10.59987/ads/2013.2.170-173
Periodontitis is a disease that affects and destroys the tissues that support teeth. Tissues damage results from a prolonged inflammatory response to an ecological shift in the composition of subgingival biofilms. Three bacterial species that constitute the red complex group, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, are considered the main pathogens involved in periodontitis. In the present study a real-time PCR based assay was designed to detect and quantify red complex species, then used to investigate 146 periodontal pocket samples from 66 periodontitis patients and 80 controls. Results demonstrated a significant higher prevalence of red complex species and increased amount of P. gingivalis and T. denticola in periodontal pocket of periodontitis patients.
application/pdf
1971-1441
137
2
en
A. Palmieri 
https://creativecommons.org/licenses/by-nc-nd/4.0
Annali di stomatologia
resorption
Oral microflora and periodontal disease: new technology for diagnosis in dentistry
Text.Serial.Journal
https://www.annalidistomatologia.eu/ads
4
Original Article
2022-08-16
2022-08-16
2013-06-01
2023-12-04
170-173
Ann Stomatol (Roma)
A. Palmieri 
2013
10.59987/ads/2013.2.170-173
170
1971-1441
2
resorption
en
173
Oral microflora and periodontal disease: new technology for diagnosis in dentistry
https://www.annalidistomatologia.eu/ads/article/download/137/120
4
1-Lauritano_- 19/06/13 10:32 Pagina 170




                                                                                                                     Original article



                   Oral microflora and periodontal disease:
                   new technology for diagnosis in dentistry




                                                                                                                                        li
                                                                                  na
                   Francesco Carinci1                                        Introduction
                   Luca Scapoli2




                                                                                io
                   Ambra Girardi2                                            Periodontitis represents a destructive chronic inflamma-
                   Francesca Cura2                                           tory disease with a bacterial infection resulting from the
                   Dorina Lauritano4                                         complex actions of a small subset of periodontal
                   Gianna Maria Nardi3                                       pathogens (1).




                                                                             az
                   Annalisa Palmieri2                                        From a pathological point of view, periodontitis can be
                                                                             defined as the presence of gingival inflammation at
                                                                             sites where there has been a pathological detachment
                   1 Department of Morphology, Surgery and Experimental      of collagen fibres from the cementum and the junctional




                                                                          rn
                     Medicine, University of Ferrara, Italy                  epithelium has migrated apically (2). The inflammatory
                   2 Department of Specialist Diagnostic and Sperimental     response of the periodontal tissues to infection is influ-
                     Medicine, University of Bologna, Italy                  enced by environmental factors as well as by genetic




                                                                       te
                   3 Department of Stomatologic and Maxillo-Facial           factors (3). The primary microbial factor contributing to
                     Sciences, University “Sapienza”, Rome, Italy            periodontitis is a shift in the content of the oral microflo-
                   4 Department of Interdisciplinary Medicine and Surgery,   ra, while the primary immunological factor is the de-
                     University of Milan-Bicocca, Milan, Italy               structive host inflammatory response (4).
                                                                    In       The microbiota associated with periodontal health and
                                                                             disease has been intensely studied for well over a cen-
                   Corresponding author:                                     tury by several generations of skilled scientists and clini-
                   Dorina Lauritano                                          cians (5, 6). Oral microbiota is an enormously complex
                   Department of Interdisciplinary Medicine and Surgery      and dynamic entity that is profoundly affected by perpet-
                                                       ni
                   University of Milan-Bicocca                               ually changing local environments and host-mediated
                   Via Cadore, 48                                            selective pressures (7). The presence of a commensal
                   20052 Monza, Italy                                        microbiota, including potential pathogens, is essential
                                                                             for the proper development of mucosal immunity (8).
                                          o

                   Phone: +39 3356790163
                   E-mail: dorina.lauritano@unimib.it                        The normal oral flora is hence in a balance between
                                                                             pathogens and commensals that requires regular
                                      izi


                                                                             cleaning to be maintained. A decrease in oral hygiene
                   Summary                                                   is quickly followed by the build-up of oral biofilms on
                                                                             tooth surfaces and, if left untreated, will progress to gin-
                                                                             gival inflammation and possibly periodontitis, alveolar
                         Ed




                   Periodontitis is a disease that affects and destroys
                   the tissues that support teeth. Tissues damage re-        bone loss and loss of teeth. It is likely that differences
                   sults from a prolonged inflammatory response to           in host-defence mechanisms, including antimicrobial
                                                                             protein profiles, determine whether bacterial coloniza-
                   an ecological shift in the composition of subgingi-
                                                                             tion progresses to overt disease (9).
                   val biofilms. Three bacterial species that constitute
                                                                             Recent data estimate that the oral cavity may contain
                   the red complex group, Porphyromonas gingivalis,
                                                                             up to 19000 bacterial phylo-types (10), but each individ-
               C




                   Tannerella forsythia, and Treponema denticola, are
                                                                             ual will only have a rate of the total numbers of
                   considered the main pathogens involved in peri-
                                                                             pathogens. Indeed, there is a substantial diversity in
                   odontitis.
    CI




                                                                             the content of the microflora between individuals (11)
                   In the present study a real-time PCR based assay
                                                                             and between different oral sites within the same individ-
                   was designed to detect and quantify red complex
                                                                             ual (12, 13). Research has indicated that dietary
                   species, then used to investigate 146 periodontal
                                                                             changes combined with poor hygiene can cause a shift
                   pocket samples from 66 periodontitis patients
                                                                             in the composition of the oral bacteria (13, 14). More-
                   and 80 controls. Results demonstrated a signifi-
                                                                             over, some evidence in recent studies suggests that
©




                   cant higher prevalence of red complex species
                                                                             the oral microbiome changes as humans age and the
                   and increased amount of P. gingivalis and T. den-
                                                                             dysbiosis in the oral cavity can lead to periodontitis (4).
                   ticola in periodontal pocket of periodontitis pa-         Several methods have been used for microbiological
                   tients.                                                   testing in periodontitis (15). However, many techniques
                                                                             have not been fully accepted due to low sensitivity or
                   Key words: tooth, ligament, periodontal, diseases, in-    specificity, moreover sometimes they are slow, expen-
                   flammation, bone, resorption.                             sive and laborious. In our laboratory (LAB srl, Ferrara,

                   170                                                                         Annali di Stomatologia 2013; IV (2): 170-173
1-Lauritano_- 19/06/13 10:32 Pagina 171




                   Oral microflora and periodontal disease: new technology for diagnosis in dentistry


                   Italy), we developed a rapid and sensitive test to detect        highly conservated sequence of the 16S ribosomal RNA
                   and quantify the three bacterial species more involved           gene. The second reaction detected and quantified the
                   in periodontitis that constituted the red complex group:         three red complex bacteria, i.e. P. gingivalis, T. forsythia
                   Porphyromonas gingivalis, Tannerella forsythia, and              and T. denticola, in a multiplex PCR. This reaction includ-
                   Treponema denticola. Both P. gingivalis and T. dentico-          ed a total of six primers and three probes that were highly




                                                                                                                                             li
                   la occur concomitantly with the clinical signs of peri-          specific for each specie. Oligonucleotide concentrations
                   odontal destruction. They appear closely ‘linked’ topo-          and PCR conditions were optimized to ensure sensitivity,




                                                                                      na
                   logically in the developing biofilm, shown an in vitro           specificity and no inhibitions in case of unbalanced target
                   ability to produce a number of outer membrane-associ-            amounts. Absolute quantification assays were performed
                   ated proteinases and are considered the first                    using the Applied Biosystems 7500 Sequence Detection
                   pathogens involved in the clinical destruction of peri-          System. The amplification profile was initiated by a 10 min




                                                                                    io
                   odontal tissues. Moreover both them and T. forsythia,            incubation period at 95°C to activate polymerase, fol-
                   show an higher prevalence in disease than in health              lowed by a two-step amplification of 15 s at 95°C and 60 s
                   suggesting that these bacterial are associated with the          at 57°C for 40 cycles. All these experiments were per-




                                                                                 az
                   local development of periodontitis (16).                         formed including nontemplate controls to exclude
                   The presence and the level of these pathogens can be             reagents contamination.
                   effectively revealed by real time polymerase chain reac-         Plasmids containing synthetic DNA target sequences
                   tion analysis using bacterial species-specific primers           (Eurofin MWG Operon, Ebersberg Germany) were
                   and probes.                                                      used as standard for the quantitative analysis. Stan-




                                                                              rn
                   Our ﬁndings support the hypothesis that detection and            dard curves for each target were constructed in a
                   quantification of red complex bacteria in crevicular fluid       triplex reaction, by using a mix of the same amount of
                   could be an appropriate tool for diagnosis and progno-           plasmids, in serial dilutions ranging from 101 to 107




                                                                           te
                   sis of periodontitis.                                            copies. There was a linear relationship between the
                                                                                    threshold cycle values plotted against the log of the
                                                                                    copy number over the entire range of dilutions (data not
                   Material and methods                                             shown). The copy numbers for individual plasmid
                                                                        In
                   A total of 146 individuals participated to the study, 66
                                                                                    preparations were estimated using the Thermo Nan-
                                                                                    oDrop spectrophotometer.
                   were affected by chronic periodontitis, while 80 consti-         The absolute quantification of total bacterial genome
                   tuted the control group. Controls include 46 healthy indi-       copies in samples allowed for the calculation of relative
                   viduals and 34 affected by a moderate gingivitis. Table 1        amount of red complex species. To prevent samples
                                                             ni
                   summarizes principal characteristics of the groups.              and polymerase chain reaction contamination, plasmid
                   A sample of the periodontal pocket microbiota was ob-            purification and handling was performed in a separate
                   tained from a single site by a paper probe. DNA was              laboratory with dedicated pipettes.
                   extracted and purified using standard protocols that in-
                                             o

                   clude two consecutive incubation with lysozyme and
                   proteinase K, followed by spin-column purification.              Statistical analysis
                                         izi



                                                                                    Descriptive statistics was performed using Microsoft
                   Real-Time Polymerase Chain Reaction                              Excel spreadsheets. The Freeman-Halton extension of
                                                                                    Fisher's exact test to compute the (two-tailed) probabili-
                           Ed




                   Primers and probes oligonucleotides were designed bas-           ty of obtaining a distribution of values in a 2x3 contin-
                   ing on 16S rRNA gene sequences of the Human Oral Mi-             gency table, given the number of observations in each
                   crobiome Database (HOMD 16S rRNA RefSeq Version                  cell. Odds ratio calculation was performed online at the
                   10.1) counting 845 entries. All the sequences were               OpenEpi web site (www.openepi.com).
                   aligned in order to find either consensus sequence or less       Absolute bacteria amount were normalized against the
                   conservate spots. Two real-time polymerase chain reac-           total bacterial load, obtaining the relative bacteria
               C




                   tion (PCR) runs were performed for each sample. The              amount (RBA). The one-way analysis of variance
                   first reaction quantified the total amount of bacteria using     (ANOVA) was used to determine whether there was
    CI




                   two degenerate primers and a single probe matching an            any significant differences between the mean RBA val-


                   Table 1. Principal characteristics of the groups.

                                                     Total              Healthy               Gingivitis                 Periodontitis
©




                   Subjects                          146                46                    34                         66
                   Males                             63                 12                    14                         37
                   Females                           83                 34                    20                         29
                   Age (means years ± SD)            39.8±18.9          31.6±18.6             34.3±15.4                  48.9±18.2
                   Sampling depht (mm±SD)            3.9±1.7            2.6±0.6               3.2±1.0                    5.0±1.7


                   Annali di Stomatologia 2013; IV (2): 170-173                                                                             171
1-Lauritano_- 19/06/13 10:32 Pagina 172




                   F. Carinci et al.


                   ue of three patients group, i.e. healthy, gingivitis and        of these three species among groups of patients with
                   periodontitis.                                                  different diagnosis - regardless of different clinical as-
                                                                                   pects that may describe severity of the disease – in or-
                                                                                   der to understand whether the presence of the red
                   Results                                                         complex species and their relative amount may be con-




                                                                                                                                                 li
                                                                                   sidered predictive factors of periodontitis.
                   Occurrence and amount of red complex bacteria from              Prevalence of the three investigated species among health,




                                                                                        na
                   crevicular fluid were evaluated in 146 individuals. A sin-      gingivitis and periodontitis patients was shown in Table 2.
                   gle specimen from each patient was analyzed by quan-            Each specie was common among healthy patients, howev-
                   titative real time PCR (LAB test LAB® s.r.l, Ferrara,           er the prevalence was roughly double in periodontitis
                   Italy) (Figs. 1, 2), obtaining measures of total bacteria       group. Intermediate values, but closer to healthy individuals




                                                                                      io
                   load and of three species involved in periodontitis, i.e.       were observes among patients affected by gingivitis.
                   P. gingivalis, T. forsythia, and T. denticola. Here we re-      The Freeman-Halton extension of Fisher's exact test in-
                   port a preliminary study focused mainly on prevalence           dicated that the prevalence of each red complex specie




                                                                                   az
                                                                                   is different among groups of patients with high degree
                                                                                   of statistical significance, P. gingivalis P value = 2x10-8,
                                                                                   T. forsythia P value = 1x10-8, and T. denticola P value =
                                                                                   2x10-4. The higher level of association with periodontitis
                                                                                   was observed for T. forsythia, indeed the observed




                                                                                rn
                                                                                   odds ratio was 6.1 (95% C.I. 3.1-11.9) when healthy in-
                                                                                   dividuals were compared to periodontitis patients, and
                                                                                   4.6 (95% C.I. 2.6-7.9) when healthy and gingivitis




                                                                             te
                                                                                   groups where combined and compared to periodontitis
                                                                                   patients.
                                                                                   Results of quantitative data indicated that the normal-
                                                                                   ized amount of P. gingivalis significantly differs among
                                                                          In       patient groups F(2, 304) = 7.77, P value = 0.001; as
                                                                                   well as for T. denticola F(2, 304) = 7.47, P value =
                                                                                   0.001. On the contrary did not vary for T. forsythia F (2,
                                                                                   304) = 1.41, P value = 0.25. The calculated mean val-
                   Figure 1. LAB®-test kit.                                        ues were plotted in Figure 3.
                                                         ni

                                                                                   Discussion
                                             o

                                                                                   The polymerase chain reaction (PCR) is the most sen-
                                                                                   sitive and rapid method to detect microbial pathogens
                                         izi


                                                                                   in clinical specimens. In particular, the diagnostic value
                                                                                   of PCR is significantly higher when specific pathogens
                                                                                   that are difficult to culture in vitro or require a long culti-
                                                                                   vation period such as for anaerobic bacteria species in-
                           Ed




                                                                                   volved in periodontitis onset. A recent improvement of
                                                                                   this technique is the real-time PCR that allow for quan-
                                                                                   titatation of DNA target using fluorogenic probes in a
                                                                                   close setup. Beside the opportunity to quantify target,
                                                                                   the advantage to perform the assay is a closed system,
                                                                                   in which the reaction tube is never opened after amplifi-
               C




                                                                                   cation, is of great value to prevent laboratory contami-
                   Figures 2 Method of sampling bacterial DNA.                     nation and false positive results. In addition the need of
    CI




                   Table 2. Association analysis between red complex bacteria and periodontitis.

                                                                                                                   OR (95% C.I.)
                                                Healthy [1]      Gingivitis [2]   Periodontitis [3]      [1] vs. [3]           [1]+[2] vs. [3]
©




                   P. gingivalis   negative     32               48               32
                                                                                                         5.1(1.8-4.3)          4.2(2.6-6.8)
                                   positive     38               32               34
                   T. forsythia    negative     49               31               22
                                                                                                         6.1(3.1-11.9)         4.6(2.6-7.9)
                                   positive     21               39               44
                   T. denticola    negative     42               38               43
                                                                                                         3.4(1.8-6.4)          2.4(1.5-3.9)
                                   positive     24               46               23


                   172                                                                                Annali di Stomatologia 2013; IV (2): 170-173
1-Lauritano_- 19/06/13 10:32 Pagina 173




                   Oral microflora and periodontal disease: new technology for diagnosis in dentistry




                                                                                                                                                           li
                                                                                       na
                                                                                     io
                   Figure 3. Plots representing the relative amount of each red complex bacterial specie in the different groups of patients.




                                                                                  az
                   a probe in addition to the two PCR primers, further in-                 review of definitions of periodontitis and methods that have
                   crease the specificity of the reaction.                                 been used to identify this disease. J Clin Periodontol 2009;
                   In the present investigation we designed and tested the                 36:458-467.




                                                                               rn
                   performance of a real-time PCR based assay to detect               3.   Kinane DF, Peterson M, Stathopoulou PG. Environmental
                                                                                           and other modifying factors of the periodontal diseases. Pe-
                   and quantify the red complex bacteria involved in peri-
                                                                                           riodontology 2000 2006; 40:107-119.
                   odontal disease. In particular we found that P. gingivalis,        4.   Berezow AB, Darveau RP. Microbial shift and periodontitis.
                   T. forsythia, and T. denticola were strongly related to pe-             Periodontology 2000 2011; 55:36-347.




                                                                            te
                   riodontitis because their prevalence was higher among              5.   Tanner ACR IJ. Tannerella forsythia, a periodontal pathogen
                   periodontitis patient. The presence of these bacterial                  entering the genomic era. Periodontology 2000 2006;
                   species can significantly increase the risk to develop pe-              42:88-113.
                   riodontitis, being the OR comprised between 6.1 (T. for-           6.   Teles RP, Haffajee AD, Socransky SS. Microbial goals of pe-
                                                                         In
                   sythia) and 3.4 (T. denticola). The results of quantitative
                                                                                      7.
                                                                                           riodontal therapy. Periodontol 2000 2006; 42:180-218.
                                                                                           Armitage GC. Comparison of the microbiological features of
                   data analysis indicated that the relative amount of P. gin-
                                                                                           chronic and aggressive periodontitis. Periodontol 2000
                   givalis and T. denticola in periodontal pocket was sensi-
                                                                                           2010; 53:70-88.
                   bly higher in affected patients. This indicated that both
                                                                                      8.   Davey ME, O'Toole GA. Microbial biofilms: from ecology to
                   the presence and relative amount of red complex bacte-
                                                             ni
                                                                                           molecular genetics. Microbiol Mol Biol Rev 2000; 64:847-867.
                   ria is relevant data in periodontal disease diagnosis.             9.   Gorr SU, Abdolhosseini M. Antimicrobial peptides and pe-
                                                                                           riodontal disease. J Clin Periodontol 38 Suppl 11:126-141.
                                                                                     10.   Slots J. Update on Actinobacillus Actinomycetemcomitans
                                             o

                   Conclusion                                                              and Porphyromonas gingivalis in human periodontal disease.
                                                                                           J Int Acad Periodontol 1999; 1:121-126.
                                                                                     11.   Nasidze I, Li J, Quinque D, Tang K, Stoneking M. Global di-
                                         izi


                   Molecular analysis of periodontal pocket microflora by
                   real-time PCR represent an effective inexpensive                        versity in the human salivary microbiome. Genome Res 2009;
                                                                                           19:636-643.
                   method to rapidly detect and quantify red complex bac-
                                                                                     12.   Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defin-
                   terial species. This test was performed in a large patient              ing the normal bacterial flora of the oral cavity. J Clin Microbiol
                          Ed




                   sample and results demonstrated that the test is a valu-                2005; 43:5721-5732.
                   able tool to improve diagnosis of periodontal disease.            13.   Avila M, Ojcius DM, Yilmaz O. The oral microbiota: living with
                                                                                           a permanent guest. DNA Cell Biol 2009; 28:405-411.
                                                                                     14.   Al-Ahmad A, Roth D, Wolkewitz M, Wiedmann-Al-Ahmad M,
                   Acknowledgenment                                                        Follo M, Ratka-Kruger P, et al. Change in diet and oral hy-
                                                                                           giene over an 8-week period: effects on oral health and oral
               C




                   This work was supported by LAB® s.r.l, Ferrara, Italy.                  biofilm. Clin Oral Investig 14: 391-396.
                                                                                     15.   Loomer PM. Microbiological diagnostic testing in the treat-
                                                                                           ment of periodontal diseases. Periodontology 2000 2004;
    CI




                                                                                           34:49-56.
                   References                                                        16.   Mineoka T, Awano S, Rikimaru T, Kurata H, Yoshida A, An-
                                                                                           sai T, et al. Site-specific development of periodontal dis-
                    1. Kuboniwa M, Inaba H, Amano A. Genotyping to distinguish             ease is associated with increased levels of Porphy-
                       microbial pathogenicity in periodontitis. Periodontology            romonas gingivalis, Treponema denticola, and Tannerel-
                       2000 2010; 54:136-159.                                              la forsythia in subgingival plaque. J Periodontol 2008;
©




                    2. Savage A, Eaton KA, Moles DR, Needleman I. A systematic             79:670-676.




                   Annali di Stomatologia 2013; IV (2): 170-173                                                                                          173
