TRANSMISSION OF PORPHYROMONAS GINGIVALIS FROM CAREGIVERS TO CHILDREN

Marieta D. Belcheva1, Angelina Kiselova-Yaneva1, Maya Rashkova2, Tsonko Uzunov3, Assya Krasteva1, Stefan Panaiotov4, Victoria Levterova4, Sashka Targova3, Milena Georgieva3, Nadia Brankova4 1) Department of Imaging and Oral Diagnostic, Faculty of Dental Medicine, Medical University, Sofia, Bulgaria 2) Department of Pediatric Dental Medicine, Faculty of Dental Medicine, Medical University, Sofia, Bulgaria 3) Department of Periodontology, Faculty of Dental Medicine, Medical University, Sofia, Bulgaria 4) National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria Journal of IMAB Annual Proceeding (Scientific Papers) 2012, vol. 18, book 2


INTRODUCTION
Periodontal diseases often are neglected in childhood and adolescence.Although gingivitis is the most common form of periodontal disease, a localized aggressive form that causes bone loss around molars and incisors occurs in a small number of subjects usually after puberty. 1 Among the several pathogens that are known to be related to periodontal disease, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Campylobacter rectus can be found in individuals with different severities of periodontitis [2][3][4] .Umeda et al. 5 evaluated the distribution of periodontal pathogens in 56 children (from 1 to 15 years old) and their parents.A PCR-based method was used to determine the prevalence of microbial organisms in saliva and dental plaque samples.A positive correlation was observed for the presence of oral pathogens between parents and their children.Thus, the authors suggested a possible vertical familiar transmission of those periodontal pathogens.In a separate study, Kobayashi et al. 6 also investigated the progress of infection by periodontal pathogens and the role of the child's mother in this transmission process.The results indicated that the colonization of periodontal bacterial in children increased with age and that the infant's flora is acquired from his or her primary caregiver. 1he present study as some other 7 that evaluate the pattern of transmission is focused on the prevalence of P. gingivalis in parents who suffer from periodontal disease and their children.

MATERIALS AND METHODS Subjects
Sixty-two parents-volunteers and their children participated in this clinical study.Permission for this study was obtained from the Ethical Committee of the Medical University of Sofia.The parents of the children received detailed information regarding the nature and the procedures involved in the study and signed informed consent forms.A complete periodontal examination of parents was performed by trained and calibrated examiners at least one week before microbial samples were collected to measure the probing pocket depth, the clinical attachment level, the plaque and bleeding upon probing scores.For the purpose of the present study and based on the obtained clinical record, participants were divided into four groups.Group A (n=31) included parents with periodontal disease, group B (n=31) -their children, group C (n=31) -healthy parents, group D (n=31) -their children.The children were examined by pediatric dentists and the plaque (PI) and PSR index were determined as well as the gingival status.
Exclusion criteria Subjects presenting with uncontrolled systemic diseases and those who were immunocompromised, who had taken antibiotics within 3 months before the clinical and microbial examination, had been undergoing periodontal treatment 3 months before the beginning of the study, were not included in the study.For control group C subjects who had no upper molars or second lower incisors were also excluded.

Specimen collection
For group A, sampled places were the deepest pockets and if they were not at the first upper molar or at the lower lateral incisor, at the closest teeth.For group C -the sulci of the first upper molar and lower lateral incisor.For group B and D sampled places were between the first and second molars in children with primary dentition; between the second primary molar and first permanent molar in children with mixed dentition; between the second premolar and first permanent molar in children with late mixed dentition or permanent dentition. 1Before the collection procedure, cotton rolls were applied to prevent contamination of the sampling area with other oral fluids.The supragingival biofilm was gently removed with sterile cotton pellets 8 and plaque samples were collected using sterile paper points inserted in sulci or pockets during 15 seconds This procedure was done for each of the four sites previously selected and the paper points of each subject were placed in a same microtube with 1 ml of reduced transport fluid (RTF) 9 .The samples were stored at -22°C until the analyses.

Culture method
From each sample 50 µl aliquot was taken and plated on Brucella (Oxoid) 5% Sheep Blood Agar with Vitamin K and Hemin (BD BBL TM ).Plates were cultivated in anaerobic atmosphere at 37°C for 48h.To obtain pure culture black pigmented colonies were recultivated at the same conditions and then a biochemical test (REMEL, Italy) was performed.For further PCR detection bacterial suspension was prepared from the rest of bacterial colonies suspended in 100 µl 0.9% NaCl.

PCR method
Bacterial genomic DNA from direct plaque samples and from plated plaque samples was extracted by using a commercially available kit (DNA-SORB-A, Sacacae Biotechnologies Srl, Italy).The species-specific polymerase chain reaction (PCR) primers used in this study are listed in Primer pairs (52 -32 ) Amplicon length (bp) P.gingivalis 404 AGG CAG CTT GCC ATA CTG CG ACT GTT AGC AAC TAC CGA TGT Fig. 1.Species-specific primers for PCR The primer sequences were selected as described by Ashimoto et al. 10 Briefly, the PCR mixture contained 1 pM/ µl of each primer, 1.5 mM MgCl2, 200 µM of deoxyribonucleotide triphosphate (Invitrogen), 1x Taq polymerase buffer, 1.25 U of Taq DNA polymerase and 5 µl of template DNA in a 20 µl reaction volume.PCR reactions were carried out in a DNA thermal cycler (Gene Amp, PCR System 9700, Aplied Biosystems).The PCR profile included an initial denaturation at 94°C for 3 min followed by 36 cyclers consisting of 94°C for 45 s, 55°C for 30 s, 72°C for 45 s and a final extension at 72°C for 10 min 11 .Following amplification, 10 µl of PCR products were analyzed by electrophoresis on a 2% agarose gel.The gel was stained with 0.5 µg/ml ethidium bromide and visualized by ultraviolet light illumination.The size of the PCR products was estimated using a molecular marker Super ladder. 11P. gingivalis W83 and ATCC 33277 were used as positive control and sterile distilled water as negative control for each reaction series.

Statistical analysis
The frequencies of bacteria in the study groups were analyzed using the chi-squared test.All tests were performed using statistical software (STATISTICA and SPSS19).Results were considered statistically significant at P-value <0.05.

RESULTS
In the present study the parents with periodontal disease (Group A) who were analyzed, were composed of 13 males and 18 females (Table 1).Their children (Group B) were 19 boys and 12 girls.The healthy parents (Group B) were 9 males and 22 females.Their children (Group D) were 16 boys and 15 girls (Table 2 Almost 80% of plated samples on Brucella 5% Sheep Blood Agar were positive for dark pigmented colonies after two days cultivation in anaerobic atmosphere but after cultivation on Blood Agar under aerobic conditions some of them survived, which means that these are not anaerobes and the other samples were negative for P.gingivalis when biochemical tests were applied. When PCR method was used 11(35,5%) patients from group A were positive for P.gingivalis, while in each of other three groups the positive cases were 2 (6,5%) (Table4).
Prevalence of P.gingivalis is greater in Group A (Ð<0.05) compared to other groups.There was no statistically significant difference between male and female patients.
According to periodontal status 17 were patients with periodontitis chronica localisata, 6 of them were positive, from 8 patients with periodontitis chronica generalisata 3 were positive and from 6 patients with gingivitis catarrhalis 2 were positive (Table5).The results above were obtained when PCR was applied to direct plaque samples (Fig. 2).PCR detected no children with healthy parents and children whose parents were with periodontal disease (Table 6).positive for P.gingivalis samples, when these were prepared from bacterial suspensions obtained from culture media.

Fig. 2. Gel electrophoresis of PCR products
After running the gel the DNA fragments can be seen with a UV light.They are separated according to their weight and positive samples are at the same level as the positive controls 1 -marker Super Ladder 2, 10 -positive control for P.gingivalis 3 -negative control 4, 5, 7, 8 -positive samples 6, 9 -negative samples

DISCUSSION
The prevalence (35.5%) of P.gingivalis in patients with chronic periodontitis according to our study is lower than the prevalence (54%) according to other study 13 using real-time PCR as detecting method.P.gingivalis was detected in 77.3% of samples from patients with early periodontitis using culture method and in 85,6% using immunofluorescence. 14Some studies report higher levels in gingivitis than in early periodontitis. 15Our study detected same prevalence in patients with periodontitis and gingivitis.
The positive samples from group D were from two eight year old boys with gingivitis localisata and mixed dentition and from group B were from two sixteen-year old boys with gingivitis ulceronecroticans and permanent dentition.The prevalence in both groups was 6.5%.Similar studies investigated the detection of periodontal pathogens by studying dental plaque and/or saliva obtained from children at different age, gender and ethnic group and reported frequency between 6.5% and 21%. 11urthermore the parents of positive for P.gingivalis children were negative and there was no evidence for transmission of the latter bacteria from caregivers to their children.Some authors using BANA-test found higher prevalence of BANA-positive bacteria (including P.gingivalis) in children whose caregivers were BANApositive as well. 1 According to other study no A.actinomycetemcomitans transmission was found between Brazilian women with severe chronic periodontitis and their children. 16he discrepancy between PCR-based and culturebased studies could be explained by the lower detection limit of PCR.For PCR, the detection limit is typically 25-100 cells, whereas, for culturing 103-104 bacteriaare needed before detection.The sensitivity of bacterial culturing is rather low, especially for non-selective media, and therefore, low numbers of a specific pathogen in a subgingival sample will remain undetected. 17Another potential source of error in the culturing procedure for anaerobic bacteria resides in the processing of samples, including transport media 9 .The agreement between culture and PCR method in detecting the absence of P.gingivalis, when PCR was performed with the bacterial suspension obtained after cultivating of plaque samples, supposed that there were no viable bacteria.Additionally, PCR will detect not only viable but also moribund and dead cells (Sanz et al. 2004) 17 .All these factors explain why in our study PCR detected P. gingivalis and microbial culturing failed, even though the same samples had been used.The same observation of a lower prevalence by culture technique was found when analysing Enterococcus faecalis in endodontic lesions and oral rinse samples. 17Some studies (Riviere et al., 1996) 15 detected P.gingivalis more frequently in early periodontitis than in health, while other (Tanner et al., 1998)  18 did not find such association, using culture method.

CONCLUSION
The primery etiology of periodontitis is the presence of specific bacteria in the subgingival plaque biofilm.Thus it seems likely that indentificationof certain species could aid in the periodontal risk assessment.Some studies found positive relations between oral microbiota in children and their parents with periodontal diseases, and some as the present study did not detect such relation.Obviously this topic depends on many factors, such as geographic population, research methods, clinical status e.t.c.Studying such correlations and standardizing methods of detection could contribute the evaluation of periodontal disease risk in adolescents. ).

Table 5 .
Prevalence of P.gingivalis in Group A according to periodontal status

Table 6 .
Prevalence of P.gingivalis in groups B and D