10.59987/ads/2017.2.59-70
Aims. The present study aimed to investigate microbial patterns associated with disease progression and coinfection by different Herpesviruses in generalized aggressive periodontitis (GAP).
Methods. Microbiological samples were obtained from active (AS) and non-active (n-AS) sites in 165 subjects affected by GAP and were analyzed for 40 bacterial species by the Checkerboard DNA-DNA Hybridization technique and for Herpes simplex 1 (HSV-1), Human Cytomegalovirus (CMV), and Epstein Bar virus (EBV) by PCR. Common Factor Analysis and Multiple Regression Analysis were applied to disclose specific microbial patterns associated with the three viruses.
Results. Herpesviruses were detected in 37.6% of subjects. Detection of each of the searched viruses was associated with specific patterns of subgingival biofilm in AS. Logistic regression analyses evidenced several virus/bacteria associations: i) EBV with Aggregatibacter actinomycetemcomitans; ii) CMV with A. actinomycetemcomitans, Veillonella parvula, Parvimonas micra and Fusobacterium nucleatum subsp. polymorphum; iii) HSV-1 with Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium periodonticum and Staphylococcus aureus.
Conclusions. Microbiological data suggest that Herpesviruses are probably not mere spectators of disease progression and that specific patterns of subgingival plaque are correlated with the presence of different Herpesviruses.
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S. Petti 
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Annali di stomatologia
disease progression
Evaluation of microbiota associated with Herpesviruses in active sites of generalized aggressive periodontitis
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Original Article
2022-08-10
2022-08-10
2017-06-01
2023-12-04
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Ann Stomatol (Roma)
S. Petti 
2017
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1971-1441
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disease progression
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Evaluation of microbiota associated with Herpesviruses in active sites of generalized aggressive periodontitis
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                                                                                               Original article


    Evaluation of microbiota associated with
    Herpesviruses in active sites of generalized
    aggressive periodontitis




                                                                                             li
                                                                                           na
    Claudio Passariello DDS1*                               monas micra and Fusobacterium nucleatum sub-
    Pierangelo Gigola MD2                                   sp. polymorphum; iii) HSV-1 with Porphyromonas
    Luca Testarelli DDS3                                    gingivalis, Tannerella forsythia, Fusobacterium




                                                                                         io
    Monica Puttini MD4                                      periodonticum and Staphylococcus aureus.
    Serena Schippa PHD1                                     Conclusions. Microbiological data suggest that
    Stefano Petti DDS1                                      Herpesviruses are probably not mere spectators




                                                                      az
                                                            of disease progression and that specific patterns

    1 Department of Public Health and Infectious
                                                            of subgingival plaque are correlated with the

    Diseases, “ Sapienza” University of Rome, Rome, Italy
                                                            presence of different Herpesviruses.

    2 Department of Medical-Surgical Specialties,




                                                                    rn
    Radiologic and Public Health Sciences University of
                                                            Key words: generalized aggressive periodontitis,

    Brescia, Brescia, Italy
                                                            herpesviruses, microbiota, disease progression.

    3 Department of Odontostomatologic and Maxillo-

    Facial Sciences, “Sapienza” University of Rome,




                                                      te
                                                            Introduction
    Rome, Italy
    4 Free Practice in Dentistry, Genova, Italy             Periodontitis is a group of infectious disorders char-
                                                            acterized by inflammation affecting periodontal tis-
                                                            sues and causing an inconstant, episodic progression
                                                    In
                                                            in breakdown of the teeth-supporting tissues. In some
    Claudio Passariello                                     patients progression of periodontitis is characterized
    Corresponding author:

    Department of Public Health and Infectious Diseases,    by shortened periods of remission, leading to a clini-
                                                            cal presentation called aggressive periodontitis, that
    “Sapienza” University of Rome
                                ni

                                                            can be either generalized or localized (1-3). The rea-
    P.le Aldo Moro 5
                                                            sons leading some patients to undergo generalized
    00185 Rome, Italy
                                                            aggressive periodontitis (GAP) are poorly under-
    E-mail: claudio.passariello@uniroma1.it
                              io


                                                            stood, and no definite useful molecular or microbio-
                                                            logical markers or predictors of disease progression
                                                            have been identified yet (4, 5). Recent reports sug-
                                                            gest moreover that atypical pathogens are frequently
                            iz



    Summary
                                                            associated with progression of periodontal lesions
                                                            and resistance to conventional treatments in aggres-
    Aims. The present study aimed to investigate mi-
                                                            sive periodontitis and have disclosed interesting as-
            Ed




    crobial patterns associated with disease progres-
    sion and coinfection by different Herpesviruses in      sociations (6, 7). These atypical pathogens, including
    generalized aggressive periodontitis (GAP).
                                                            and different members of the family Enterobacteri-
                                                            Staphylococcus aureus, Pseudomonas aeruginosa
    Methods. Microbiological samples were obtained
    from active (AS) and non-active (n-AS) sites in         aceae, are known to possess virulence factors that
    165 subjects affected by GAP and were analyzed          could account for the accelerated tissue destruction
                                                            characterizing GAP.
 IC




    for 40 bacterial species by the Checkerboard
    DNA-DNA Hybridization technique and for Herpes          Recent studies by our group have shown higher
                                                            Staphylococcus aureus oral carriage rates in peri-
                                                            odontitis affected patients as compared to healthy
    simplex 1 (HSV-1), Human Cytomegalovirus

                                                            controls (8). Moreover, we showed that isolation of S.
    (CMV), and Epstein Bar virus (EBV) by PCR.
C




                                                            aureus from subgingival sites of GAP positively corre-
    Common Factor Analysis and Multiple Regression

                                                            lates with disease progression, with higher levels of
    Analysis were applied to disclose specific micro-

                                                            inflammatory mediators and with faster periodontal
    bial patterns associated with the three viruses.

                                                            breakdown (9).
    Results. Herpesviruses were detected in 37.6% of
©




                                                            Subgingival isolates of S. aureus from active sites of
    subjects. Detection of each of the searched virus-

                                                            GAP were shown to possess a set of genes encoding
    es was associated with specific patterns of sub-

                                                            for pathogenicity and virulence factors (9) which are
    gingival biofilm in AS. Logistic regression analy-

                                                            already known to be involved in the pathogenesis of a
    ses evidenced several virus/bacteria associa-

                                                            variety of severe staphylococcal infections (10).
    tions: i) EBV with Aggregatibacter actino-

                                                            Among factors potentially able to trigger and amplify
    mycetemcomitans; ii) CMV with A. actino-
    mycetemcomitans, Veillonella parvula, Parvi-

    Annali di Stomatologia 2017;VIII (2):59-70                                                                 59
    C. Passariello et al.


    tissue destruction in GAP, co-infection by different         pesviruses can influence the qualitative and quantita-
    Herpesviruses has been given wide consideration in           tive composition of subgingival plaque and play a role
    the last decades.                                            in the progression of lesions in GAP.
    Studies started about three decades ago have shown           The present study was consequently designed as an
    that HSV-1, EBV and CMV are detected with high               observational study aimed to characterize the subgin-
    prevalence and at high copy counts in progressive            gival microbiota of active sites of GAP and to corre-
    periodontal diseases (11-13).                                late microbial patterns with the presence/absence of




                                                                                                    li
    Association and immunologic studies have provided a          three species of Herpesviruses.




                                                                                                  na
    solid base of evidence supporting a periodontopathic
    role of Herpesviruses, although at present the specif-
    ic molecular and cellular mechanisms which enable
    these viruses to exacerbate periodontitis are still to
                                                                 Materials and methods

    be clarified.




                                                                                                io
    In a recent review of the literature Slots in summariz-      Patients with a clinical diagnosis of GAP (1) and with
                                                                 Studied population and samples

    ing results from studies performed by different groups       at least one active site were selected for the present
    in different countries evidenced that mean percent-          observational study as described previously (9).




                                                                           az
    ages of sites of aggressive periodontitis reported to        Briefly, microbiological samples were obtained from
    be positive for detection of Herpesviruses are 49% for       subjects attending 12 private dental practices for peri-
    CMV, 45% for EBV and 63% for HSV (11). A detailed            odontal problems.
    analysis of available data evidenced the existence of        All patients were recruited from structures located in




                                                                         rn
    significant differences among published studies and a        highly urbanized areas (Genua, Brescia, Piacenza,
    possible bias coming from the fact that in most stud-        Savona, Milan) and consisted prevalently of subjects
    ies sampling was performed regardless of disease             with high school or university degrees. Overall, 214
    activity.                                                    patients were selected during first visits, basing on an




                                                          te
    In a recent report Stein et al. failed to find any associ-   initial diagnosis of generalized aggressive periodonti-
    ation between aggressive periodontitis and the pres-         tis. The following criteria were used for inclusion in
    ence of Herpesviruses in a sample of patients in Ger-        the study: age ranging ≥ 18 - ≤ 39 years; presence of
                                                        In
    many (14).                                                   ≥ 20 teeth in the mouth; presence of at least 6 teeth
    Sunde et al. found that 40% and 12% of periodontitis         with Periodontal Probing Depth (PPD) ≥ 5 mm and
    sites were positive, respectively, for EBV and CMV,          with bleeding on probing (including at least one in-
    although viral loads were close to detection limits          cisor and one first molar). The following criteria were
    (15). Consequently they considered a role for these          used for exclusion from the study: presence of sys-
                                ni

    viruses in the pathogenesis of periodontal lesions un-       temic diseases or conditions known to affect antibac-
    likely and, possibly, only the consequence of contam-        terial defences, and/or soft tissues and epithelial bar-
    ination by blood or saliva or of an accumulation of          rier integrity; subjects who had assumed antibiotics,
                              io


    lymphoid cells harbouring viruses in the inflamed tis-       and/or topic antimicrobials, and/or who underwent pe-
    sue.                                                         riodontal treatment in the previous 6 months; preg-
    On the other hand many other Authors found a posi-           nant or breast feeding subjects.
                            iz



    tive correlation between detection of Herpesviruses          Periodontal status was assessed in terms of Visible
    and severity and progression of periodontal lesions of       Plaque Index (VPI), Gingival Bleeding Index (GBI),
    both chronic and aggressive periodontitis (11, 16-19).       and Periodontal Probing Depth (PPD) at the begin-
           Ed




    Histomolecular analyses demonstrated that gingival           ning of the study (T0), and after 45 days (T45). Peri-
    epithelial cells of periodontitis affected sites are fre-    odontal sites showing a significant increase in both
    quently infected by one or more Herpesviruses and            PPD and GBI between T0 and T45 were considered
    that viral loads positively correlate with disease           as active sites (AS) and were distinguished from non-
    severity (20, 21).                                           active sites (n-AS).
    Overall, experimental data collected by different re-        VPI and GBI were evaluated separately at the four
 IC




    search groups in different geographical areas sug-           main aspects of each tooth, while PPD was evaluated
    gest that human Herpesviruses could play a relevant          at six sites for each tooth, using a Michigan periodon-
    role in both chronic and aggressive periodontitis (12-       tal probe with Williams markings.
    16), although some Authors hypothesize that the              Enrolment in the study was confirmed upon detection
C




    prevalence of HSV, CMV, and EBV in periodontal le-           of at least one AS between T0 and T45.
    sions could vary also on a geographical and ethnic           Each patient was examined at T0 and T45 by the
    base (22).                                                   same examiner.
    It is well known that many viruses are able to act as        All clinical evaluations were performed separately by
©




    strong promoters of bacterial pathogenicity and viru-        two experienced clinicians (P.G. and M.P.), calibrated
    lence, thus facilitating the onset and progression of        to provide consistent diagnoses as follows: twenty
    acute, aggressive infections in many districts of the        subjects were selected among those participating in
    human body (23-25). It is consequently conceivable           the study. Each examiner recorded values of PPD at
    that, as a consequence of specific molecular mecha-          the 6 aspects of 6 selected teeth (2 molars, 2 premo-
    nisms of cooperation, co-infection by different Her-         lars and 2 incisors) for each subject; measures were

    60                                                                              Annali di Stomatologia 2017;VIII (2):59-70
    Herpesviruses and microbiota in periodontitis


    repeated after 48 hours at the same sites. Values         kit (Fermentas). Quantitative determinations were
    recorded by the two examiners at the same time were       performed following detection by a densitometric
    used to calculate inter-examiner reproducibility. Val-    scanner (BioRad GS800 calibrated densitometer) us-
    ues recorded by each examiner at different times          ing the Quantity One® analysis software (BioRad)
    were used to calculate intra-examiner reproducibility.    and expressed as percent of total DNA counted in the
    Inter- and intra-examiner reproducibility was mea-        sample. Samples giving no signal were all reported
    sured through Cohen’s weighted kappa which result-        as scoring 0.1% of total bacterial DNA in the sample,




                                                                                                li
    ed 0.70 (intra-examiner 1), 0.88 (intra-examiner 2)       to account for limits of sensitivity of the test.




                                                                                              na
    and 0.75 (inter-examiner), suggesting substantial to
    almost perfect agreement (26).
    One hundred and sixty-five of 214 patients (77.1%)        A second set of samples was obtained from each se-
                                                              Detection of Herpesviruses

    (mean age 32.3±3.8 years, ages ranging 18 to 39)          lected AS and n-AS, as described above, to detect
    were selected for microbiological sampling. Of these,     the presence of Herpes simplex type 1 (HSV-1), Hu-




                                                                                            io
    81 were males (mean age 32.4±3.6 years, ages rang-        man Cytomegalovirus (CMV) and Epstein Bar virus
    ing 21 to 39) and 84 were females (mean age               (EBV), using previously described specific PCR
    32.3±4.1 years, ages ranging 18 to 39).                   methods (30, 31).




                                                                        az
    The sampled patients participated voluntarily, were       Patients were split into four groups according to posi-
    explained the nature of the study and invited to sign     tivity for detection of HSV-1 (HSV+), EBV (EBV+),
    an informed consent in conformity with the Helsinki       CMV (CMV+), and negativity for detection of any of
    Declaration on Ethical Principles for Medical Re-         the searched viruses (HHV-).




                                                                      rn
    search Involving Human Subjects. During the 45 days
    of observation all patients were invited to maintain
    their standard oral hygiene procedures.                   The Student’s t-test for paired samples was per-
                                                              Statistics

    The study design was approved by the ethical com-         formed to detect AS, i.e. those sites showing signifi-




                                                       te
    mittee of the University of Brescia.                      cant differences in clinical variables (PPD and GBI)
                                                              between T0 and T45.
                                                              Significance of differences in the percentages of
                                                     In
    Following clinical evaluation at T45, samples of sub-     species-specific DNAs between AS and n-AS and,
    Microbiological sampling

    gingival biofilm were obtained from one AS and one        within AS, among the four groups (i.e. HSV+, EBV+,
    n-AS in each subject. Selection of AS and n-AS in         CMV+ and HHV-) were evaluated through the one-
    each patient was made paying attention to select two      way analysis of variance (ANOVA) for dependent
    sites resembling each other as much as possible with      samples. The Tukey’s HSD test was performed as
                                ni

    regard to position and PPD at the time of sampling.       post hoc test to detect differences between pairs of
    Samples of subgingival biofilm were collected follow-     groups.
    ing careful removal of supragingival biofilm by a ster-   In order to investigate the relative frequencies of sub-
                              io


    ile Columbia Universal curette: a sterile nr. 40 en-      gingival microbiota potentially associated with EBV,
    dodontic paper point was inserted into the pocket and     CMV and HSV-1 in AS, three logistic regression
    maintained in place for 60 seconds. Samples were          analyses were designed using EBV, CMV and HSV-
                            iz



    then transferred into 1.5 ml screw cap vials contain-     1, dichotomized into detected/undetected, as re-
    ing 0.5 ml of sterile DNAse free molecular grade wa-      sponse variables and the relative frequencies as ex-
    ter, vortexed for 2 minutes, stored at -80° C and         planatory variables, adjusted for patient’s age and
            Ed




    processed within 48 hours from sampling.                  gender, VPI and GBI as confounders.
                                                              Preliminarily the existence of inter-correlated bacteri-
                                                              al species was investigated by performing the corre-
    Total bacterial DNA was extracted and purified from       lation matrix with the Pearson’s correlation coeffi-
    Purification of bacterial DNA

    each sample as described previously (27) using the        cients “r” among species (threshold value was set at
    Nucleospin Genomic DNA purification Kit (Macherey-        r≥0.5). Common Factor Analysis (CFA) was then
 IC




    Nagel GmbH Düren, Germany) according to instruc-          used to reduce the number of bacterial variables (32-
    tions of the manufacturer. Purified DNA was quanti-       34).
    fied by the QubitTM quantitation system (Invitrogen,      CFA generated a small set of non-inter-correlated
    Milan, Italy) and stored at -80° C until used.            factors (i.e. groups of inter-correlated bacterial
C




                                                              species).
                                                              The association between each generated factor and
    Forty selected bacterial species (listed in Tab. 1)       EBV, CMV and HSV-1 was initially explored with lo-
    Quantitation of specific bacterial DNA in samples

    were quantitatively detected in total DNA samples by      gistic regression analysis. Factors resulting more
©




    a modified checkerboard DNA-DNA hybridization             strongly associated with EBV, CMV, and HSV-1 were
    technique (CKB) (28, 29).                                 first tested for multi-collinearity and then chosen for
    Species specific probes were prepared by the Biotin       the multiple logistic regression analysis (35).
    DecaLabel™ DNA Labeling Kit (Fermentas, Thermo            The goodness of fit of the models was assessed
    Fisher Scientific), and detected after high stringency    through the likelihood ratio c2 test (p<0.05) and pseu-
    hybridization using the Biotin Chromogenic detection      do-R 2 . The analysis was validated by splitting the

    Annali di Stomatologia 2017;VIII (2):59-70                                                                     61
    C. Passariello et al.


    Table 1. Bacterial strains used to prepare specific species probes for the modified checkerboard DNA-DNA Hybridization
    analysis.

    Bacterial Taxa                                                       Strain
    Actinomyces gerencseriae                                             CCUGa 32936T
                                                                         CCUG 18307T




                                                                                                     li
    Actinomyces israelii
                                                                         CCUG 18310T




                                                                                                   na
    Actinomyces naeslundii
    Actinomyces oris                                                     DSMb 23056
    Actinomyces odontolyticus                                            DSM 19120
                                                                         DSM 2008




                                                                                                 io
    Veillonella parvula
    Streptococcus gordonii                                               DSM 6777
    Streptococcus intermedius                                            DSM 20573
                                                                         DSM 12643




                                                                            az
    Streptococcus mitis
    Streptococcus oralis                                                 DSM 20623
    Streptococcus sanguinis                                              DSM 20567
                                                                         DSM 8324




                                                                          rn
    Aggregatibacter actinomycetemcomitans
    Capnocytophaga gingivalis                                            DSM 3290
                                                                         DSM 7171




                                                        te
    Capnocytophaga ochracea
    Capnocytophaga sputigena                                             DSM 7273
    Eikenella corrodens                                                  DSM 8340
                                                                         DSM 19528
    Campylobacter gracilis
                                                      In
    Campylobacter rectus                                                 DSM 3260
    Campylobacter showae                                                 DSM 19458
                                                                         DSM 3993
                                ni
    Eubacterium nodatum
    Fusobacterium nucleatum ss. nucleatum                                DSM 15643
                                                                         DSM 20482
                              io

    Fusobacterium nucleatum ss. polymorphum
    Fusobacterium nucleatum ss. vincentii                                DSM 19507
    Fusobacterium periodonticum                                          ATCCc 33693
                            iz



    Parvimonas micra                                                     ATCC 33270
    Prevotella intermedia                                                ATCC 25611
           Ed




    Prevotella nigrescens                                                ATCC 33563
    Streptococcus constellatus                                           DSM 20575
    Tannerella forsythia                                                 ATCC 43037
    Porphyromonas gingivalis                                             ATCC 33277
                                                                         DSM 14222
 IC




    Treponema denticola
    Eubacterium saburreum                                                ATCC 33271
    Gemella morbillorum                                                  DSM 20572
                                                                         DSM 1135
C




    Leptotrichia buccalis
    Neisseria mucosa                                                     DSM 17611
    Prevotella melaninogenica                                            ATCC25854
                                                                         DSM 20563
©




    Streptococcus anginosus
    Selenomonas noxia                                                    DSM 19578
    Treponema socranskii                                                 ATCC 35536
    Staphylococcus aureus                                                ATCC 12600



    62                                                                               Annali di Stomatologia 2017;VIII (2):59-70
    Herpesviruses and microbiota in periodontitis


    sample into two subsamples of equal size, and as-                  EBV+,CMV+, and HHV-.
    sessing the coefficients using one subsample and the               At T0, AS of CMV+ subjects showed significantly
    goodness of fit using the other subsample.                         higher mean VPI values as compared to those of
    Statistical analysis was made by a blinded statistician            HHV- subjects (Tab. 2).The AS of both HSV+ and
    (S.P.) who was not aware of the bacterial and viral                CMV+ subjects showed significantly greater increase
    taxa at the moment of analysis. The statistical soft-              in PPD between T0 and T45 as compared to those of
    ware StatView 5.0.1 (SAS® Institute Inc., NC, USA)                 HHV- subjects (Tab. 2).




                                                                                                         li
    was used. The level of significance was set at 95%.




                                                                                                       na
                                                                       The quantitative detection of 40 bacterial species in
                                                                       Characterization of subgingival biofilm

                                                                       subgingival biofilm samples by the CKB technique, as
                                                                       expected, showed the existence of several significant
    Results

    By adopting the selection criteria described in the                differences between AS and n-AS (Fig. 1). AS were




                                                                                                     io
    materials and methods section, 165 AS and 165 n-AS                 overall characterized by the presence of significantly
    were selected for microbiological sampling. Both the               higher amounts of Aggregatibacter actinomycetem-
    AS group and the n-AS group comprised 45 upper




                                                                                 az
    molars, 41 lower molars, 19 upper premolars, 16 low-               tum, Fusobacterium nucleatum subsp. nucleatum,
                                                                       comitans, Campylobacter rectus, Eubacterium noda-

    er premolars, 31 upper incisors and 13 lower incisors.
    Overall, according to clinical values recorded at T0,
                                                                       Prevotella intermedia, Prevotella nigrescens, Tan-

    AS and n-AS were comparable in terms of PPD                        ma denticola, Treponema socranskii, and S. aureus
                                                                       nerella forsythia, Porphyromonas gingivalis, Trepone-

    (mean values being 5.5 mm and 5.6 mm respectively,                 as compared to n-AS (Fig. 1). As opposite, Actino-




                                                                               rn
    P = 0.24), VPI (mean values being 1.6 and 1.5 re-
    spectively, P = 0.35), and GBI (mean values being
                                                                       myces gerencseriae, Actinomyces naeslundii, Veil-

    1.7 and 1.6 respectively, P = 0.39). In accordance
                                                                       lonella parvula, Streptococcus mitis, Capnocythopha-




                                                             te
    with criteria adopted for selection, AS alone showed a
                                                                       ga gingivalis, Capnocythophaga ochracea, Eikenella

    significant increase in both PPD and GBI between T0                lorum, and Neisseria mucosa, were represented in
                                                                       corrodens, Eubacterium saburreum, Gemella morbil-

    and T45, while values of VPI did not change signifi-               significantly lower amounts in AS as compared to n-
    cantly.                                                            AS (Fig. 1).
                                                           In
                                                                       Significant differences were also detected in the
                                                                       quantitative composition of subgingival biofilm from
    Sixty-two out of 165 subjects (37.6%) resulted posi-               AS of HSV+, EBV+, CMV+ subjects as compared to
    Detection of Herpesviruses in subgingival plaque

    tive for the detection of one of the 3 searched virus-             HHV- subjects (Fig. 2).
                                ni

    es. Prevalence was 14.5, 13.9 and 9.1% for HSV-1,                  In fact, sites of HSV+ subjects were characterized by
    EBV, and CMV respectively. None of the studied                     significantly higher amounts of T. forsythia, P. gingi-
    sites resulted positive for detection of more than one             valis, and S. aureus and by significantly lower
                              io


    of the searched viruses. No difference was observed                amounts of A. actinomycetemcomitans, C. showae,
    in positivity for the three searched viruses between               E. nodatum, F. nucleatum subsp. nucleatum, P. inter-
    AS and n-AS in the single patients. No statistically               media, P. nigrescens and G. morbillorum as com-
                            iz



    significant differences were observed with regard to               pared to HHV- subjects (Fig. 2).
    age and gender among subjects divided in HSV+,                     Sites of EBV+ subjects were characterized by signifi-
            Ed




    Table  2. Distribution of theCytomegalovirus
                                  sample according to positivity
                                                         or no for  detection of type 1 HerpesMean
                                                                                               Simplex Virus
                                                                                                          of (HSV+),  Epstein Bar
    Table 2. Distribution of the sample according to positivity for detection of type 1 Herpes Simplex virus (HSV+), Epstein Bar

    Virus (EBV+) and Human Cytomegalovirus (CMV+) or no Human Herpesvirus (HHV-). Mean values of Visible Plaque Index
    virus (EBV+)   and Human                     (CMV+)          Human  Herpesvirus   (HHV-).      values    Visible Plaque Index

    (VPI), Gingival Bleeding Index (GBI) and periodontal probing depth (PPD) detected in AS at T0 and their mean variations
    (VPI), Gingival Bleeding Index (GBI) and periodontal probing depth (PPD) detected in AS at T0 and their mean variations
    between T0 and T45 (∆VPI, ∆GBI, ∆PPD).
    between T0 and T45 (!VPI, !GBI, !PPD).
 IC




          Patient group            Number          VPI        GBI            PPD          !VPI            !GBI         !PPD
        according to virus          out of       mean        mean          mean mm      mean score        mean          mean
            detection                165         score       score          (± SD)       T45-T0          score          mm
C




                                                 (± SD)      (± SD)                                      T45-T0        T45-T0
     HSV+                              24         1.5          1.5            5.6           0.1            0.5          1.5*
                                                 (±0.5)       (±0.5)         (±0.7)        (±0.3)         (±0.5)       (±0.5)
©




     EBV+                              23          1.4         1.5            5.5           0.1            0.5          1.2
                                                 (±0.50)      (±0.5)         (±0.6)        (±0.3)         (±0.5)       (±0.4)

     CMV+                              15         1.7*         1.6             5.5          0.1            0.5          1.3*
                                                 (±0.5)       (±0.5)         (±0.5)        (±0.5)         (±0.5)       (±0.5)

     HHV-                             103          1.5         1.7            5.6           0.0            0.4          1.1
                                                 (±0.50)      (±0.5)         (±0.6)        (±0.3)         (±0.5)       (±0.3)


    Annali di Stomatologia 2017;VIII (2):59-70                                                                                  63
    C. Passariello et al.




                                                                                                        li
                                                                                                      na
                                                                                                    io
                                                                               az
                                                                             rn
                                                          te
                                                        In
    Figure 1. Plot of differences of percentages of total DNA probe count of the 40 test species in subgingival biofilm samples
    between 165 AS and 165 n-AS of generalized aggressive periodontitis. Significant differences between AS and n-AS for
    each species are indicated with an asterisk. Species were ordered and grouped according to the complexes described by
                                ni

    Socransky et al., 1998 (28).
                              io


    cantly higher amounts of A. actinomycetemcomitans               with factor 8 and inversely associated with factor 1
    and by significantly lower amounts of F. nucleatum              (Tab. 3).
    subsp. vincentii, F. periodonticum, T. forsythia, P.            According to the simple logistic regression analyses,
                            iz



    gingivalis, and E. saburreum as compared to HHV-                three factors (factors 2, 4, and 8) were marginally
    subjects (Fig. 2).                                              (i.e., 0.20<p<0.05) or significantly (i.e., p≤0.05) as-
    Sites of CMV+ subjects were characterized by signifi-           sociated with EBV, three (factors 5, 8, and 11) with
           Ed




    cantly higher amounts of A. actinomycetemcomitans               CMV, and three (factors 1, 8, and 10) with HSV
    and by significantly lower amounts of F. nucleatum              (Tab. 4).
    subsp. vincentii, T. forsythia, P. gingivalis, and E.           Multiple logistic regression analysis showed that: 1)
    saburreum as compared to HHV- subjects (Fig. 2).                factor 8 was the only one being significantly and in-
                                                                    versely associated with EBV; 2) factor 11 was signifi-
                                                                    cantly and directly associated with CMV, while factors
    Analysis of the association of subgingival microbiota
 IC




    Analysis of values of skewness and kurtosis showed              5 and 8 were significantly and inversely associated
    with Herpesviruses.

    that percentages of total DNA probe counts of the               with CMV; 3) factor 8 was significantly and directly
    studied bacterial species followed a reasonably nor-            associated with HSV-1, while factors 1 and 10 were
    mal distribution (data not shown). Analysis of the              significantly and inversely associated with HSV-1
C




    Pearson’s correlation coefficients between relative             (Tab. 5). The three regression models were not dis-
    frequencies of bacterial species strongly justified the         turbed by multi-collinearity, (VIFs = 0.10). The likeli-
    use of CFA (data not shown). CFA generated 19 fac-              hood c2 tests resulted highly significant (P<0.0001),
    tors; of these, 15, showing eigenvalues >1, were se-            while the Pseudo-R2 values were all 0.6 for the three
©




    lected as meaningful and subjected to factor rotation.          models. These tests suggest that the variables in-
    After oblique and orthogonal rotation, the 15 selected          cluded in the regression models were highly predic-
    factors accounted for variance proportions ranging              tive.
    between 4.1 and 13.2% (Tab. 3). All bacterial taxa              Following validation analysis, factors 2 and 4 were no
    were included in only one factor, excluding P. gingi-           longer associated with EBV, factor 5 was no longer
    valis and T. forsythia which were directly associated           associated with CMV, and factor 10 was no longer

    64                                                                                  Annali di Stomatologia 2017;VIII (2):59-70
    Herpesviruses and microbiota in periodontitis




                                                                                                          li
                                                                                                        na
                                                                                                      io
                                                                                   az
                                                                                 rn
                                                                te
    Figure 2. Mean percentage (±standard deviation) of the total DNA probe count of the 40 test species in subgingival biofilm
                                                              In
    samples from 165 AS of generalized aggressive periodontitis distinguished according to positivity for detection of HSV-1
    (HSV+), EBV (EBV+), CMV (CMV+) or no Herpesvirus (HHV-) specific sequences. Significance of differences for each
    species between AS of HSV+, or EBV+, or CMV+, and HHV- sites is indicated by replicating the symbol corresponding to
    each category at the top of the plot. Species were ordered and grouped according to the complexes described by Socran-
                                ni

    sky et al., 1998 (28).
                              io


    Table 3. Meaningful factors generated by Common Factor Analysis and bacterial species resulting strongly directly or
    inversely associated with each dimension (i.e. factor loading assessed with Pearson’s correlation coefficients between
    bacterial species and dimension ≥0.33).
                            iz



    Factor      Accounted                          Bacterial species associated with dimension
             Ed




                Variance (%)        Directly                                            Inversely

    1           13.2                E.nodatum, P.intermedia, P.nigrescens                S.aureus, T.forsythia, P.gingivalis
    2           10.4                A.israelii, T.denticola, T.socranskii
    3           7.2                 C.rectus, S.noxia
    4           7.0                 A.oris, N.mucosa
 IC




    5           7.1                 E.suburreum, F.nucleatum subsp vincentii
    6           6.4                 C.gracilis, S.anginosus
    7           6.5                 A.odontolyticus, C.gingivalis
    8           8.7
C




                                    P.gingivalis, T.forsythia, F.periodonticum           A.actinomycetemcomitans
    9           6.9                 S.intermedius, P.melaninogenica, S.constellatus
    10          4.7                 G.morbillorum                                        L.buccalis
    11          4.3                 P.micra, F.nucleatum subsp. polymorphum
©




                                                                                         V.parvula
    12          4.8                 E.corrodens, S.sputigena                             S.sanguinis
    13          4.1                 A.gerencseriae, A.naeslundii                         S.gordonii
    14          4.1                 C.showae, C.ochracea
    15          4.6                 S.mitis, S.oralis



    Annali di Stomatologia 2017;VIII (2):59-70                                                                                 65
    C. Passariello et al.


    Table 4. Association (OR with 95% confidence interval -95CI) between each single factor and EBV, CMV and HSV-1,
    adjusted for confounders and assessed through logistic regression analysis. Factors with p<0.20 were included in the fi-
    nal logistic regression models.

    Factor                                               Association with OR (95CI)




                                                                                                            li
                             EBV                                      CMV                               HSV-1

    1                                                                                                   Inverse 0.1 (0.1-0.3) **




                                                                                                          na
    2                        Direct 1.4 (0.9-2.2)*

    4                        Direct 1.4 (0.9-2.2) *

    5                                                                 Inverse 0.6 (0.4-1.1) *




                                                                                                        io
    8                        Inverse 0.1 (0.03-0.2) **                Inverse 0.1 (0.05-0.3) **         Direct 4.1 (1.7-10.1) **

    10                                                                                                  Inverse 0.7 (0.4-1.0) *




                                                                                  az
    11                                                                Direct 2.2 (1.2-4.1) **

    * 0.20<p<0.05: marginally associated; included in the final model.
    ** p≤0.05: significantly associated; included in the final model.




                                                                                rn
    Table 5. Multivariate association (OR with 95% confidence interval -95CI) between the meaningful factors and Epstein
    Bar Virus (EBV), Human Cytomegalovirus (CMV), and type 1 Herpes Simplex Virus (HSV-1), adjusted for confounders




                                                              te
    and assessed through logistic regression analysis.

    Factor
                                                            In  OR (95CI)

                            EBVa                                CMVb                               HSV-1c

    1                                                                                              0.1 (0.04-0.3) – p<0.0001

    2                       1.8 (0.8-4.0) – p=0.14d
                                ni

    4                       2.0 (0.9-4.5) – p=0.08d

    5                                                           0.4 (0.2-0.9) – p=0.02d
                              io


    8                       0.05 (0.01-0.2) – p<0.0001          0.07 (0.02-0.3) – p<0.0001         12.7 (2.1-75.5) – p=0.005

    10                                                                                             0.5 (0.2-1.0) – p=0.05d
                            iz



    11                                                          4.6 (1.6-13.6) – p=0.005

    Whole model goodness of fit:
             Ed




    a likelihood-ratio χ2 =84.16; p<0.0001. Pseudo-R2=0.63
                         7df
    b likelihood-ratio χ2 =59.99; p<0.0001. Pseudo-R2=0.60
                         7df
    c likelihood-ratio χ2 =83.22; p<0.0001. Pseudo-R2=0.61
                         7df
    d validation analysis did not confirm the statistically significant association
 IC




    associated with HSV-1. The likelihood c 2 tests re-                ing able to trigger the activation of sites are still de-
    mained highly significant (P<0.0001).                              bated (36, 37).
                                                                       Most Authors agree to recognize in a limited number
                                                                       of microbial species, grouped in a few complexes, the
C




                                                                       principal and most frequent etiologic agents of com-
                                                                       mon clinical forms of periodontal disease28. Neverthe-
    Discussion

    Powerful molecular methods of investigation intro-                 less, it has been suggested that non typical bacterial
    duced during the last few decades enabled a sub-                   species and some Herpesviruses might also cooper-
©




    stantial improvement of our knowledge on different                 ate to accelerate disease progression and its suscep-
    aspects of the oral and periodontal microbiota and on              tibility to periodontal treatments (6, 7, 9, 11, 16, 17,
    its interactions with host defenses.                               19, 22, 38).
    Nevertheless, the etiopathogenesis of different clini-             The role of Herpesviruses in the etiopathogenesis of
    cal forms of periodontitis, the specific mechanisms                periodontal diseases is still the object of discussion
    accounting for its episodic progression and those be-              and different Herpesviruses have been detected with

    66                                                                                      Annali di Stomatologia 2017;VIII (2):59-70
    Herpesviruses and microbiota in periodontitis


    variable prevalence in a number of different studies,        to data reported in other studies (11, 15, 16, 19, 22).
    starting to be published about three decades ago.            The fact that we found full correspondence with re-
    Large part of these studies evidenced that HSV-1,            gard to positivity/negativity for detection of viruses
    EBV and CMV are detected with high prevalence in             between experimental (AS) and control sites (n-AS) is
    periodontal lesions, that they directly infect gingival      not surprising, nor in contrast with previous studies in
    epithelial cells and that viral loads positively correlate   which experimental, diseased sites were compared to
    with disease severity (11-13, 16-21).                        control healthy sites demonstrating the existence of




                                                                                                    li
    Although some recently published studies failed to           significant differences in viral loads between the two




                                                                                                  na
    find any association between aggressive periodontitis        groups (11, 15-17, 19-22).
    and the presence of Herpesviruses (14), or detected          Analysis of clinical data of patients, aggregated ac-
    the three viruses at very low levels, and suggested          cording to positivity/negativity for detection of each of
    that their detection could possibly be the conse-            the three searched viruses, evidenced that the four
    quence of contamination by blood or saliva or of lym-        groups were comparable with regard to age and gen-




                                                                                                io
    phoid cells (15), available data, considered altogether      der. At T0 they were also comparable with regard to
    suggest that human Herpesviruses probably play a             VPI, GBI and PPD (Tab. 2). Exception to this were
    relevant role in both chronic and aggressive peri-           CMV+ ASs showing significantly higher VPI values at




                                                                           az
    odontitis (12-16), and that differences can in large         T0 as compared to those of HHV- subjects (P=0.04).
    part be explained on the basis of geographical and           HSV-1+ and CMV+ ASs showed significantly greater
    ethnic variability (22).                                     increase of PPD between T0 and T45 as compared to
    In a critical review of the literature that was published    HHV- ASs (P<0.01). These data suggest that at least




                                                                         rn
    recently, Slots evidenced that the possibility to corre-     CMV and HSV-1 are not mere spectators of the pro-
    late the presence of Herpesviruses with periodontal          gression of GAP lesions.
    disease progression is biased by the fact that in most       The second sample from each studied site was used
    studies sampling was performed regardless of dis-            to analyze composition of the subgingival biofilm by




                                                          te
    ease activity.                                               means of a modified CKB technique (28, 29); main
    Recently we demonstrated that subjects affected by           modifications regarded inclusion in the panel of an S.
    periodontal diseases show higher prevalence of S.            aureus specific probe, and column purification of
                                                        In
    aureus in their oral microbiota and that they common-        metagenomic DNA, instead of simple alkaline lysis, in
    ly host highly toxigenic strains of such an opportunis-      order to obtain more efficient DNA extraction from
    tic pathogen (9).                                            Gram positives and to reduce interference due to mi-
    Moreover, we showed that the isolation of S. aureus          crobial proteins in crude lysates.
    from subgingival sites of GAP correlates with disease        The comparative, semiquantitative analysis of the
                                ni

    progression.                                                 metagenome from subgingival biofilm samples ob-
    Having cumulatively considered the above observa-            tained from AS and n-AS (Fig. 1) substantially con-
    tions, data on composition of the subgingival micro-         firmed previous observations by other Authors and
                              io


    biota of GAP, and data on detection of Herpesviruses         reinforced evidence of the role played by certain
    from subgingival sites of GAP, we designed the pre-          known periodontopathogens and atypical pathogens
    sent study to characterize the subgingival microbiota        (namely S. aureus in our study) in the progression of
                            iz



    of AS of GAP and to correlate microbial patterns             lesions of GAP (4-7, 9).
    found in these sites with the presence/absence of            Data presented here confirm previous reports and
    Herpesviruses.                                               suggest that the presence of Herpesviruses affects
            Ed




    To this purpose, one AS and one n-AS were selected           composition of the subgingival biofilm in GAP and
    in a population of subjects affected by GAP. Attention       prevalence of certain periodontopathogens (22).
    was dedicated at selecting two sites that differed al-       Moreover, they show that virus specific microbial pat-
    most only with regard to activity of the lesion; basing      terns characterize both AS and n-AS and that, during
    on a protocol of repeated clinical examination extend-       active progression of lesions, the prevalence of cer-
    ing over a period of 3 months the time interval of 45        tain bacterial species in the subgingival biofilm
 IC




    days was found as the most appropriate to detect ac-         changes in a virus specific manner.
    tively progressing sites in the studied population (9).      To our knowledge this is the first investigation aiming
    Two samples of subgingival biofilm were obtained             to characterize microbial patterns associated with ac-
    from each selected site.                                     tive progression of lesions of GAP and to evaluate at
C




    One sample for each studied site was processed to            the same time if the presence/absence of three
    detect the presence of HSV-1, EBV, or CMV by stan-           species of Herpesviruses is associated with specific
    dard PCR methods. The analyzed sites were conse-             microbial patterns.
    quently distinguished in four groups according to pos-       In order to disclose specific microbial patterns associ-
©




    itivity for detection of HSV-1, EBV, CMV or negativity       ated with the presence/absence of each of the three
    for Herpesviruses (HHV-).                                    Herpesviruses and with active progression of lesions
    Adoption of standard PCR methods and the fact that           required an articulated analysis of data, in which the
    only two samples were analyzed from each subject             relative frequencies of the searched bacterial species
    could account for the relatively low number of sites         were evaluated considering the potential influence of
    that resulted positive for Herpesviruses as compared         general variables (age, gender, hygienic skills, reac-

    Annali di Stomatologia 2017;VIII (2):59-70                                                                         67
    C. Passariello et al.


    tivity of the host, local anatomy) and specific vari-        munity, on the expression of proteins at the surface
    ables (co-variation of groups of inter-related bacterial     of epithelial cells, and on the expression of adhesins
    species) (34).                                               and toxins by microorganisms (28, 40-43).
    Consequently data obtained by CKB from subgingival           Recent researches showed that polymicrobial syner-
    biofilm samples of AS, divided according to positivi-        gy can occur during infection and that these interac-
    ty/negativity for detection of HSV-1, EBV, or CMV            tions can affect health and disease (44).
    were preliminary analyzed by CFA to disclose the ex-         The high prevalence of S. aureus in HSV-1+ sites




                                                                                                          li
    istence of bacterial taxa whose relative frequencies         and its correlation to lesion activity deserves consid-
                                                                 eration. A consistent mass of experimental and clini-




                                                                                                        na
    varied among samples in an inter-correlated manner.
    CFA enabled to reduce the number of microbial vari-          cal data shows that polymicrobial infections involving
    ables and to prevent the negative impact of inter-cor-       S. aureus exhibit enhanced disease severity and
    relation on results of logistic regression analysis (33-     morbidity (44).
    35).                                                         S. aureus possesses and uses strategies to survive




                                                                                                      io
    At the end of CFA a small set of non-inter-correlated        in many different ecological niches and to counterat-
    factors was generated, each factor being character-          tack the competing bacteria. Such cooperative and
    ized by a group of inter-correlated bacterial species.       competitive interactions enable this flexible oppor-




                                                                            az
    The association between each single factor and EBV,          tunist to evolve rapidly towards persistent phenotypes
    CMV or HSV-1, adjusted for individual characteristics        characterized by multidrug resistance and increased
    and clinical indices, was explored with logistic regres-     virulence (45).
    sion analysis.                                               With regard to the possible role of S. aureus in the
                                                                 progression of lesions of GAP, we have previously




                                                                          rn
    Those factors resulting more strongly associated with
    EBV, CMV, or HSV-1 were chosen for multiple logis-           shown that subgingival isolates from AS of GAP are
    tic regression analysis.                                     characterized by a richer armamentarium of virulence
    Such an analytical process of microbiological and            and pathogenicity factors as compared to supragingi-




                                                          te
    clinical data was able to return reliable information on     val isolates (9).
    bacterial species that are significantly associated with     Altogether data presented here and data of the recent
                                                                 literature suggest that although the presence of Her-
    the three viruses and how.
                                                                 pesviruses is not necessary for the progression of pe-
                                                        In
    This information can be summarized as follows: I)
                                                                 riodontal lesions of GAP, it can facilitate it, possibly
    coinfection by EBV is directly associated with a high-
                                                                 by promoting pathogenicity and virulence of periodon-
    er prevalence of A. actinomycetemcomitans and in-
                                                                 topathogenic and other bacteria in a virus and bacter-
    versely associated with that of P. gingivalis, T. for-
                                                                 ial species dependent manner. Furthermore they sug-
    sythia and F. periodonticum; II) coinfection by CMV is
                                ni

                                                                 gest existence of a complex cooperative interaction
    directly associated with a higher prevalence of A.
                                                                 promoted by HSV-1 infection, involving S. aureus and
    actinomycetemcomitans, V. parvula, P. micra and F.
                                                                 the periodontopathogens P. gingivalis, T. forsythia,
    nucleatum subsp. polymorphum and inversely associ-
                              io


                                                                 and F. periodonticum, that could promote an acceler-
    ated with that of P. gingivalis, T. forsythia and F. peri-
                                                                 ate progression of lesions of GAP.
    odonticum; III) coinfection by HSV-1 is directly asso-
                                                                 Further studies will be necessary to demonstrate and
    ciated with a higher prevalence of P. gingivalis, T. for-    characterize this cooperation at the cellular and mole-
                            iz



    sythia, F. periodonticum and S. aureus and inversely         cular level.
    associated with that of A. actinomycetemcomitans, E.
    nodatum, P. intermedia and P. nigrescens.
           Ed




    The observation that changes in the subgingival
    biofilm are virus specific suggests that they cannot be
                                                                 Acknowledgements
    explained simply by the immunosuppressive effect of          This work was supported by a grant from MIUR (Pro-
    Herpesviruses and are probably consequence of spe-           ject PRIN 2007LXNYS7 003) to CP. The Authors de-
    cific mechanisms of cooperation between each of the          clare that they have no conflict of interests with re-
    3 viruses and some bacterial species.                        gard to this work.
 IC




    The existence of cooperative interactions involving
    different viruses and bacterial species, including S.
    aureus, is well known and can depend on different
    cellular and molecular mechanisms (23-25). Coopera-
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