromsubgroup B1 subjects (A1 Ω45.4∫31.8% vs. A2 Ω26.2∫23.7%, B1 Ω16.2

∫12.1% or B2 Ω16.8∫13.2%, PΩ0.036, Bonferroni multiple com-parison).

A total of 68 different organismswere identified, comprising facultativeor obligatory anaerobic gram-positivecocci (23 species), gram-positive rods(16 species), gram-negative cocci (onespecies), gram-negative rods (19 spe-cies), gram-negative fusiforms (sevenspecies) and gram-negative motile rods(two species). The frequently isolatedbacterial species (defined here as havinga frequency isolation 15%, nΩ20) areshown in Table4. Streptococcus constel-latus was more prevalent in group Aand its percentage proportion, togetherwith that of Streptococcus salivarius sal-ivarius, was also higher. When datawere grouped under genera, the percen-tage proportions of total Staphylococ-cus, Streptococcus and Porphyromonasspecies were significantly higher ingroup A than in group B (9.1∫15.7%vs. 1.4∫4.7%, 18.1∫18.2% vs. 4.5∫7.0% and 4.7∫12.0% vs. 0.0∫0.17%,respectively, P0.05). Rothia dentoca-riosa was present only in group Asamples but was undetectable by culturein group B samples, while Actinomycesisraelii and Campylobacter rectus wereundetectable by culture in group Asamples. The isolation frequency offusiform species was found to be statis-tically lower in group A than in groupB (27.3% vs. 66.7%, PΩ0.04, Fisher’sexact test). The prevalence of lost/un-identified species was high in bothgroups, while many isolates were lost(Table4, Fig.1). A significantly higher

Subgingival flora post-renal transplant 41

Fig. 1. Relative mean proportion of predominant cultivable bacterial types from subgingival plaque samples of renal transplant recipients.Group A Ω plaque samples from sites without periodontal breakdown; Group B Ω plaque samples from sites with periodontal breakdown.*The percentage proportion of gram-positive facultative anaerobic cocci was significantly different between the two groups (P Ω 0.006, analysisof variance).

proportion of total Staphylococcus spp.were isolated from the subgingivalsamples of subgroup A1 subjects thanfrom those of subgroup B1 subjects(A1 Ω16.8∫19.6% vs. A2 Ω4.8∫12.6%, B1 Ω0.9∫3.6% or B2 Ω2.8∫7.1%, PΩ0.038, Bonferroni multiplecomparison), while a significantlyhigher proportion of total Fusobacteri-um spp. were observed in subgroup B1

subjects (A1 Ω1.8∫3.5%, A2 Ω0∫0%,B1 Ω4.3∫12.3%, and B2 Ω0.41∫1.1%,P0.0001, Bonferroni multiple com-parison).

The 48 bacterial species that were iso-lated at lower frequencies were as fol-lows. Facultative anaerobic gram-posi-tive cocci: Abiotrophia adiacens, Lactoc-occus lactis cremoris, Leuconostoc spp.,Micrococcus spp., Staphylococcus aur-icularis, Staphylococcus cohnii cohnii,Staphylococcus epidermidis, Staphylo-coccus sciuri, Staphylococcus xylosus,Staphylococcus spp., Streptococcus aci-dominimus, Streptococcus intermedius,Streptococcus oralis, and Streptococcuspneumoniae; facultative or obligatoryanaerobic gram-positive rods: Acti-nomyces odontolyticus, Actinomyces me-yeri, Actinomyces viscosus, Lactobacil-lus acidophilus, Clostridium hastiforme,Eubacterium aerofaciens, asaccharoyticEubacterium, Propionibacterium acnes,and Propionibacterium propionicum;obligatory anaerobic gram-negativecocci: Veillonella spp.; facultative or

obligatory anaerobic gram-negativerods: Kingella dentrificans, Neisserialactamica, Neisseria mucosa, Pasteurellapneumotropica/haemolytica, Bacteroidesfragilis, Bacteroides stercoris, Bacter-oides vulgatus, Porphyromonas asacch-arolyticas, Porphyromonas gingivalis,Prevotella bivia, Prevotella buccae, Pre-votella loescheii, Prevotella melaninog-enica, Prevotella oralis, and Prevotellaoris; facultative or obligatory anaerobicgram-negative fusiforms: Capnocyto-phaga sputigena, Capnocytophaga spp.,Fusobacterium motiferum, Fusobacteri-um necrophorum, and Fusobacteriumnucleatum; and an obligatory anaerobicgram-negative motile rod: Campylo-bacter showae. Of all bacteria speciesisolated, 15% were isolated only ingroup A, and 47% were isolated only ingroup B. No differences were found inthe proportion of individual cultivablespecies detectable when groups A and Bwere further subdivided into subgroups.

Aerobic and facultative anaerobic gram-negative rods and yeasts

Aerobic and facultative anaerobicgram-negative rod and yeast specieswere isolated at a low prevalence and inlow quantities in the subgingival plaquesamples of the renal transplant recipi-ents. Pseudomonas fluorescens/putidawas isolated from one sample of bothgroups. An aerobic and facultatively

anaerobic gram-negative rod was iso-lated from one of the group B samplesbut it failed to survive the subculturing.Of all the specimens, only one samplefrom group B was found to contain ayeast, Candida parapsilosis.

Discussion

Since 1977, renal medicine in HongKong’s public hospitals has advancedsteadily, with the aim of improving themedical care and quality of life of end-stage renal failure patients. A recentstudy showed that 0.05% of the localpopulation was affected by end-stagerenal failure and about 28% of theaffected individuals received renaltransplantation (5). The reported mor-tality rate was 7.1% over 6years (9).Medical care, however, is not comple-mented by oral health service provision.Our earlier clinical report showed that65% of the cohort surveyed had notseen a dentist for at least 24months be-fore the study (6). This probably ex-plained why only a very small numberof periodontally healthy individualswithout gingival overgrowth could berecruited for this study. Such a lack oforal health care for survivors of severesystemic diseases is common in HongKong (27) because of the very limiteddental service offered by the local pub-lic health care system.

The two clinical groups in this report

42 Leung et al.

Table 4. Prevalence of microbes isolated and the corresponding mean percentage isolation from subgingival plaque samples of renal transplantrecipientsa

P

Group A Group B Prevalenceb Percentage isolationc

Gram-positiveFacultative anaerobic cocci

Gemella haemolysans 18.2 (1.7)d 7.4 (1.5) NS NSGemella morbillorum 27.3 (3.2) 40.7 (4.6) NS NSStreptococcus constellatus 36.4 (5.5) 3.7 (0.1) 0.019 0.009Streptococcus mitis biovar 1 27.3 (2.0) 18.5 (1.3) NS NSStreptococcus sanguis 18.2 (3.0) 7.4 (0.2) NS NSStreptococcus salivarius 27.3 (3.6) 7.4 (0.7) NS 0.002

Anaerobic cocciPeptostreptococcus micros 27.3 (3.0) 25.9 (5.1) NS NSPeptostreptococcus prevotiie 27.3 (2.1) 11.1 (0.6) NS NS

Facultative anaerobic rodsActinomyces georgiae/gerencseriae 0 (0) 22.2 (2.6) NS NSActinomyces naeslundii 18.2 (3.0) 11.1 (0.6) NS NSArachnia propionica 18.2 (1.2) 25.9 (4.2) NS NSLactobacillus jenseniie 9.1 (4.4) 18.5 (1.7) NS NSRothia dentocariosa 18.2 (3.8) 0 (0) NS NS

Gram-negativeAnaerobic rods

Actinomyces israelii 0 (0) 18.5 (3.7) NS NSAnaerobic rods

Campylobacter gracilis 27.3 (2.2) 33.3 (5.2) NS NSCampylobacter rectus 0 (0) 22.2 (3.7) NS NSPrevotella corporis 9.1 (3.4) 33.3 (1.5) NS NSPrevotella intermedia 9.1 (0.6) 18.5 (1.1) NS NS

Facultative fusiformsCapnocytophaga gingivalis 18.2 (1.5) 22.2 (3.9) NS NSCapnocytophaga ochracea 0 (0) 22.2 (2.4) NS NS

Non-oralf 36.4 (15.6) 51.9 (10.1)g NS NSLost/unidentified spp. 63.6 (17.9)h 81.5 (25.6)i NS NSaOnly species with frequency of isolation 15% in any one group are included. Group A Ω plaque samples from subjects without periodontaldestruction; Group B Ω plaque samples from subjects with periodontal destruction. Except where indicated otherwise, all median values Ω0%.bFisher’s exact test.cAnalysis of variance.dData shown are percentage prevalence and mean (in parentheses).eMicrobes that are not normally considered as members of the oral or oropharyngeal flora.fData including species with frequency of isolation 15%.gMedian Ω3.1%.hMedian Ω7.1%.iMedian Ω14.7%.

had similar backgrounds in terms of re-nal transplant, immunosuppressantused and post-transplantation duration.The only substantial difference was thatthe renal transplant recipients who hadsigns of periodontal destruction (groupB) were older than those without peri-odontal destruction (group A) (Table1).The recruits were irregular attendants atdental clinics and their periodontal his-tory could not be traced. It was uncer-tain whether the recruits’ periodontaltissue destruction occurred before orafter the renal transplant. Nevertheless,no difficulty was experienced in at-taining expected periodontal healing re-sponses in patients after treatment (6),showing that transplant status does not

necessarily affect the subject’s responseto periodontal treatment (11).

The present study describes, for thefirst time, the subgingival microbiologyof renal transplant recipients receivingthe most common combination of im-munosuppressive agents, namely corti-costeroids together with a calcineurininhibitor. Data were obtained on peri-odontally healthy and diseased speci-mens from this special patient group.The results support the idea that con-siderable changes in the subgingivalflora of renal transplant recipients canoccur. A high prevalence and a moder-ate percentage proportion of non-oralspecies were identified (Table4). Higherthan average amounts of lost/unidenti-

fied isolates were detected in anaerobicculture (Table4, Fig.1).

Colonization of subgingival sites bynon-oral species has previously been re-ported in special patient groups, includ-ing those with failing hydroxyapatite-coated implants (25), compromisedmedical conditions (22, 30), human im-munodeficiency virus infection (24, 37)and head and neck irradiation (16). Theprevalence of such species in subgingi-val samples from these special groupsis usually low (30%) and the diseaseconditions do not seem to have a sig-nificant effect on the normal subgingi-val flora. The data of the present studyseem to confirm the findings of previousstudies; however, the prevalences and

Subgingival flora post-renal transplant 43

relative quantities of these non-oral mi-crobes, as shown in Table4, were con-siderably higher.

A striking finding was that a moder-ate proportion of the predominantlycultivable isolates were subsequentlylost (Table4). This observation alonetriggered a review of the already strin-gent anaerobic laboratory process andquality control procedures. No anom-aly was reported from a regular surveil-lance of the facilities and no concurrentincrease in unexpected sample loss wasreported from other specimens. A qual-ity check yielded negative findings, andno similar situations were observed inother studies conducted during thesame period (36). From these obser-vations, we conclude that the findingsof loss and non-identification weregenuine.

An animal study is available in theliterature on the effect of cyclosporin Aon ligature-induced periodontal break-down in ferrets (8). An increase in theproportion of spirochetes to 4% wasnoted in cyclosporin A pre-medicated28-day ligature-induced periodontalbreakdown in ferrets. Another reportdescribed the subgingival microbiologyof nifedipine-associated gingival over-growth in humans (20), and in anotherstudy a partial microbial evaluation ofplaque at gingival margins with pheny-toin-related gingival overgrowth wascarried out (32). A high proportion ofspirochetes was also observed in thepresent study (Table3), comparable tofindings for samples from periodontitispockets (1) of normal patients. Thesubgingival cultivable microbial com-position observed in the current study,however, contrasted with that associ-ated with gingival overgrowth inducedby nifedipine (20). In that study, theauthors reported a significantly higherprevalence of C. sputigena and F. nucle-atum from gingival enlargement lesions.Despite the fact that most of the cur-rent subjects also received calcium anta-gonists such as nifedipine to attenuatethe nephrotoxicity of calcineurin inhibi-tors, the current anaerobic culture re-sults, within the limitations of the study,did not support the hypothesis that C.sputigena and F. nucleatum are associ-ated with gingival enlargement lesionsas a result of immunosuppression ther-apy post-renal transplant. Instead, Cap-nocytophaga species, especially C. gingi-valis, were prevalent in both groups,while a significantly lower prevalence offusiform species was found in samples

from subjects without periodontal de-struction, regardless of gingival over-growth. This observation implies thatother factor(s) might be dictating thecomposition of the predominantly culti-vable subgingival plaque flora in thecurrent cohort.

In the present study, gram-stainedsmears of the samples were also evalu-ated. The microscopic evaluation of thesamples before the bacterial culture wasrelevant because spirochetes could notbe isolated with routine anaerobic cul-ture and the available spirochete iso-lation techniques could only purify afew treponemes (37). The importance ofthe complimentary or supportive roleof gram-stained smears in analysis ofroutine anaerobic culture results hasbeen discussed in an earlier publication(16).

Not counting the lost isolates, ap-proximately 50% of the isolates wereobligate anaerobes, which is similar tothe proportion found in an earlier workfrom our group on the subgingival mi-croflora of various periodontal con-ditions (15, 16). This observation alsosupports the argument that the anaer-obic facilities were probably func-tioning normally, otherwise a largerproportion of facultative anaerobeswould have been observed. We postu-late that causes other than failure in an-aerobiosis are responsible for the in-crease in lost isolates. If there had beena failure in anaerobiosis, many fewer oreven no obligate anaerobes would beavailable after the primary culture andthey probably would not survive andgrow to form a colony. An alternate ex-planation for our observation is a lossof satellite bacterial colonies after theyhad been subcultured. Microbial sym-biosis or satellite growth was reportedfor oral and respiratory flora (2, 26, 34).The special environment of the renaltransplant subgingival niche might havefostered transformation of the subgin-gival microbial biofilm. The fact thatthe subgingival biofilm is much morecomplex than can be appreciated fromculture alone (21) indirectly suggeststhat such a proposition should not beoverlooked.

In the subjects we studied, given thefindings of our earlier study (6), it seemsthat bacterial or fungal pathogens suchas Nocardia, Listeria, Aspergillus, andCryptococus, which often cause post-transplantation infections in local renaltransplant recipients, were not recover-able from the subgingival niche.

Further investigations with more con-trol groups, such as pre-renal graft indi-viduals as well as normal control sub-jects, are needed to determine whetherrenal transplant recipients might habora different or unique subgingival micro-flora. Specific nonculture-sensitive tech-niques such as bacterial identificationusing 16S rRNA (21) may be more ap-propriate in anticipation of the samefrequency of loss of isolates as was en-countered in this study.

In conclusion, a high prevalence ofgingival enlargement was observed inthe present cohort of kidney transplantrecipients. The subgingival microfloraof the kidney transplant recipients, whowere affected by periodontitis, com-prised gram-negative rods and spiro-chetes. However, a considerable pro-portion (up to 25%) of the subgingivalflora from the renal transplant recipi-ents were not recoverable by anaerobicculture and a high prevalence of non-oral microbes was found. Furtherstudies with a larger renal transplant re-cipient patient group and periodontalstatus-, age-, and sex-matched controls,utilizing anerobic culture as well as cul-ture-independent bacterial identifi-cation methods, are required to confirmthe preliminary findings of this report.

Acknowledgments

The authors are grateful to Drs Thom-as Fu and Helena Wong who assistedwith the clinical part of the study. Wealso thank the staff hygienists of thePeriodontology Clinic, Prince PhilipDental Hospital, who took good care ofthe recruits’ periodontal health duringthe study, and Dr Esmonde F. Corbetfor help with the manuscript.

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