|Year : 2017 | Volume
| Issue : 4 | Page : 543-550
Profiling of Aggregatibacter actinomycetemcomitans Serotypes B and C and the genotypes in periodontal health and disease
Swati Setty1, Tanvee Wadikar1, SS Suprith1, Kishore Bhat2, Srinath Thakur1
1 Department of Periodontology and Oral Implantology, SDM College of Dental Sciences and Hospital, Dharwad, India
2 Department of Microbiology, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India
|Date of Web Publication||1-Feb-2018|
Dr. Swati Setty
Department of Periodontology and Oral Implantology, SDM College of Dental Sciences and Hospital, Sattur, Dharwad, Karnataka
Source of Support: None, Conflict of Interest: None
Background: A. actinomycetemcomitans is prevalent in periodontitis but is found in some periodontally healthy individuals as well. Certain serotypes of the organism have shown to determine severity of the disease. The distribution of serotype and genotype is affected by geographic and ethnic variation. Therefore, the present study was aimed to detect serotypes b & c of A. actinomycetemcomitans and the genotypes and find its correlation with periodontal status. Materials and Methods: A total of 75 subjects (25 aggressive periodontitis, 25 chronic periodontitis and 25 periodontally healthy) in age range of 14-55 yrs were included. Subgingival plaque samples were collected and checked for the presence of A. actinomycetemcomitans. Following isolation of the organism, detection of the serotype b or c was done by multiplex PCR. Genotyping of A. actinomycetemcomitans was done by arbitrarily primed PCR(polymerase chain reaction). Results: Out of 75 plaque samples, 35(46.66%) tested positive for A. actinomycetemcomitans. Serotype c was detected in 19/35 (54.28%), whereas serotype b alone was not detected in any of the samples. Two samples were positive for both the serotypes (b and c) (5.71%) and 14 (40%) were untypeable. 14 different arbitrarily primed PCR genotypes were obtained among 35 A. actinomycetemcomitans isolates. Conclusion: Serotype c was predominant in periodontally diseased as well as periodontally healthy individuals. An association could be present between genotype – serotype and genotype – periodontal status.
Keywords: Aggressive periodontitis, genotype, polymerase chain reaction, serotype
|How to cite this article:|
Setty S, Wadikar T, Suprith S S, Bhat K, Thakur S. Profiling of Aggregatibacter actinomycetemcomitans Serotypes B and C and the genotypes in periodontal health and disease. Indian J Med Microbiol 2017;35:543-50
|How to cite this URL:|
Setty S, Wadikar T, Suprith S S, Bhat K, Thakur S. Profiling of Aggregatibacter actinomycetemcomitans Serotypes B and C and the genotypes in periodontal health and disease. Indian J Med Microbiol [serial online] 2017 [cited 2019 Dec 12];35:543-50. Available from: http://www.ijmm.org/text.asp?2017/35/4/543/224419
| ~ Introduction|| |
The most common organisms associated with periodontal diseases are Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Campylobacter rectus, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, as well as the treponemes. At the 1996 World Workshop on Clinical Periodontics, A. actinomycetemcomitans, T. forsythia and P. gingivalis were identified as specific periodontal pathogens as causative agents in periodontal disease.
The majority of studies on periodontal microflora in different populations found a common pattern in the bacterial component in patients with periodontitis. However, there are differences in the reported prevalence of the specific microorganisms associated with chronic and aggressive periodontitis amongst various populations.
A. actinomycetemcomitans is one of the major pathogens in periodontal disease.
Six serotypes (a, b, c, d, e and f) of A. actinomycetemcomitans have been described based on the composition of structurally and antigenically distinct O-polysaccharides of their lipopolysaccharides. In addition, a novel serotype g has recently been proposed. There also exist phenotypically non-serotypeable strains of A. actinomycetemcomitans that lack expression of serotype-specific polysaccharide antigen. Serotyping can be done by ELISA, polymerase chain reaction (PCR) or by using specific antisera in immunodiffusion assay.
Certain serotypes are associated with specific forms of periodontitis, extraoral infections and periodontal health. Data on the prevalence of A. actinomycetemcomitans serotypes in populations from geographically distant regions showed a predominance of three serotypes a, b and c and a minor frequency of two serotypes (d and e) amongst A. actinomycetemcomitans oral isolates.
Strains of the same serotypes appear to represent distinct clones, which may be further distinguished by various genotyping methods. Several lines of evidence have suggested that A. actinomycetemcomitans clones exhibit variable virulence potentials. A serotype b JP-2 clone, characterised by a 530-bp deletion in the promoter region of the leucotoxin operon, has been shown to be highly virulent and considered as a risk factor for aggressive periodontitis and active periodontal tissue breakdown. On the other hand, serotype b was isolated from some periodontally healthy individuals also; confirming the genetic heterogeneity amongst the same serotype. Genotyping can be performed by restriction endonuclease analysis, ribotyping, arbitrarily primed-PCR and 16S rRNA sequencing. To the best of our knowledge, no study, yet, has reported serotype and genotype distribution of A. actinomycetemcomitans in India. Thus, the present study was aimed to detect the presence of A. actinomycetemcomitans and its serotypes b and c along with the genotypes and find its correlation with periodontal status.
| ~ Materials and Methods|| |
This study was conducted in the dental college from March 2014 to August 2014. It was a cross-sectional study including 75 individuals. The ethical clearance was obtained from the institutional ethical committee. Informed consent was obtained from all the participants before participating in the study. A detailed medical and dental history was recorded to rule out certain participants. All the study participants were included according to the following selection criteria: systemically healthy individuals who had not received periodontal treatment for at least 6 months prior to sampling, were aged from 14 to 55 years, had all permanent teeth erupted with presence or absence of third molars clinically and non-smokers, non-pregnant and non-lactating mothers and who have not taken any systemic antibiotic therapy in the previous 3 months.
Individuals having probing depth or clinical attachment loss ≤3 mm and no radiographic signs of bone loss were considered periodontally healthy. Participants were diagnosed as chronic periodontitis and aggressive periodontitis according to analytical criteria of American Academy of Periodontology (1999).
The selected participants were categorised into three groups: group I having 25 periodontally healthy controls, Group II having 25 patients with chronic periodontitis and Group III having 25 patients with aggressive periodontitis.
In each quadrant, deepest pockets were selected for sample collection. After removal of supragingival plaque, the sampling site was air dried and isolated with cotton rolls. Subgingival plaque samples were collected from the four deepest pockets in four different quadrants using curettes and then pooled and transferred into a sterile vial containing reduced transport fluid. In periodontally healthy controls, samples were collected in a similar manner from the gingival sulcus. The samples were sent to laboratory and processed within 24 h.
In laboratory, the plaque samples were vortexed, followed by inoculation in the culture medium using an inoculating loop according to the culture requirements of the microorganism.
Technique to culture Aggregatibacter actinomycetemcomitans
Dentaid agar was used as the selective medium for A. actinomycetemcomitans. Following inoculation in the culture medium, plates were incubated at 37°C in 5%–10% CO2 jar for 48–72 h. Since A. actinomycetemcomitans is a capnophile, it requires CO2 for its growth.
After completion of incubation, the plates were removed; colony characteristics of the A. actinomycetemcomitans were identified. A. actinomycetemcomitans colonies were seen as white translucent colonies. Colony count of the microbes was done for quantification and recorded as colony-forming units. The colony was observed under the stereo-microscopy and observed for star-shaped structure in the colony. These organisms were further confirmed by Gram-staining and key biochemicals. A. actinomycetemcomitans was found to be negative for sucrose, lactose, trehalose and melibiose. A. actinomycetemcomitans had sugar-fermenting properties for glucose, galactose and maltose. After identification, bacterial DNA was extracted for further analysis.
DNA extraction procedure
It was done by modified proteinase K method. The colonies were transferred to the tube containing TE buffer and the tube was centrifuged at 5000 rpm for 5 min. The supernatant was discarded. With the help of a micropipette, 500 μL fresh TE buffer was added to the tube and centrifuged for 3–4 min. The procedure was repeated 3–4 times with fresh TE buffer. The supernatant was discarded and 50 μL of lysis buffer I was added. The sample in the tube was vortexed and kept for 5 min. Again, 50 μL lysis buffer II and 10 μL proteinase – K (100 ug/ml) were added and vortexed vigorously. It was kept in water bath for 2 h and then in boiling water bath for 10 min. The obtained DNA was stored at −20°C.
Serotyping by multiplex polymerase chain reaction
The extracted DNA was used to detect serotypes b and c. Primers used for serotypes b and c were as follows; serotype b and c: forward = 59-ARA AYT TYT CWT CGG GAA TG-39 (R = A/G; Y = C/T and W = A/T), serotype b: reverse = 59-TCTCCACCATTT TTG AGT GG-39 (333 bp) and serotype c: reverse = 59-GAAACCACTTCTATT TCT CC-39 (268 bp).
Using a thermal cycler, amplifications were performed in 25-ml volumes, each containing 0.5 pmol primer, 0.2 mM deoxynucleoside triphosphates, 1-fold reaction buffer with 2.5 mM MgCl2, 1 U native Taq polymerase and 2.5-ml template DNA. The PCR was performed with an initial denaturation at 94°C for 3 min for 35 cycles, with one cycle consisting of a denaturation at 94°C for 15 s, annealing at 55°C for 30 s, a polymerisation at 72°C for 30 s and a final extension at 72°C for 2 min. The amplified PCR products were analysed by electrophoresis on a 1% agarose gel. The sensitivity and specificity of the method were checked with the help of well-characterised bacterial control strains of each serotype.
Genotyping by arbitrarily primed polymerase chain reaction
The primer used was OPA-13: 5'-GCACCCAC3'.
A PCR master mix was prepared which contained 0.5 U/μL Taq polymerase, reaction buffer, 4 mM MgCl2, 0.4 mM of each dNTP and was aliquoted into each tube in a final volume of 50 μl/aliquot. After thawing, it was gently vortexed and briefly centrifuged. A thin-walled PCR tube was placed on ice and the following components were added for each 50 μl reaction: 25 μl of PCR Master mix, 1 μl (20 pico mole) of primer, 1 μl (10 pg/μg) of template DNA and 50 μl of water.
After gently vortexing and spinning down, the tubes were placed in a thermal cycler. The temperature profile in a thermocycler (Applied Biosystems, USA) was 35 cycles of 94°C for 1 min, 32°C for 2 min and 72°C for 2 min. The initial denaturation was carried out at 94°C for 5 min, and the final extension was carried out at 72°C for 5 min. Amplification products were analysed electrophoretically in a 1% (w/v) agarose gel containing ethidium bromide (0.5 mg/ml) and were visualised under ultraviolet light illuminator. The bands obtained were interpreted with the help of DNA ladder and were assigned as particular genotype.
The proportions of A. actinomycetemcomitans were calculated in terms of percentage using descriptive statistics. Chi-square test was used to find association between serotypes and genotypes in individual group.
| ~ Results|| |
Presence of Aggregatibacter actinomycetemcomitans
Out of 75 plaque samples, 35 (46.66%) were tested positive for A. actinomycetemcomitans. Among 25 plaque samples from periodontally healthy controls, 7 (28%) were tested positive for A. actinomycetemcomitans. Out of 25 plaque samples from chronic periodontitis patients, A. actinomycetemcomitans was detected in 12 (48%) samples. Among 25 plaque samples from aggressive periodontitis patients, 16 (64%) were tested positive for A. actinomycetemcomitans.
Serotypes of Aggregatibacter actinomycetemcomitans
Serotype c was detected with the highest frequency of 19/35 samples (54.28%), whereas serotype b alone was not detected in any of the samples. Two samples were positive for both the serotypes b and c (5.71%) and 14 (40%) were untypeable.
In periodontally healthy group, out of seven A. actinomycetemcomitans-positive samples, serotype c was detected in four samples (57.14%) whereas three samples (42.86%) were untypeable. In chronic periodontitis group, out of 12 A. actinomycetemcomitans-positive samples, five samples (41.66%) tested positive for serotype c whereas seven samples (58.33%) were non-typeable. In aggressive periodontitis group, out of 16 A. actinomycetemcomitans-positive samples, serotype c alone was detected in 10 samples (62.5%). Both the serotypes b and c were detected in two samples (12.5%) whereas four samples (25%) were serologically untypeable [Table 1].
|Table 1: Distribution of Aggregatibacter actinomycetemcomitans serotypes in relation to periodontal status|
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Genotype assessment of Aggregatibacter actinomycetemcomitans
The OPA-13 primer distinguished 14 different arbitrarily primed-PCR profiles amongst 35 A. actinomycetemcomitans isolates. Genotype VI was the most commonly detected genotype in six isolates and was found only in aggressive periodontitis group. Genotype XII was detected in five isolates from chronic periodontitis group. Genotype XIII was detected in four isolates from periodontally healthy group. One genotype in chronic periodontitis group was non-typeable. Genotype VII was detected in aggressive periodontitis as well as chronic periodontitis [Table 2]. A specific genotype was found in a particular periodontal status without any overlap; except Genotype VII which was found in more than one periodontal status (chronic and aggressive periodontitis).
|Table 2: Distribution of Aggregatibacter actinomycetemcomitans genotypes in relation to periodontal status|
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The relationship between genotypes and serotypes was studied using Pearson's Chi-square test [Table 3]. In aggressive periodontitis group, the most frequently detected genotype VI was associated with serotype c. Genotypes I, IV and VII were also associated with serotype c. Serotypes of Genotypes II and VII were non-typeable. The isolates containing both the serotypes b and c were associated with genotypes III and V. Pearson's Chi-square test showed a statistically significant association (P < 0.05) between serotypes and genotypes in aggressive periodontitis group.
|Table 3: Distribution of genotypes in relation to serotypes in aggressive periodontitis, chronic periodontitis and periodontally healthy groups|
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In chronic periodontitis group, the most frequently detected Genotype XII and Genotype IX were serologically untypeable. Genotypes VII, X and XI were associated with serotype c. Pearson's Chi-square test showed a statistically significant association (P < 0.05) between serotypes and genotypes in chronic periodontitis group.
In periodontally healthy group, the most frequently detected Genotype XIII was associated with serotype c. Serotype of Genotype XIV was untypeable. Pearson's Chi-square test showed a statistically significant association (P < 0.05) between serotypes and genotypes in periodontally healthy group.
| ~ Discussion|| |
The study participants included were selected based on convenience sampling i.e., patients visiting the department of periodontics of the dental institute, and therefore may not be entirely representative of our population.
Numerous techniques have been used in microbiologic investigations over the past 100 years to identify and enumerate species in subgingival biofilm samples. Regardless of the differences in these techniques, the sampling step is crucial and presents a potential source of error. Curette sampling was used in the present study since it has shown to provide a reliable and reproducible method to obtain subgingival samples. Curette samples are likely to be more representative of the tooth-associated biofilms, but can include the loosely bound and the epithelial-associated biofilms.
Pooled sample yields more quantity of plaque, so it can be used for multiple analyses. It increases the probability of detection of an organism as sample is collected from multiple sites. Pooled sample is considered the representative sample of an oral cavity.
Various methods have been used for serotyping such as PCR or serological test using specific antisera in immunodiffusion assay. A DNA-based method using PCR amplification of serotype-specific sequences detects A. actinomycetemcomitans serotypes more sensitively than serotyping by serological methods; therefore, multiplex PCR was the method of choice in the present study. In order to track bacterial infections, discrimination between individual isolates is necessary. Arbitarily primed-PCR is distinct from the classic PCR in the use of a single primer and a low annealing temperature has been used to detect genomic polymorphism at strain level. Arbitarily primed-PCR has several advantages over phenotyping and other genotyping methods: it is universally applicable since it needs no prior knowledge of DNA sequence and yields fast and reliable results at low cost. Hence, arbitarily primed -PCR was the method of choice in the present study for genotyping.
The overall detection frequency of A. actinomycetemcomitans in the present study was 46.66%. In aggressive periodontitis group, 64% of samples tested positive for A. actinomycetemcomitans, which was similar to that in Chinese population (62%) but lesser than that in Brazilian population (72%) and Korean population (74%). The reported prevalence of A. actinomycetemcomitans in aggressive periodontitis in South Indian population was 61%, which was almost similar to the present study. Thus, the detection frequency of A. actinomycetemcomitans in aggressive periodontitis group in this study was similar to that in other Asian populations.
In chronic periodontitis individuals, in the present study, 48% of samples tested positive for A. actinomycetemcomitans. It was more than that reported in Indonesia (40%) and Brazil (41%) but lesser than that in Korea (74%).
A very few studies have reported the detection frequency of A. actinomycetemcomitans in periodontally healthy controls. In the present study, amongst periodontally healthy controls, 28% of samples tested positive for A. actinomycetemcomitans, which was more than that in Korean population (8%) and Brazilian population (15%). A prevalence of 5% was reported in South Indian population in periodontally healthy controls which was much lesser than that of the present study.
Among seven serotypes of A. actinomycetemcomitans (a, b, c, d, e, f and g), a, b and c are more commonly found in oral infections than rest. Serotype f was usually seen to be associated with non-oral infections. Especially in Asian population, serotype c is the most common and is present in both periodontally healthy and disease. Though serotype b is less common in Asian population, the highly leucotoxic JP2 clone of serotype b conceivably accounts for the observed high prevalence of aggressive periodontitis. Therefore, the present study evaluated for serotypes b and c.
The predominance of serotype c was reported in Chinese, Korean  and Thai  populations with a detection frequency of 57.75%, 62% and 57%, respectively. A high prevalence of serotype c was noted in Japan  and Indonesia  too. On the other hand, serotype b was more commonly seen in populations of New York, Finland, Germany  and Greece. A very low detection frequency of serotype b was noted in Chinese (7%), Korean (5%) and Thai (7%) populations. No study, to the best of our knowledge, yet, has reported serotype distribution of A. actinomycetemcomitans in Indian population. In the present study, amongst 35 A. actinomycetemcomitans-positive isolates, serotype c was detected in 19/35 (54.28%) of isolates; being the predominant one; whereas, serotype b alone was not detected in any of the samples. The results of the present study are similar to the results of most of the studies conducted in Asian populations as mentioned above.
There are limited studies in the past evaluating serotype distribution pattern of A. actinomycetemcomitans amongst periodontally healthy individuals. In the present study, serotype c was predominantly seen amongst periodontally healthy individuals with detection frequency of 57.14%. Similarly, studies conducted in China, Japan  and Finland  have reported predominance of serotype c amongst periodontally healthy individuals. Compared to the present study, a lesser prevalence was reported in Japan (26.66%) whereas more prevalence was reported in China (75%). A study conducted in Finland (Caucasian population) had reported a prevalence of 54% and 20% for serotypes c and b, respectively, amongst periodontally healthy individuals, whereas in Greece, serotype a was predominant amongst periodontally healthy individuals.
In the present study, serotype c was predominantly present amongst periodontally diseased population too, with a proportion of 41.66% in chronic periodontitis and 62.5% in aggressive periodontitis. Various studies conducted in Asian population have shown predominance of serotype c in periodontitis-affected individuals. Proportions of serotype c amongst periodontally diseased individuals were 57%, 62% and 57% in China, Korea and Thailand, respectively.,, However, in New York (52%), Germany (33%) and the USA, serotype b was seen to be associated with periodontal disease.
Yang et al. have reported a predominance of serotype b in chronic periodontitis as well as aggressive periodontitis. Proportions of serotypes b and c were 52.46% and 19.67%, respectively, in chronic periodontitis whereas 49.15% and 25.42%, respectively, in aggressive periodontitis. In Finland, proportions of serotypes b and c were 45% and 19%, respectively, in chronic periodontitis and 48% and 13%, respectively, in aggressive periodontitis, showing a predominance of serotype b. In the USA, proportions of serotypes b and c were 29% and 28%, respectively, in chronic periodontitis and 61% and 21%, respectively, in aggressive periodontitis, indicating a predominance of serotype b amongst aggressive periodontitis.
The pattern of serotype distribution in the present study is remarkably similar to that of several studies conducted in Asian population, thus suggesting the influence of race/ethnicity and geographic regions on serotype distribution pattern of A. actinomycetemcomitans.
In the present study, serotype c was predominant amongst aggressive periodontitis, chronic periodontitis as well as periodontally healthy individuals, thus suggesting a probable association between serotype c and periodontal status of the individual. However, studies from Asia showed no significant relationship between serotypes and the extent or severity of periodontal disease.,, On the contrary, studies from the USA have shown that serotype b was more often isolated from patients with aggressive periodontitis than from periodontally healthy controls., In Finnish individuals, serotype b was dominant in patients with aggressive and/or chronic periodontitis, whereas serotype c was the most common serotype in periodontally healthy controls.
The observation that individuals carrying serotype b were associated with more severe disease in some populations, but not in others, may indicate virulence differences within this serotype, or differences in host susceptibility amongst different populations. Similarly, the fact that serotype c strains were associated with aggressive and chronic periodontitis as well as periodontally health indicates virulence differences amongst A. actinomycetemcomitans serotype c strains or, alternatively, differences in the host susceptibility of different ethnic populations.
Furthermore, antibody reaction against A. actinomycetemcomitans serotypes b and c has been suggested to be race dependent. In addition, periodontitis is a polymicrobial infection. In different races, the genetic type of immunological response may modulate the bacterial flora, harbouring the oral cavity. It could be due to synergism of certain clones of A. actinomycetemcomitans with other organisms playing role in the pathogenesis of periodontal disease as the frequency of serotype c was shown to be significantly higher in P. gingivalis-positive sites than in P. gingivalis-negative sites.
Patients are usually seen to be infected by only one serotype and colonisation is stable over time. It is possible that the colonisation of additional A. actinomycetemcomitans strains may be difficult due to competition between the resident strain and the invading strain or due to the suppression of the invading strain from the host immune response to the resident strain.
In the present study, amongst 35 A. actinomycetemcomitans-positive samples, 5.71% of samples were positive for both the serotypes b and c. The samples positive for both the serotypes were of aggressive periodontitis. The majority of the studies showed that amongst A. actinomycetemcomitans-positive individuals, 5%–20% of the individuals were infected by more than one clonal type.,,, An exception is the study by Yoshida et al. in which 33% of the individuals were infected by more than one clone of A. actinomycetemcomitans. The probability of multiple clone infection appears to be governed by the random chance of infection by different clonal types of A. actinomycetemcomitans. It was suggested that multiple serotypes in localised juvenile periodontitis patients may also represent a serotype 'shift' during a lengthy infection or 'burnout' of the disease. The infrequent isolation of multiple serotypes may reflect an interference phenomenon, previous successful treatment or immune elimination of other serotypes.
The serotype distribution and stability of A. actinomycetemcomitans were studied over an 8-year period in untreated Indonesian individuals. Multiple serotypes were found in 12.2% in 1994 and 17% in 2002. Finding the same serotype does not exclude the possibility of reinfection by a different genotype, which can only be established by more detailed DNA fingerprinting of the isolates. Furthermore, not finding a certain serotype does not necessarily indicate the absence of a particular serotype, as it may be present under the level of detection by the used method. This shift suggests an opportunistic character of A. actinomycetemcomitans.
In the present study, 14/35 (40%) isolates were untypeable. As this study has checked for the presence of serotypes b and c only, the untypeable ones may belong to any of the remaining a, d, e, f and g serotypes.
Variation reported in results could be due to certain factors that play important roles in governing the microbial composition of plaque samples. These factors include the number or type of sampling sites, age of patient, transportation, clinical status of sampling sites (probing depth, bleeding index, etc.) and racial and geographic variations.
OPA-13 primer distinguished 14 different arbitarily primed-PCR profiles amongst 35 A. actinomycetemcomitans isolates. Similarly, 10–15 different genotypes were distinguished by arbitarily primed–PCR in various studies.,, These results demonstrated genetic diversity amongst the investigated population and a clonal nature in a periodontal patient of A. actinomycetemcomitans by arbitarily primed-PCR.
Genotype VI was the most commonly detected in six isolates and was found only in aggressive periodontitis group. Genotype XII was detected in five isolates from chronic periodontitis group. Genotype XIII was detected in four isolates from periodontally healthy group. A specific genotype was found in a particular periodontal status without any overlap, except Genotype VII which was found in more than one periodontal status (chronic and aggressive periodontitis), suggesting an association between genotype and periodontal status. Asikainen et al. also reported a possible relationship between some A. actinomycetemcomitans clones and periodontal disease after analysing the clinical isolates by arbitarily primed-PCR. In a study by He et al., some arbitarily primed -PCR genotypes (Groups II and VIII, corresponding to serotypes a and e, respectively) were more dominant in deep periodontal pockets. Hence, according to He et al., certain type (s) of A. actinomycetemcomitans other than serotype b could be important in the pathogenesis of periodontal disease.
One genotype in chronic periodontitis group was non-typeable. This could be due to the use of only one primer for arbitarily primed-PCR. There is a possibility that additional use of some other primers would have helped in typing the one.
In the present study, Genotype VII was detected in aggressive periodontitis as well as chronic periodontitis. This could be due to the difficulty in distinguishing between generalised aggressive periodontitis (GAP) and severe form of chronic periodontitis as clinical presentation of both is more or less similar. The patient diagnosed as chronic periodontitis was 36 years old, adding to the diagnostic dilemma. Thus, the patient diagnosed as chronic periodontitis could be a borderline case of GAP.
In the present study, Pearson's Chi-square test showed a statistically significant association (P < 0.05) between serotypes and genotypes in aggressive periodontitis, chronic periodontitis and periodontally healthy group. A similar finding that isolate of a given arbitarily primed -PCR genotype usually belongs to the same serotype is reported by Asikainen et al. and He et al.
Small sample size, detection of only two serotypes b and c out of seven serotypes and lack of reproducibility of arbitarily primed -PCR are some of the limitations of the present study. Large sample size and evaluation for all the seven serotypes can give a clearer picture about serotype distribution of A. actinomycetemcomitans in Indian population.
Collectively, these studies strongly indicate that A. actinomycetemcomitans is a regular member of the oral microbiota in several Asian populations irrespective of disease activity. Although serotype distributions of A. actinomycetemcomitans have been found to be related to periodontal status in some populations,, there is no evidence that the serotype antigen per se is a determinant of virulence. Owing to the clonal population structure of the species, which implies coupling between genes (i.e., linkage disequilibrium), the different serotypes and, in some cases, individual evolutionary lineages within the serotype clusters are likely to differ in other ways that may contribute to potential differences in virulence. Presence of the same serotype in diseased as well as periodontally healthy conditions indicates the clonal nature of the bacteria and instils the importance of genotyping.
| ~ Conclusion|| |
A stronger association could be present between serotype c and aggressive periodontitis than chronic periodontitis and periodontally healthy as it was detected in all groups with highest frequency. An association could also be present between conjugated presence of serotypes b and c and aggressive periodontitis. Fourteen different arbitarily primed -PCR profiles were found. A statistically significant association between serotypes and genotypes was present. An association could be present between genotype and periodontal status as a particular genotype was detected in a specific periodontal status (except for Genotype VII).
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Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Offenbacher S, Zambon JJ. Consensus report for periodontal diseases: Pathogenesis and microbial factors. Ann Periodontol 1996:1:926-32.
Lafaurie GI, Contreras A, Barón A, Botero J, Mayorga-Fayad I, Jaramillo A, et al.
Demographic, clinical, and microbial aspects of chronic and aggressive periodontitis in Colombia: A multicenter study. J Periodontol 2007;78:629-39.
Könönen E, Müller HP. Microbiology of aggressive periodontitis. Periodontol 2000 2014;65:46-78.
Kim TS, Frank P, Eickholz P, Eick S, Kim CK. Serotypes of Aggregatibacter actinomycetemcomitans
in patients with different ethnic backgrounds. J Periodontol 2009;80:2020-7.
Asikainen S, Chen C, Slots J. Actinobacillus actinomycetemcomitans
genotypes in relation to serotypes and periodontal status. Oral Microbiol Immunol 1995;10:65-8.
Asikainen S, Lai CH, Alaluusua S, Slots J. Distribution of Actinobacillus actinomycetemcomitans
serotypes in periodontal health and disease. Oral Microbiol Immunol 1991;6:115-8.
Sakellari D, Katsikari A, Slini T, Ioannidis I, Konstantinidis A, Arsenakis M, et al
. Prevalence and distribution of Aggregatibacter actinomycetemcomitans
serotypes and the JP2 clone in a Greek population. J Clin Periodontol 2011;38:108-14.
Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999;4:1-6.
Vanpelt E, Belkum VA, Hays JP. Principles and Technical Aspects of PCR Amplification. The Netherlands: Springer Science + Business Media BV; 2008.
Teles FR, Haffajee AD, Socransky SS. The reproducibility of curet sampling of subgingival biofilms. J Periodontol 2008;79:705-13.
Strand P, Palmer RM, Wilson RF. Sampling of subgingival plaque: A comparison of two methods using Darkfield microscopy. Oral Microbiol Immunol 1987;2:142-4.
Dufresne L. Field application of oral fluid sampling and testing in swine. Am Assoc Swine Veterinarians 2011;8:5-8.
Suzuki N, Nakano Y, Yoshida Y, Ikeda D, Koga T. Identification of Actinobacillus actinomycetemcomitans
serotypes by multiplex PCR. J Clin Microbiol 2001;39:2002-5.
He T, Hayashi J, Yamamoto M, Ishikawa I. Genotypic characterization of Actinobacillus actinomycetemcomitans
isolated from periodontitis patients by arbitrarily primed polymerase chain reaction. J Periodontol 1998;69:69-75.
Mombelli A, Gmür R, Lang NP, Corbert E, Frey J. Actinobacillus actinomycetemcomitans
in Chinese adults. Serotype distribution and analysis of the leukotoxin gene promoter locus. J Clin Periodontol 1999;26:505-10.
Lee JW, Choi BK, Yoo YJ, Choi SH, Cho KS, Chai JK, et al.
Distribution of periodontal pathogens in Korean aggressive periodontitis. J Periodontol 2003;74:1329-35.
Mahalakshmi K, Krishnan P, Chandrasekaran SC, Panishankar KH, Subashini N. Prevalence of periodontopathic bacteria in the subgingival plaque of a South Indian population with periodontitis. J Clin Diagn Res 2012;6:747-52.
Timmerman MF, Van der Weijden GA, Arief EM, Armand S, Abbas F, Winkel EG, et al.
Untreated periodontal disease in Indonesian adolescents. Subgingival microbiota in relation to experienced progression of periodontitis. J Clin Periodontol 2001;28:617-27.
Cortelli JR, Cortelli SC, Jordan S, Haraszthy VI, Zambon JJ. Prevalence of periodontal pathogens in Brazilians with aggressive or chronic periodontitis. J Clin Periodontol 2005;32:860-6.
Choi BK, Park SH, Yoo YJ, Choi SH, Chai JK, Cho KS, et al.
Detection of major putative periodontopathogens in Korean advanced adult periodontitis patients using a nucleic acid-based approach. J Periodontol 2000;71:1387-94.
Trevilatto PC, Tramontina VA, Machado MA, Gonçalves RB, Sallum AW, Line SR, et al.
Clinical, genetic and microbiological findings in a Brazilian family with aggressive periodontitis. J Clin Periodontol 2002;29:233-9.
Rylev M, Kilian M. Prevalence and distribution of principal periodontal pathogens worldwide. J Clin Periodontol 2008;35:346-61.
Bandhaya P, Saraithong P, Likittanasombat K, Hengprasith B, Torrungruang K. Aggregatibacter actinomycetemcomitans
serotypes, the JP2 clone and cytolethal distending toxin genes in a Thai population. J Clin Periodontol 2012;39:519-25.
Yoshida Y, Suzuki N, Nakano Y, Shibuya K, Ogawa Y, Koga T, et al.
Distribution of Actinobacillus actinomycetemcomitans
serotypes and Porphyromonas gingivalis
in Japanese adults. Oral Microbiol Immunol 2003;18:135-9.
van der Reijden WA, Bosch-Tijhof CJ, van der Velden U, van Winkelhoff AJ. Java project on periodontal diseases: Serotype distribution of Aggregatibacter actinomycetemcomitans
and serotype dynamics over an 8-year period. J Clin Periodontol 2008;35:487-92.
Zambon JJ, Slots J, Genco RJ. Serology of oral Actinobacillus actinomycetemcomitans
and serotype distribution in human periodontal disease. Infect Immun 1983;41:19-27.
Yang HW, Huang YF, Chan Y, Chou MY. Relationship of Actinobacillus actinomycetemcomitans
serotypes to periodontal condition: Prevalence and proportions in subgingival plaque. Eur J Oral Sci 2005;113:28-33.
Yang HW, Asikainen S, Doǧan B, Suda R, Lai CH. Relationship of Actinobacillus actinomycetemcomitans
serotype b to aggressive periodontitis: Frequency in pure cultured isolates. J Periodontol 2004;75:592-9.
Dahlén G, Widar F, Teanpaisan R, Papapanou PN, Baelum V, Fejerskov O, et al. Actinobacillus actinomycetemcomitans
in a rural adult population in Southern Thailand. Oral Microbiol Immunol 2002;17:137-42.
Saarela M, Asikainen S, Alaluusua S, Pyhälä L, Lai CH, Jousimies-Somer H, et al.
Frequency and stability of mono- or poly-infection by Actinobacillus actinomycetemcomitans
serotypes a, b, c, d or e. Oral Microbiol Immunol 1992;7:277-9.
Chen C, Wang T, Chen W. Occurrence of Aggregatibacter actinomycetemcomitans
serotypes in subgingival plaque from United States subjects. Mol Oral Microbiol 2010;25:207-14.
Roman-Torres CV, Aquino DR, Cortelli SC, Franco GC, Dos Santos JG, Corraini P, et al.
Prevalence and distribution of serotype-specific genotypes of Aggregatibacter actinomycetemcomitans
in chronic periodontitis Brazilian subjects. Arch Oral Biol 2010;55:242-8.
Chung HJ, Chung CP, Son SH, Nisengard RJ. Actinobacillus actinomycetemcomitans
serotypes and leukotoxicity in Korean localized juvenile periodontitis. J Periodontol 1989;60:506-11.
Feng X, Zhang L, Xu L, Meng H, Lu R, Chen Z, et al.
Detection of eight periodontal microorganisms and distribution of Porphyromonas gingivalis
fimA genotypes in Chinese patients with aggressive periodontitis. J Periodontol 2014;85:150-9.
Paju S, Carlson P, Jousimies-Somer H, Asikainen S. Heterogeneity of Actinobacillus actinomycetemcomitans
strains in various human infections and relationships between serotype, genotype, and antimicrobial susceptibility. J Clin Microbiol 2000;38:79-84.
Armitage GC, Cullinan MP. Comparison of the clinical features of chronic and aggressive periodontitis. Periodontol 2000 2010;53:12-27.
Zambon JJ. Actinobacillus actinomycetemcomitans
in human periodontal disease. J Clin Periodontol 1985;12:1-20.
[Table 1], [Table 2], [Table 3]