Transmission of Aggregatibacter actinomycetemcomitans between Brazilian Women with Severe Chronic Periodontitis and their Children

1Department of Clinical Dentistry, School of Pharmacy, Dentistry and Nursing, Federal University of Ceará, Fortaleza, CE, Brazil 2Department of Physiology and Pathology, School of Dentistry of Araraquara, São Paulo State University, Araraquara, SP, Brazil 3Department of Clinical, Toxicologic and Bromatologic Analysis, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil 4Department of Diagnosis and Surgery, School of Dentistry of Araraquara, São Paulo State University, Araraquara, SP, Brazil


INTRODUCTION
The initial event in periodontal diseases is the transmission of periodontopathogens, usually from the oral cavity of a family member to another.Tissue destruction factors produced by these pathogens can cause loss of connective tissue attachment and alveolar bone by evading host defense mechanisms (1).
Aggregatibacter actinomycetemcomitans (Aa) is a recognized virulent periodontopathogen that has been associated with the etiology and pathogenesis of periodontal diseases (2,3).Aa transmission between family members with different profiles of periodontal diseases and in different geographic areas of the world has been demonstrated (4)(5)(6)(7)(8).Tinoco et al. (4) evaluated Aa transmission in Brazilian families of aggressive periodontitis patients.However, as far as it could be ascertained, there seems to be no available studies about Aa transmission between Brazilian families of chronic periodontitis patients.
Studies on the occurrence of periodontopathogens and prevalence of periodontal diseases in different populations are relevant due to geographic location, socioeconomic conditions and genetic heterogeneity.

Braz Dent J (2007) 18(3): 220-224
These characteristics have a relationship with both the prevalence of periodontal diseases and the pathogens related to them (9)(10)(11)(12)(13)(14)(15).Furthermore, studies about the transmission of periodontopathogens are valuable to elucidate its significance as a risk factor for the establishment of periodontal diseases and to search available ways for their prevention (16).
This study evaluated Aa transmission between Brazilian women with chronic periodontitis and their children.

Subjects
This study comprised 30 women with severe chronic periodontitis, who had been referred to the Periodontics Service of the School of Dentistry of Araraquara, UNESP, Brazil, for periodontal treatment.The selected patients had at least 3 sites with 6 mm of probing depth and clinical attachment loss and had at least one child aged 7 to 16 years.In order to evaluate mother-child transmission, the children were included in the study when their mothers were colonized by Aa.
The following conditions were considered as exclusion criteria for both mothers and children: periodontal treatment within the previous 6 months or antibiotic therapy within the previous 3 months; systemic alterations that could have interfered with the periodontal conditions or acute periodontal diseases; use of any medication associated with gingival overgrowth; be pregnant or in lactation periods.
The study protocol was approved by the Ethics in Human Research Committee of the School of Dentistry of Araraquara, UNESP, Brazil.All subjects were given full information about the study design and procedures and all signed a written informed consent form.

Clinical Examination
All patients were examined by the same calibrated observer.Measurements were made on all totally erupted teeth, except for third molars.The following measurements were recorded: presence of visible plaque and gingival bleeding, dichotomously obtained from the plaque and gingival indexes (17); probing depth (PD); clinical attachment loss (CAL); and bleeding on probing (BOP).PD, CAL and BOP were evaluated at 6 sites, at the mesiobuccal, buccal, distobuccal, distolingual, lingual and mesiolingual positions.Clinical examination was performed with a PCP-UNC 15 periodontal probe (Hu-Friedy Mfg.Co. Inc., Chicago, IL, USA).

Microbiological Procedure
The women were screened by bacterial culture for the presence of subgingival Aa.Samples of the children were collected when their mothers were colonized by the periodontopathogen.When present both in the mother and in her child, Aa colonies of each one were randomly chosen, evaluated by arbitrarily-primed polymerase chain reaction (AP-PCR) and compared in order to investigate bacterial transmission.

Bacterial Sampling and Culture
In the mothers, subgingival bacterial samples were collected with a periodontal curette from 3 sites that presented the worst periodontal destruction, with at least 6 mm of PD and CAL, and BOP.In the children, the samples were also collected with a periodontal curette from 3 sites that presented the worst periodontal destruction, but in the absence of this factor, samples were collected mesially from 3 permanent first molars.
Prior to sampling, supragingival plaque and calculus at the sample sites were removed and the respective areas were isolated with sterile cotton rolls.Each patient's samples were pooled and immediately immersed in reduction transport fluid (RTF) (18).All samples were processed within 2 to 6 h after sampling, being dispersed by vortexing and 10 -1 to 10 -3 dilutions were then prepared with RTF from each sample.These dilutions were plated onto tryptic soy-serum-bacitracin-vancomycin (TSBV) agar (19) and incubated in 10% CO 2 in air at 37° C for 5 days.Aa was identified based on colony morphology, presence of a star-like inner structure, Gram staining, positive catalase test and negative oxidase test.

DNA Extraction
Aa isolates were cultured in 6 mL brain heart infusion broth, supplemented with yeast extract (0.6%) and L-cystine (0.02%), for 24 h in an atmosphere of 10% CO 2 in air at 37°C.Bacterial cells were harvested by centrifugation (10.000 x g for 10 min), washed twice on TE buffer (10 mM Tris, pH 7.6, 1 mM EDTA) and resuspended in 567 μL of TE, 30 μL of 10% SDS and 3 μL of 20 mg/mL proteinase K.This solution was incubated at 37°C for 60 min to induce cell lysis.Next, 150 μL of 5 M NaCl and 80 μL of CTAB/NaCl solution (10% hexadecyltrimethylammonium bromide / 0.7 M NaCl) were added and incubated at 65°C for 20 min.
Samples were extracted twice with equal volumes of chloroform-isoamyl alcohol (24:1) and twice with phenol-chloroform-isoamyl alcohol (25:24:1).DNA was precipitated by adding 600 μL of isopropanol and centrifuged (12.000 x g for 30 min).DNA precipitate was rinsed once with 600 μL of cold 70% ethanol.After centrifugation (12.000 x g for 15 min), ethanol was removed and DNA resuspended in 200 μL of TE buffer.
The thermocycle (GeneAmp PCR System 2400, Perkin Elmer, New Jersey, NY, USA) profile was initiated with a 5-min denaturation at 94ºC, followed by 45 cycles of 30 s at 94ºC, 30 s at 42ºC and 1 min at 72ºC, with final extension for 3 min at 72ºC.Amplification products were analyzed eletrophoretically on 1.4% agarose gels stained with ethidium bromide solution (0.003%) and photographed (Kodak Digital Science; Eastman Kodak Company, Rochester, NY, USA) under UV light.Photographs were analyzed with specific software (1D Kodak Image Analysis; Eastman Kodak Company) for gel analysis.Aa transmission was based on the similarity of the amplitypes generated from colonies of each mother and her respective child.

RESULTS
Patients' sex, age, race and smoking status data are shown on Table 1.
For analysis of Aa transmission, only children whose mothers had been colonized by the bacterium were evaluated.Aa was found in 8 mothers (27%), so 1 child per mother was evaluated using the same clinical and microbiologic tests.Aa was found in only 2 children, a 7-year-old white boy and a 13-year-old white girl.Aa colonies of these children and their mothers were evaluated by AP-PCR.The generated amplitypes did not show similarities between the strains of each mother-child pair (Fig. 1).
Table 2 shows the mean values of the periodontal clinical parameters found in the mothers and children.According to the American Academy of Periodontology classification of periodontal diseases (20), 9 mothers suffered from severe generalized chronic periodontitis (more than 30% of sites with CAL>5 mm) and 21 from severe localized chronic periodontitis (less than 30% of sites with CAL>5 mm) -data not shown.CAL was not found in the periodontal sites of the children.The distribution of PD and CAL means of the sites sampled for microbiologic analysis are show in Table 3.

DISCUSSION
Several studies have supported the occurrence of Aa transmission between individuals of the same family, but only two of them have evaluated specifically Aa transmission between chronic periodontitis patients and their children: one in The Netherlands (5) and one in the USA (6).
Petit et al. ( 5) studied the possibility of Aa transmission in 13 families among which at least one of the parents were colonized by Aa.Five out of 26 children had the bacterium and in 4 of these 5, Aa clones were the same as those found in at least one of their parents.Preus et al. ( 6) evaluated Aa vertical transmission in 9 families of individuals with chronic periodontitis, colonized by this bacterium and that had one child was also colonized by the pathogen.Ten children were colonized by Aa and 5 showed the same amplitype as their mother or father.These authors selected the study subjects in different ways.Preus et al. ( 6) evaluated parent-children pairs previously screened for the presence of Aa.On the other hand, Petit et al. ( 5), as we did, selected the families on the basis of Aa presence in a proband (one of the parents) and then screened the children for its presence.If it were present in the children, transmission would be evaluated.In this study, however, only mothers were enrolled.The bacterium could have been transmitted from the fathers to their children, but this was not investigated.
Previous studies have shown that, usually, each person is infected by a single Aa clone (7,8).In our study, Aa colonies to be evaluated by AP-PCR were chosen randomly.The presence of more than one clone per person was not assessed.If the subjects were colonized by more than one clone, the mother-child pairs would share the same clones, but this could not be detected.The subjects could have other clones, but not those that were evaluated.Tinoco et al. ( 4) evaluated Aa transmission among Brazilian families of aggressive periodontitis patients.Likewise, they found no Aa transmission pattern among the subjects, probably due to polyclonal bacterial infection.
Asikainen and Chen (2) hypothesized that in developing countries like Brazil, subjects may be more susceptible to polyclonal colonization by periodontopathogens.They are more likely exposed to a variety of sources than individuals in Europe or in the United States, and might be colonized by a larger number of microorganisms due to the higher incidence of childhood diseases in their countries, which can compromise their immune status (3).Hence, both children colonized by Aa in the present study could have acquired it from another person, not their mothers.
Another possibility is due to a technical issue.Molecular biology assays have been used to examine periodontopathogen transmission -restriction endonuclear analysis (5), ribotyping (7) and AP-PCR (4,6,8).This study used AP-PCR, which allows evaluating the similarity between bacterial clonal types and then suggest their transmission.However, primers can target different regions of bacterial genomic DNA in identical clones.Thus, it is possible that transmission had occurred and that the clonal types were identical.Though, it is not possible to show the similarity between them within the different amplitypes generated from different DNA segments (1,6).
It is possible that if the prevalence of Aa had been higher, more transmission could have been found.A prevalence of 27% was observed among the women with severe chronic periodontitis.However, the prevalence of Aa is variable worldwide.In Europe, Sanz et al. (14) found a significant difference between the presence of Aa in periodontal patients from Spain (2.3%) and The Netherlands (23.3%).Dahlen et al. (11) and Mombelli et al. (15) found prevalences of 88% and 63%, in Southern Thailand and China, respectively.These authors suggest that Aa is a common constituent of the normal microbiota in these populations.Like the present study, all these authors used culture methods to process their samples.
In Brazil, Colombo et al. (12) observed, using checkerboard DNA-DNA hybridization, the presence of

Table 2 .
Distribution (%) of the periodontal clinical parameters among the patients.

Table 1 .
Demographic characteristics of subjects.

Table 3 .
Means of probing depth (PD) (in mm) and clinical attachment loss (CAL) (in mm) of the sampled sites.