A microbiological study of Papillon-Lefèvre syndrome in two patients

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J Clin Pathol 2001;54:371–376                                                                                                       371

                               A microbiological study of Papillon-Lefèvre
                               syndrome in two patients
                               K L Robertson, D B Drucker, J James, A S Blinkhorn, S Hamlet, P S Bird

                               Abstract                                              pockets form leading to increased tooth mobil-
                               Aim—To analyse the microflora of subgin-              ity.6 Usually, both deciduous teeth7 and perma-
                               gival plaque from patients with Papillon-             nent teeth6 are lost prematurely.
                               Lefèvre syndrome (PLS), which is a very                  PLS is a systemic disease with immune
                               rare disease characterised by palmar-                 system defects,8 which might result in the pro-
                               plantar hyperkeratosis with precocious                liferation of certain periodontopathogens.
                               periodontal destruction.                              Microbiological studies of the oral microflora
                               Methods—Bacterial isolates were identi-               of patients with PLS have shown that the pre-
                               fied using a combination of commercial                dominant organisms in the periodontal sites
                               identification kits, traditional laboratory           are Gram negative anaerobic rods,9 including
                               tests, and gas liquid chromatography.                 Porphyromonas gingivalis, Prevotella intermedia,
                               Some isolates were also subjected to                  Prevotella loescheii, Bacteroides gracilis,10 and
                               partial 16S rDNA sequencing. Plaque                   Fusobacterium nucleatum.10 11 Eikenella corro-
                               samples were also assayed for the pres-               dens, capnocytophaga,11 veillonella, anaerobic
                               ence of Porphyromonas gingivalis, Prevo-              streptococci,12 and spirochaetes have also been
                               tella intermedia, and Actinobacillus                  reported.13 14 Although the presence of cultivat-
                               actinomycetemcomitans in a quantitative               able Actinobacillus actinomycetemcomitans and
                               enzyme linked immunosorbent assay                     raised serum antibody titres to A actinomyc-
                               (ELISA) using monoclonal antibodies.                  etemcomitans have been found,10 12 13 15 16 this is
                               Results—The culture results showed that               not always the case.11 17
                               most isolates were capnophilic and facul-                The aims of our study were to identify the
                               tatively anaerobic species—mainly Cap-                subgingival plaque microflora of two patients
                               nocytophaga spp and Streptococcus spp.                with PLS and to assess the amounts of the
                               The latter included S constellatus, S ora-            periodontopathic bacteria, A actinomycetem-
                               lis, and S sanguis. Other facultative bac-            comitans, P gingivalis, and P intermedia using a
                               teria belonged to the genera gemella,                 quantitative enzyme linked immunosorbent
                               kingella, leuconostoc, and stomatococcus.             assay (ELISA).
                               The aerobic bacteria isolated were species
                               of neisseria and bacillus. Anaerobic spe-
                               cies included Prevotella intermedia,                  Methods
                                                                                     PLAQUE SAMPLES
                               P melaninogenica, and P nigrescens, as
                               well as Peptostreptococcus spp. ELISA                 Two patients with PLS attended the University
                               detected P gingivalis in one patient in all           of Manchester Dental Hospital. Patient 1 had
                               sites sampled, whereas A actinomycetem-               only six teeth remaining and subgingival
                               comitans was detected in only one site                plaque samples were taken from each tooth. A
                               from the other patient. Prevotella inter-             further five subgingival plaque samples were
                               media was present in low numbers.                     also collected from the molars and canine teeth
                               Conclusions—Patients with PLS have a                  of patient 2. All samples were collected with a
                               very complex subgingival flora including              curette and (1) placed into reduced transport
                               recognised periodontal pathogens. How-                fluid for rapid transportation to Manchester
Oral Microbiology              ever, no particular periodontopathogen is             Royal Infirmary for initial bacterial cultivation;
Laboratory, University
                               invariably associated with PLS.                       (2) resuspended in phosphate buVered saline
of Manchester Dental                                                                 (PBS) containing 0.01% thiomersal, frozen,
                               (J Clin Pathol 2001;54:371–376)
School, Higher                                                                       and sent to the University of Queensland, St
Cambridge Street,              Keywords: Papillon-Lefèvre syndrome;
Manchester M15 6FH,
                                                                                     Lucia, Australia for analysis with specific
                               periodontopathogens                                   monoclonal antibodies by means of ELISA.
K L Robertson
J James                        Papillon-Lefèvre syndrome (PLS) was first             ENZYME LINKED IMMUNOSORBENT ASSAY
A S Blinkhorn                  described in 1924 by Papillon and Lefevre.1 It        Plaque samples were thawed, six to eight 1 mm
D B Drucker
                               is a rare autosomal recessive disease,2 with an       glass beads added to each vial, and the bacteria
University of                  incidence of 1–4 cases/million people,3 and           dispersed by vortexing and then sonicated for
Queensland School of           with consanguinity between parents seen in            five seconds. Samples were diluted in an equal
Dentistry, Brisbane            one third of cases.4 The disease is characterised     volume of 0.1 M carbonate buVer pH 9.6. A
Qld 4072, Australia            by palmar-plantar hyperkeratosis, a thickening        100 µl volume of each sample was pipetted into
S Hamlet                       of the skin on the palms of the hands and the         triplicate wells of a 96 well Maxisorp microtitre
P S Bird
                               soles of the feet.5 In addition, precocious perio-    plate (Nunc, Roskilde, Denmark). A known
Correspondence to:             dontal destruction of both the deciduous and          concentration of bacterial cells of either
Dr Drucker                     permanent teeth is seen.6 The gingiva become          A actinomycetemcomitans Y4, P gingivalis FDC-
David.Drucker@man.ac.uk        red and inflamed and may be ulcerated5 and a          381, or P intermedia ATCC 25611 (ranging
Accepted for publication       form of rapid, severe periodontitis occurs.4          from 9 to 150 × 104 cells/ml) in carbonate
20 November 2000               Alveolar bone is resorbed and deep periodontal        buVer were assayed on each plate with the

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372                                                                                 Robertson, Drucker, James, et al

          plaque samples. Microtitre plates were incu-        use of schemes in the Wadsworth anaerobic bac-
          bated overnight at 4°C and washed three times       teriology manual and Virginia Polytechnic Insti-
          with PBS/Tween 20 (0.05%; PBS-T). Non-              tute anaerobe laboratory manual.18 19 Bacterial
          specific binding was blocked with PBS-T con-        culture supernatants from Gram positive bac-
          taining 1% fetal calf serum (Commonwealth           teria were analysed for the presence of
          Serum Laboratory, Melbourne, Australia) and         non-volatile acids.18 For gas chromatographic
          the plates were incubated for one hour at room      analysis of end products, established methods
          temperature (RT). After washing (×3) with           were followed,18 except that supernatant fluid
          PBS-T, diluted horseradish peroxidase labelled      from 48 hour gas liquid chromatography
          monoclonal antibodies specific to either A ac-      (GLC) broth (Lab M) cultures was used. Sam-
          tinomycetemcomitans, P gingivalis, or P interme-    ples (2 µl) were analysed on a column of 6%
          dia were added to all the coated wells and incu-    Carbowax 20M TPA on Chromosorb W
          bated for two hours at RT. After further            AWDMCS (Phase Separations, Queensferry,
          washing (×3) with PBS-T, colour development         UK), whether ether extracts of volatile end
          was achieved by adding 150 µl of 2.5 mM             products or chloroform extracts of methylated
          ó-tolidine (Kodak Eastman, Rochester, New           non-volatile acids. The injector and detector
          York, USA) in 100 mM phosphate citrate              temperatures of the chromatograph (AI, Cam-
          buVer (pH 3.5) containing 0.025 mM EDTA             bridge, UK) were both 150°C. The initial tem-
          and activated by 3% H2O2. The colour                perature of the column was 65°C, which was
          development was stopped after 10 minutes by         held for 30 seconds before being increased lin-
          the addition of 50 µl of 1 M HCl. The plates        early to 115°C over two minutes, and held for
          were read in a Bio-Rad microplate reader            six minutes and 20 seconds. The equilibration
          model 3550 (Bio-Rad Laboratories, Hercules,         time between samples was three minutes.
          California, USA) at 450 nm and 655 nm.                 When satisfactory identification could not be
                                                              achieved, the partial sequence of the 16S rRNA
          DETECTION OF SPIROCHAETES                           gene was determined and compared with
          Smears were prepared from the plaque sam-           library data for known organisms.
          ples, air dried, and stained with a modified
          Gram stain, where the counter stain was 20%
                                                              DNA EXTRACTION: GRAM NEGATIVE BACTERIA
          carbol fuchsin, applied for five minutes. Slides
                                                              Bacterial isolates were grown to purity on FAA.
          were viewed under a Zeiss (Jena, Germany),
                                                              Isolated colonies were subcultured into fastidi-
          Axioplan light microscope at ×1000 magnifica-
                                                              ous anaerobe broth and DNA was extracted
                                                              from a 72 hour culture. Briefly, 5 ml of each
                                                              bacterial culture was centrifuged at 3000 ×g for
                                                              10 minutes and the cells resuspended in
          Plaque samples were plated on to Columbia
                                                              sucrose/Tris/EDTA (STE). Lysozyme (Sigma,
          blood agar plates supplemented with 5% defi-
                                                              Poole, Dorset, UK) was added to give a final
          brinated horse blood (Oxoid, Basingstoke,
                                                              concentration of 2 mg/ml and incubated at
          UK) and fastidious anaerobe agar (FAA; Lab
                                                              37°C for 30 minutes. Proteinase K (0.3 mg/ml)
          M, Bury, UK) plates supplemented with 5%
                                                              and sodium dodecyl sulphate (SDS; 1% final
          defibrinated horse blood (Oxoid). Columbia
                                                              concentration) were added and incubated at
          agar plates were incubated in an aerobic
                                                              55°C for one hour, to induce cell lysis. An
          atmosphere for three days at 37°C. The latter
                                                              equal volume of phenol/chloroform/isoamyl
          were incubated in a Compact M anaerobic
                                                              alcohol (25/24/1, vol/vol/vol) was added to each
          cabinet (Don Whitley Scientific, Shipley, UK)
                                                              suspension and vortexed. After centrifugation
          in an atmosphere of 10% hydrogen, 10%
                                                              at 16 000 ×g for 10 minutes, 24 µl 5 M NaCl
          carbon dioxide, and 80% nitrogen for three to
                                                              and 600 µl propan-2-ol were added. Gentle
          five days at 37°C. Selected isolated colonies
                                                              mixing and incubation at 0°C for 10 minutes
          were subcultured.
                                                              were followed by centrifugation at 16 000 ×g
                                                              for 10 minutes. The pellet was resuspended in
                                                              water and one volume of ammonium acetate
          Plaque contains many species; thus, we se-
                                                              was added (final concentration of 2.5 M). The
          lected the predominant organisms for further
                                                              suspension was then cooled at 0°C for one hour
          study. All isolates were Gram stained and
                                                              then centrifuged at 16 000 ×g for 20 minutes.
          tested for catalase, oxidase activity, aerobic
                                                              The supernatant was removed and the DNA
          growth, and anaerobic growth. In some cases,
                                                              was reprecipitated by the addition of 600 µl of
          other tests were used such as Hugh and
                                                              chilled ethanol (−85°C). The DNA was
          Leifson’s oxidation fermentation test, nitrate
                                                              pelleted by centrifugation at 16 000 ×g for 10
          reduction, extracellular polysaccharide pro-
                                                              minutes and then washed in 500 µl 70% chilled
          duction on TYC agar, and spore production.
                                                              ethanol (−85°C). The ethanol was removed
          Isolates were identified using a range of
                                                              and the DNA was resuspended in 100 µl Tris/
          commercial identification kits, namely: API 20
                                                              EDTA (TE) and stored at 4°C.
          NE; Rapid ID 32 Strep; API Coryne; Rapid ID
          32A (BioMérieux, Basingstoke, Hampshire,
          UK), and the Rapid ANA II system (Prolab,           DNA EXTRACTION: GRAM POSITIVE BACTERIA
          Liverpool, UK). Microcodes generated using          DNA was extracted from all Gram positive
          API 20 NE, ID 32 Strep, API Coryne, or ID           clinical isolates using the Puregene DNA
          32A were analysed by BioMérieux. Microcodes         isolation kit (Flowgen Instruments, StaVord-
          generated using Rapid ANA II were analysed          shire, UK), according to the manufacturer’s
          by Prolab. Anaerobe identification also made        instructions.

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Microbiological study of PLS                                                                                                                  373

                               MEASUREMENT OF DNA                                    Sequences resulting from amplification with
                               Samples (5 µl) of DNA were stained with             primer RE-RTU3 were reverse transcribed and
                               ethidium bromide (1 µg ml−1 in 1 × trisphos-        changed to the complement. Results from each
                               phate EDTA (TPE)) after electrophoresis in an       primer were then aligned using GAP function
                               0.8% agarose gel and then visualised by             and checked twice against each other to
                               ultraviolet light (ë = 254 nm) and photo-           produce a consensus sequence. The consensus
                               graphed on Polaroid 667 film. The amount of         sequence was submitted for a FASTA search to
                               DNA was determined by visual comparison             determine the most likely relation.
                               with known amounts of uncut ë DNA (Sigma;
                               40–400 ng µl−1).
                               PARTIAL 16S rRNA GENE SEQUENCE ANALYSIS             A total of 108 pure cultures of predominant
                               Bacterial DNA was amplified in a Crocodile IIJ      organisms was obtained for identification and
                               thermal cycler (Appligene, County Durham,           comprised the genera and species listed in
                               UK). The reaction mix (total volume, 50 µl)         tables 1–3. The bacterial species isolated
                               consisted of 2 µl of DNA, Taq polymerase (1.5       (including those determined by ELISA) were
                               units), 0.2 mM of each deoxynucleoside              separated into aerobic, anaerobic, facultative,
                               triphosphate (dATP, dCTP, dGTP, dTTP),              and capnophilic species and 17 genera were
                               MgCl2 (1.5 mM), 1× buVer IV, and 0.3 µM             found to be present. The facultative micro-
                               of both primers: RE-TPU1 (5'-AGA                    organisms identified consisted mainly of spe-
                               GTTTGATCMTGGCTCAG)                     and   RE-    cies of streptococcus, including Streptococcus
                               RTU3       (5'-GWATTACCGCGGCKGCG)20                 anginosus, S bovis, S constellatus, S mitis, and
                               (Oligonucleotide Synthesising Service, School       S sanguis. Species of capnocytophaga and
                               of Biological Sciences, University of Manches-      gemella were also identified. The obligately
                               ter, UK). The reaction mix was covered with an      anaerobic flora consisted mostly of prevotella
                               equal volume (20 µl) of mineral oil to prevent      species, namely: Prevotella intermedia, P nigres-
                               evaporation. Negative controls contained all        cens, P melaninogenica, and “unidentified”
                               components of the reaction mix except the           prevotella (formerly “PINLO”). In addition,
                               bacterial template DNA. The thermal cycling         Peptostreptococcus anaerobius, Pstr micros and
                               profile was as follows: one cycle of four minutes   species of mobiluncus were identified. Table 1
                               at 94°C, one minute at 55°C, one minute at          gives a comprehensive list of species cultured
                               72°C; 29 cycles of one minute at 94°C, one          for each patient. Gemella spp, Neisseria spp,
                               minute at 57°C, one minute at 72°C; one cycle
                               of one minute at 94°C, one minute at 57°C,          Table 1 Complete list of bacterial species isolated from two
                                                                                   patients with Papillon-Lefèvre syndrome, displaying
                               and five minutes at 72°C. Polymerase chain          association with patient
                               reaction (PCR) products (3 µl) were resolved
                               in 1.0 % agarose gels, stained with ethidium                                                  Patient 1   Patient 2
                               bromide, and visualised under ultraviolet light     Obligate aerobes
                               (ë = 254 nm). A ë Pst1 digest was used as a           Neisseria sp                            2           0
                               size marker (270 ng/µl). Gels were photo-             N cinerea                               1           1
                                                                                     N elongata                              0           1
                               graphed on Polaroid 665 and 667 films and the         N flavescens                            0           2
                               presence of the correct size fragment con-            Neisseria sp possibly N subflava        1           0
                               firmed. QIAquick PCR purification kit (Qia-         Obligate anaerobes
                                                                                     Eubacterium sp                          0           1
                               gen, Crawley, Sussex, UK) was used to clean           Porphyromonas gingivalis                1           0
                               the PCR product. The quantity of the product          Prevotella sp possibly P oralis         1           0
                               was estimated by visual comparison in a 0.8%          P intermedia                            1           1
                                                                                     P melaninogenica                        0           2
                               agarose gel with a known amount of ë DNA.             P nigrescens                            0           2
                               Concentrations of PCR products (30–90 ng)             Peptostreptococcus sp                   1           0
                                                                                     Pstr anaerobius                         0           1
                               were amplified further in a Perkin Elmer              Pstr micros                             1           0
                               (Warrington, UK) model 2400 thermal cycler.         Facultative
                               Two reaction mixes were set up for each PCR           Actinobacillus actinomycetemcomitans    0           1
                                                                                     Actinomyces sp                          1           0
                               product, one with each primer. The reaction           A israelii                              1           0
                               mix had a total volume of 20 µl, which                Bacillus cereus                         1           0
                               consisted of 8 µl ABI PRISM™ terminator               Gemella haemolysans                     1           0
                                                                                     G morbillorum                           1           1
                               cycle sequence ready reaction mix (Perkin             Kingella denitrificans                  1           0
                               Elmer) or 4 µl ABI PRISM™ BigDye™ termi-              Leuconostoc sp                          0           1
                               nator cycle sequence ready reaction mix               L mesenteroides                         0           1
                                                                                     Mobiluncus sp                           1           0
                               (Perkin Elmer) and either RE-RTU3 or                  Streptococcus sp (to genus only)        1           1
                               RE-TPU1 (0.15 µM). The PCR product was                S anginosus                             0           2
                               added and the volume made up with water.              S bovis II                              1           0
                                                                                     S constellatus                          3           0
                               The thermal cycling profile was as follows: 25        S gordonii                              0           1
                               cycles of 10 seconds at 96°C, five seconds at         S intermedius                           0           1
                                                                                     S milleri group                         1           1
                               50°C, and four minutes at 60°C. The cycle             S mitis                                 2           1
                               sequencing product was purified by ethanol            S oralis                                1           0
                               precipitation. The automated sequence analy-          S salivarius subspecies P salivarius    1           1
                                                                                     S sanguis                               3           1
                               sis was performed by Oswals DNA Service,              Stomatococcus mucilaginosus             1           0
                               University of Southampton. Sequences were             Suttonella indologenes                  1           0
                               manipulated using the Genetics Computer             Capnophilic
                                                                                     Capnocytophaga sp                       1           0
                               Group (GCG) package version 8.0 (Wiscon-
                               sin, USA).                                          The numbers refer to the total number of sites with the isolate.

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374                                                                                                    Robertson, Drucker, James, et al

          Table 2 Predominant species isolated from patient 1                   “Control” sites were not studied because in the
          according to site sampled                                             case of severe PLS there are no healthy control
          Site A (upper right deciduous canine)
            Actinomyces sp               Peptostreptococcus sp                      Aerobes identified were species of bacillus
            Bacillus cereus              Prevotella sp                          and neisseria. The Gram positive, spore former
            Capnocytophaga sp            Streptococcus constellatus
            Kingella denitrificans       S mitis
                                                                                Bacillus cereus is not considered part of the nor-
            Neisseria cinerea            S sanguis                              mal oral flora, although it has been reported in
            Neisseria sp                 Streptococcus sp                       plaque samples.20 With no reports of an associ-
          Site B (lower left deciduous canine)
            Actinomyces israelii         S milleri group                        ation with periodontal disease and because of
            Mobiluncus sp                S oralis                               its universal occurrence and the formation of
            Neisseria sp                 S sanguis                              spores, the possibility that this is a contaminant
            Streptococcus constellatus
          Site C (upper left deciduous 1st molar)                               cannot be ruled out. Neisseria spp—N sicca/
            Streptococcus bovis II       Stomatococcus mucilaginosus            subflava10 and N pharyngis21—have been re-
            S constellatus               Neisseria subflava                     ported previously in other patients with PLS.
            S sanguis
          Site D (lower left deciduous 1st molar)                               In our study, the identification of isolates as
            Gemella haemolysans          Peptostreptococcus micros              either species of neisseria or prevotella was sec-
            G morbillorum                Streptococcus sanguis
            Prevotella intermedia
                                                                                ond only to identification as streptococcus.
          Site E (lower right deciduous canine)                                     Five genera of obligately anaerobic bacteria
            Suttonella indologenes       Capnocytophaga sp                      were identified—eubacterium, peptostrepto-
            Actinomyces meyeri
          Site F (lower right deciduous 1st molar)
                                                                                coccus, porphyromonas, prevotella, and
            Streptococcus mitis          S salivarius subspecies salivarius     treponema. Species of eubacterium have been
                                                                                isolated from subgingival plaque22 and are
          Table 3 Predominant species isolated from patient 2                   associated with oral health, gingivitis,23 and
          separated according to site of sample                                 periodontal disease in humans,24 25 although
                                                                                little evidence is available for an association
          Site A (upper right permanent 1st molar)                              with PLS. One study13 has failed to isolate
            Eubacterium sp                P nigrescens
            Leuconostoc sp                Peptostreptococcus anaerobius         eubacterium from plaque samples despite the
            Neisseria cinerea             S anginosus                           use of selective media, although low numbers
            N elongata                    S intermedius
            N flavescens                  S salivarius subspecies savlivarius
                                                                                were cultured from mouth rinse samples,
            Prevotella melaninogenica     Actinomyces meyeri                    suggesting that eubacterium was present in the
            Streptococcus sanguis                                               oral cavity of patients with PLS. Unidentified
          Site B (upper right permanent canine)
            P melaninogenica              S milleri group
                                                                                Peptostreptococcus spp have been isolated from
            P nigrescens                  S mitis                               the subgingival plaque of patients with perio-
            Streptococcus gordonii        Streptococcus sp                      dontal disease25 and Pstr micros has been
          Site C (lower right permanent canine)
            Leuconostoc mesenteroides                                           associated with gingivitis.23 Peptostreptococcus
          Site D (lower left canine)                                            micros10 and unspeciated peptococcaceae13 have
            Peptostreptococcus micros                                           also been identified previously from patients
          Site E (lower left first molar)
            Actinomyces meyeri                                                  with PLS.
                                                                                    Porphyromonas gingivalis is an accepted peri-
                                                                                odontal pathogen,26 27 which was detected by
          Peptostreptococcus spp, P intermedia, and Strepto-                    monoclonal antibodies in one of the patients.
          coccus spp were isolated from both patients.                          The use of monoclonal antibodies to P gingiva-
          Actinomyces spp, Bacillus spp, Capnocytophaga                         lis in a previous study10 detected this species in
          spp, Kingella spp, Mobiluncus spp, P gingivalis,                      one of the two patients. It has also been isolated
          and Stomatococcus spp were isolated only from                         previously from patients with PLS using
          patient 1, whereas A actinomycetemcomitans,                           culture methods.10 13
          Eubacterium spp, Leuconostoc spp, P nigrescens,                           Prevotella intermedia, P melaninogenica, P ni-
          and P melaninogenica were isolated only from                          grescens, and P oralis were identified in our
          patient 2. Tables 2 and 3 show the correlation                        present study. Prevotella oralis, P oris, P loe-
          between the species identified and the site                           schii,10 P intermedia,10 13 and other unspeciated
          sampled in the patients.                                              black pigmented anaerobes9 13 17 have been
             Culture provided qualitative data only. How-                       associated previously with PLS. In earlier stud-
          ever, monoclonal antibodies provided quanti-                          ies, P nigrescens was probably misidentified as
          tative data and detected P gingivalis in low                          P intermedia. Prevotella melaninogenica has not
          numbers in all six sites sampled in patient 1;                        been isolated previously from PLS samples but
          A actinomycetemcomitans was detected in one                           it occurs in subgingival plaque28 and has been
          site in low numbers in patient 2. Prevotella                          associated with gingivitis26 and periodontal dis-
          intermedia was detected in low numbers from                           ease29. Prevotella intermedia was detected both
          both patients—in one of six sites from patient 1                      by culture and ELISA in our study, methods by
          and three of six sites from patient 2.                                which it has previously been detected in PLS.10
             The occurrence of spirochaetes in the plaque                           Unspeciated spirochaetes were seen within
          was confirmed by microscopy.                                          gingival smears during our study and have been
                                                                                reported previously in patients with PLS.13 14
                                                                                    Actinobacillus actinomycetemcomitans is an
          Discussion                                                            accepted periodontopathogen,23 27 30 which has
          A range of bacteria was found in the subgingi-                        been associated particularly with prepubertal
          val plaque of both these patients with PLS. In                        periodontitis31 and localised juvenile periodon-
          addition, the periodontopathic bacteria P gin-                        titis.32 It has been associated with PLS on many
          givalis, P intermedia, and A actinomycetemcomi-                       occasions10 12 13 15 16 and is considered an impor-
          tans were detected, albeit in low numbers.                            tant pathogen for the periodontal component

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Microbiological study of PLS                                                                                                                     375

                               of PLS.32 We detected this organism using             actinomyces because of a positive Gram
                               monoclonal antibodies, but only in one sample         reaction and a similar GLC profile.
                               from patient 2. Therefore, the presence of               Members of the genus capnocytophaga have
                               A actinomycetemcomitans might not be neces-           been frequently associated with gingivitis,23
                               sary for PLS to progress. This argument could         periodontal disease,27 and juvenile periodonti-
                               also apply to P gingivalis, which was only            tis.29 Species of this organism have been
                               detected in patient 1.                                reported in patients with PLS.10 11 Capnocyto-
                                  We identified three actinomyces species that       haga ochracea10 has been isolated, but most
                               have been associated with PLS previously.10           studies have identified isolates only to the
                               Actinomyces spp form part of the resident oral        genus level. Paradoxically, capnocytophaga has
                               microflora of humans33 and are therefore              also been found to be associated negatively
                               isolated from the healthy oral cavity and             with periodontal disease.40
                               associated with gingivitis.23                            Species not isolated here but reported in
                                  Gemella morbillorum has been isolated previ-       other studies10 11 13 include Fusobacterium spp,
                               ously from patients with PLS and is also asso-        Eikenella corrodens, veillonella, and Bacillus gra-
                               ciated with a healthy mouth,23 whereas no             cilis. The recognised periodontal pathogens
                               association with periodontal disease or PLS has       P gingivalis and A actinomycetemcomitans were
                               been reported for G haemolysans. The likeli-          found in low numbers in our patients with
                               hood of gemella causing similar infections to         PLS. It is possible that A actinomycetemcomitans
                               those caused by viridans streptococci has been        acts together with human herpesviruses in the
                               noted,34 so that the isolation of this micro-         development of the syndrome,41 but our
                               organism from patients with PLS is not                present study did not include virological exam-
                               surprising.                                           ination. Furthermore, a “massive occurrence
                                  Kingella species include known upper respi-        of A actinomycetemcomitans” has been noted in
                                                                                     periodontal pockets in PLS, although the same
                               ratory tract commensals35; however, there are
                                                                                     species was present also in the mouths of
                               no reports of the isolation of members of this
                                                                                     siblings and a parent without PLS.42 Similarly,
                               genus from patients with PLS and only limited
                                                                                     in a group of 12 Saudi-Arabian adolescents
                               associations with periodontal disease. The pri-
                                                                                     with PLS, there was no PLS specific profile of
                               mary habitat of K oralis is human dental plaque
                                                                                     the subgingival infection because the bacterial
                               and it been isolated from supragingival and           composition resembled that characterising
                               subgingival plaque taken both from healthy            deep pockets in adult patients with periodonti-
                               oral cavities and patients with periodontitis.36      tis.43 It is possible that recognised periodontal
                                  The genus leuconostoc contains species             pathogens might be involved in PLS. However,
                               similar to streptococci and often identified ini-     the severity of periodontitis in patients with
                               tially as S sanguis type II as well as S salivarius   PLS cannot be explained simply by the
                               and S pneumoniae, when using automated                presence of any of the bacteria found in this
                               identification systems.37 Leuconostoc has not         study, even the recognised periodontopatho-
                               been previously identified in PLS samples.            gens. These patients are known to have a higher
                               However, this genus has been reported to be a         risk of developing disease when compared with
                               cause of bacteraemia in patients already              other individuals, suggesting that host factors
                               critically ill with acute leukaemia, renal failure,   determine the individual’s disease susceptibil-
                               and human immunodeficiency virus infec-               ity. The inherited components of PLS induce
                               tion.37 It has been suggested that the incorrect      both immune and epithelial defects.41 We con-
                               identification of leuconostoc as viridans strep-      clude that PLS occurs in genetically suscepti-
                               tococci means that the pathogenic associations        ble individuals whose periodontal disease is
                               of this genus have been underestimated.37 Dur-        associated with periodontal pathogens but the
                               ing our study, both viridans streptococci             species of periodontopathogen is not of over-
                               (S constellatus, S mitis, S oralis, and S sanguis)    riding importance.
                               and S salivarius were identified using Rapid ID
                               32 Strep; however, additional testing would be        Thanks to M Armstrong at the Manchester Royal Infirmary for
                               required to check that none of these isolates         initial cultivation of samples.
                               was a species of leuconostoc.37 Of the ten
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                               organism may have been misidentified as                   disease. Curr Opin Periodontol 1993:18–27.

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                      A microbiological study of Papillon-Lefévre
                      syndrome in two patients
                      K L Robertson, D B Drucker, J James, A S Blinkhorn, S Hamlet and P S Bird

                      J Clin Pathol 2001 54: 371-376
                      doi: 10.1136/jcp.54.5.371

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