Prevalence and Comparison of Periodontopathic Bacteria in Aggressive And Chronic Periodontitis Patients- A Microbiological Study

INTRODUCTION

Periodontitis, one of the most prevalent dental diseases, is a complex disease with primary bacterial etiology¹ . As recently as in 1970's it was thought that virtually any organism present in the subgingival plaque was responsible for periodontal destruction. However, the studies carried out later have questioned this hypothesis and suggested a finite list of possible periodontal pathogen² . Accordingly, the consensus report on the microbiology of periodontal disease have designated A.actinomycetemcomitans, P.gingivalis, and T.forsythensis as most important species in the initiation of periodontitis, P.intermedia, E.corrodens, C.rectus, F.nucleatum and spirochetes to be closely related to periodontitis³ . Specific bacterium is considered to be causative for specific type of periodontitis and is isolated in higher numbers in those types of periodontitis. The culture method plays a key role in the characterization of the microbiota of the dental plaque and is still considered to be gold standard⁴ .

Studies have shown variation in the prevalence of specific periodontopathogens in different populations and geographical area. Although some microbiological studies are conducted in Indian population, very few studies compare the microflora in aggressive and chronic periodontitis. In the present study an attempt is made to isolate the major periodontopathogens using anaerobic culturing, determine and compare the proportion of these organisms in aggressive and chronic periodontitis patients.

MATERIALS AND METHODS

Fourty patients in the age group of 17-45yrs, who visited Department of Periodontics, PMNM Dental collage and hospital, Bagalkot, for various treatment needs were selected and divided into two groups of chronic(CGP) and aggressive periodontitis (AgP) based on the criteria established by 1999 International Workshop for classification of periodontal diseases. Patients with aggressive periodontitis were further subdivided into localized (LAgP) and generalized (GAgP) based on the distribution of bone loss. Patients with systemic diseases, pregnant women, patients who had undergone oral prophylaxis and who had taken antibiotics within 3 months prior to study were excluded from the study. After obtaining written informed consent, a brief case history was recorded followed by detailed intraoral clinical and periodontal examination. The tooth with deepest pocket in each quadrant was selected for sampling of subgingival plaque. Following isolation, the plaque sample was collected using a sterile currette, pooled and transferred immediately to 1ml thioglycolate medium with hemin and vit K and sent to laboratory for further culturing and analysis. The samples were cultured within 48hrs of collection. The given sample is diluted to 5l aliquots. Each aliquot of 5l is inoculated into blood agar with Columbia agar base, Brewer anaerobic agar and Bacteriods bile esculin agar plates and placed in an anaerobic jar for 72 hours at 37°C. The type and number of the colonies of the organisms so cultured are noted. Further identification of the species is done through colony morphology, hemolysis, pigmentation, Gram stain morphology and certain key biochemical reactions^5,6,7 .

Statistical analysis

Patients were used as the experimental unit of observation. Periodontal pathogens were calculated for each subject. Means and standard deviations were determined. Student paired 't' test was employed to compare the microbiological data. The difference in the prevalence of bacteria among groups was tested by Fischer's exact test and pair wise comparison were made by multiplicity adjusted Fischer's exact test. p-values <0.05 were considered statistically significant. The detection frequencies and ratio of target organisms were analyzed by ANOVA.

RESULTS

Out of 40 patients, 17 were diagnosed GAgP, 19 CGP and 4 LAgP. P.ginigvalis were isolated in higher proportion from CGP and GAgP (50% and 45.45% respectively). A.actinomycetemcomitans was isolated from both GAgP and LAgP but not from a single sample of CGP. P.intermedia was isloated at about 61.54% and 30.77% respectively from CGP and GagP. F.nucleatum was isolated from 12 subjects belonging to CGP forming 70.59%. T.forsythus was cultivated from the samples of subjects with GagP and CGP accounting for 44.44% and 55.56%. Only P.gingivalis, A.actinomycetemcomitans and P.intermedia were isolated from subjects with LagP(Graph I). When the counts in all three types of disease were compared, the difference was found to be statistically non-significant (p-value 0.1199) (table I & II) (graph II). However, there was statistically significant difference in mean counts between CGP and LAgP. The difference in the mean counts of all the five organisms was statistically significant (p-value 0.0088)(table III & IV).

DISCUSSION

Dental plaque is a complex microbial biofilm comprised of as many as 500 different bacterial species organized in the supragingival and subgingival locations⁸ . The microbial composition of subgingival plaque shows diversity under various clinical periodontal conditions and many studies have attempted to clarify the relationship between a specific bacterium and the pathosis of periodontitis⁹ . Historically, culture methods have been widely used in studies aimed at characterizing the composition of the subgingival microflora and are still considered the reference method (gold standard) when determining the utility of a new microbial diagnostics in periodontics. The main advantage of this method is the possibility to obtain relative and absolute counts of cultured species and assess the antibiotic susceptibility of the grown bacteria. However, culture methods can only grow viable bacteria requiring strict sampling and transporting conditions. Further, it requires specific laboratory equipment and experienced personnel besides being relatively time consuming and expensive¹⁰ .

Variations in the global distribution of periodontopathic bacteria could be important in epidemiology and treatment of periodontal disease¹¹ .The present study evaluated the subgingival microflora in subjects with aggressive periodontitis and chronic periodontitis. The patients were selected from those seeking dental care and according to the inclusion criteria used they were likely to reflect the 'natural' untreated situations. 

There is much discussion in the dental literature whether patient microbiological data should be analyzed on individual  tooth site locations or whole patient data¹² . In the present study, pooled sample from the deepest pocket in each quadrant was used for microbiological analysis. Sampling from deepest pocket in each quadrant, as outlined by Savitt et al⁹ , result in the successful recovery of A.a, P.gingivalis and P.intermedia in subjects who harbored these species. According to Socransky et al¹³ , ideally, a plaque sample should be taken at a peak of disease activity because detection of pathogens in the inactive phase of the disease may confuse the contribution of those pathogens to the lesion. The most reliable procedure to detect activity in periodontal lesion is to measure changes in clinical attachment level or progression in alveolar bone destruction between two points of time. Leaving patients untreated involve ethical questions. Four sites were selected for sampling in each patient in the present study. While this is rather restrictive in terms of ascertaining the true prevalence of each species, it does provide a comparison of the relative level of each species within this population. While it is generally accepted that deep pockets are not a prerequisite for colonization by putative pathogens, these species are generally been recovered more frequently and in higher numbers from deeper sites and from diseased subjects¹⁴ . 

In the present study, only P.gingivalis, A.a and P.intermedia 13 were detected in patients with LAgP. In contrast Lopez et al in the study of occurrence of certain bacterial species and morphotypes in juvenile periodontitis found higher prevalence of F.nucleatum, P.intermedia, E.corrodens, P.gingivalis, C.rectus, A.a in LJP affected sites.

A.actinomycetemcomitans is considered the primary pathogen of aggressive periodontitis especially in localized form even when known as juvenile periodontitis or EOP. This organism is isolated in periodontal lesions from 75-100% in LAgP⁹ . It is in par with this study where these organisms were detected at 75%. Tinoco et al¹¹ has also found the prevalence of A.a to be 80% in 25 adolescents with LJP. Slots et al¹⁵ also found greater proportion of A.a in localized lesions. This may be an evidence for bacterial specificity. Immunologic response to A.a has been associated with race may be one of the factors that determine changes in the frequency of this bacterium in LJP¹³ . However, in the present study no A.a was detected from any of the samples of patients with CGP. Similar results were obtained by Dowett¹⁴ in a study of microbiology of plaque samples from indigenous Indians of Guatemela. This organism is shown to occur infrequently or less frequently. Similar results were also reported by Tanner et al¹⁴ who failed to detect A.a in US sample. In contrast, Gajardo et al found the prevalence of A.a to be higher in chronic periodontitis cohort than aggressive group. Dahlen et al¹⁴ found a low prevalence of A.a in elderly Chinese. Probably a detailed sampling would be required for the detection of A.a. A  systematic review by Mombelli et al¹¹ reported that presence or absence of A.a could not discriminate subjects with aggressive from those with chronic periodontitis. 

Apart from A.a, P.gingivalis (20%) and P.intermedia (20%) were detected in one patient each with LAgP in the present investigation. Some reporters have reported the presence of P.gingivalis not only in the generalized form, but also in localized form of AgP. However, the prevalence of P.gingivalis is thought to increase with subject age and this bacterium seems to assume greater importance with increasing age. This was in par with the present study where the prevalence of P.gingivalis was higher in GAgP than LAgP. Thus the role of P.gingivalis in periodontitis of younger people was regarded as less important as that of A.a. P.intermedia has been associated with AgP in a number of studies⁹ .

Zambon proposed that T.forsythus was the main etiologic agent of periodontitis and has been associated with various clinical forms of periodontitis. In the present study, the number (mean count 88888.89) and proportion of T.forsythus was higher in chronic periodontitis. In contrast, Zambon has reported that T.forsythus was predominant bacterium in AgP. The reason may be because of the amount of sample obtained and the strict anerobic environment required by the organism for the survival. Tanner et al⁹ reported mean levels of T.forsythus elevated at sites showing active lesions and this bacterium was associated with sites converting from periodontal health to diseases, thus playing an important role in chronic periodontitis.

Similar prevalence in relation to P.gingivalis was found between chronic (57.89%) and aggressive periodontitis (52.38%) in the present investigation. Hence the question arises as to what factors strongly relate to severe and rapid periodontal breakdown in aggressive periodontitis. The possible explanation may be the involvement of high pathogenic strains and individual host immune response⁹ . 

In the present study F.nucleatum was found at higher frequency (12 patients/63.15%) in chronic generalized periodontitis. Studies have shown that this bacterium seems to appear in too many gingivitis and non-diseased sites. Further it is considered to be the commensal of subgingival plaque microflora that increases in number in diseased sites ¹⁶.

In the present study the proportion and prevalence of organisms detected except for A.a was greater in chronic rather than aggressive periodontitis. In contrast, the study by Cortelli et al¹¹ to determine the prevalence of periodontal pathogens in Brazilians with aggressive or chronic periodontitis showed the prevalence of each organism higher in aggressive periodontitis compared with chronic periodontitis. The variation in low recovery may be due to varying criteria for patient selection, microbiological sampling and culture methods used or true geographically based difference.

In the present study, when pair wise comparison of each organism was evaluated only three out of 40 patients had both P.gingivalis and A.a detected. These two organisms were identified together infrequently. The difference in the mean count of these organisms was statistically significant (P-value 0.0174). These two organisms produce mutual antagonistic products, or a third organism dependent on the presence of either A.a or P.gingivalis exerts antagonistic activity against the bacterium, or the organism may compete for the same nutritional factors or receptor sites. P.intermedia and A.a colonized periodontal sites independently of each other. This was in par with that of the findings in a study by Slots et al¹⁷ .

In a systematic review by Mombelli et al, there was no distinction between subjects with chronic and aggressive periodontitis based on presence/absence of periodontal pathogens (P.gingivalis, A.actinomycetemcomitans, P.intermedia, T.forsythia and C.rectus). Further no distinction was observed between localized and generalized aggressive periodontitis¹⁸ .

In a study of diverse population of Columbia by Lafaurie et al to compare the relative frequencies of putative pathogens and enteric rods in the subgingival microbiota of individuals with chronic and generalized aggressive periodontitis by PCR method to detect presence/absence of periodontal pathogens and culture medium selective for gram negative enteric rods, found no difference in the percentage of patients harboring P. g i n g i v a l i s, T. f o rs y t h i a , C .r e c t u s a n d A. actinomycetemcomitans for chronic periodontits and generalized aggressive periodontitis¹⁹ .

Approximately 70 taxa were found prevalent, in 10 young adult patients diagnosed with generalized aggressive periodontitis using culture independent molecular technique. Forty of these taxa belonged to phylotypes for which no cultivated isolate have been reported. The two most prevalent genera detected in all patients were selenomonas and streptococcus. It was noteworthy that none of the red complex periodontal pathogens were found at any sites samples in any of the subjects²⁰ . 

Rief et al used oligonucleotide probes to compare the subgingival prevalence of A.a, P.gingivalis, P.intermedia, T.forsythia, Treponema g roup II, Treponema lecithinolythicum, C.rectus, Capnocytophaga ochracea, Fusobacterium species and F.nucleatum in patients with generalized aggressive periodontitis and chronic periodontitis. Statistically significant difference between two groups was the higher prevalence of T.lecithinolytium in generalized aggressive periodontitis subjects²¹ . 

In a longitudinal study on the microbiota associated with initiation of localized aggressive periodontitis in 428 young individuals from Morocco for 2yrs, demonstrated JP2 clone of A.a as important member of the subgingival microbial community lead to development of localized aggressive periodontitis in some patients²² .

The issue of association between various microorganisms in periodontitis being complex, the evaluation of factors involved in this association is beyond the scope of this study. Further studies are needed in this direction.

CONCLUSION

No difference was found in the microflora of the patients with CGP and GAgP. However a significant difference was found in the microflora of the patients with LAgP and CGP. Thus it is not possible to differentiate the patients with chronic and aggressive periodontitis based on the microbiology of the subgingival plaque alone.

The utilization of microbiologic monitoring in the management of periodontal disease may be a very valuable adjunct to conventional periodontal therapy, particularly when treatment based on non-specific plaque control fails to achieve the desired results. However, ascertaining cause and effect in periodontal disease microbiology is beyond the capability of culture studies.

The ability of clinicians to predict periodontal disease activity will undoubtedly increase considerably in the future with advanced knowledge of the periodontopathic microbiota and important host defense molecules and with further development of practical tests to assess critical risk factors in periodontitis.