|Year : 2021 | Volume
| Issue : 2 | Page : 88-92
Comparison of Porphyromonas gingivalis count and salivary immunoglobulin A against lipopolysaccharide of Porphyromonas gingivalis in periodontal health and disease
Abhinav Subhashchandra Baheti1, Pushpa S Pudakalkatti1, Kishore G Bhat2
1 Department of Periodontology, Maratha Mandal's Nathajirao G Halgekar Institute of Dental Sciences and Research Center, Belgaum, Karnataka, India
2 Department of Microbiology and Immunology, Maratha Mandal's Nathajirao G Halgekar Institute of Dental Sciences and Research Center, Belgaum, Karnataka, India
|Date of Submission||25-Apr-2021|
|Date of Decision||07-Sep-2021|
|Date of Acceptance||07-Sep-2021|
|Date of Web Publication||30-Nov-2021|
Abhinav Subhashchandra Baheti
Maternity Home, Junapress, Tal at Post Shevgaon, Dist Ahmednagar, Dwarka - 414 502, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Porphyromonas gingivalis (P. gingivalis) have been implicated to play a role in the pathogenesis of periodontal disease. Aim: The aim of this study was to compare P. gingivalis in subgingival plaque samples and salivary (immunoglobulin A [IgA]) antibodies against lipopolysaccharide (LPS) of P. gingivalis in periodontal health and disease. Materials and Methods: Totally sixty subjects were included, thirty subjects in healthy group and thirty subjects in chronic periodontitis group. Subgingival plaque and unstimulated saliva samples were obtained from each study subject. P. gingivalis (colony-forming units [CFUs]) were detected using culture method, and salivary IgA levels were assessed using enzyme-linked immunosorbent assay technique. Comparisons between the healthy and chronic periodontitis groups were done for P. gingivalis CFUs/ml (CFUs/milliliter) as well as for salivary IgA levels using Mann–Whitney U-test. Correlation between CFUs/ml of P. gingivalis and salivary IgA levels was also assessed in both the groups. Results: Although P. gingivalis CFUs/ml count was more in the chronic periodontitis group, difference was not statistically significant. Salivary IgA levels were significantly higher in the chronic periodontitis group compared to the healthy group. No correlation was observed between CFUs/ml of P. gingivalis and salivary IgA levels in the healthy group as well as the chronic periodontitis group. Conclusions: Increased levels of specific salivary antibodies (IgA) against LPS from P. gingivalis are associated with periodontal disease. Salivary IgA level against LPS from P. gingivalis can be a promising indicator in the serological diagnosis of periodontal disease.
Keywords: Colony-forming units/milliliter, lipopolysaccharide, periodontal disease, Porphyromonas gingivalis, salivary immunoglobulin A
|How to cite this article:|
Baheti AS, Pudakalkatti PS, Bhat KG. Comparison of Porphyromonas gingivalis count and salivary immunoglobulin A against lipopolysaccharide of Porphyromonas gingivalis in periodontal health and disease. Dent Med Res 2021;9:88-92
|How to cite this URL:|
Baheti AS, Pudakalkatti PS, Bhat KG. Comparison of Porphyromonas gingivalis count and salivary immunoglobulin A against lipopolysaccharide of Porphyromonas gingivalis in periodontal health and disease. Dent Med Res [serial online] 2021 [cited 2022 Dec 5];9:88-92. Available from: https://www.dmrjournal.org/text.asp?2021/9/2/88/331403
| Introduction|| |
Porphyromonas gingivalis (P. gingivalis) is considered a major periodontal pathogen. Antibodies against periodontal pathogens have been studied in blood, saliva, and gingival crevicular fluid samples from subjects with periodontitis. The preponderant immunoglobulin found in saliva is immunoglobulin A (IgA).
In several studies, levels of antibodies against whole cells of P. gingivalis were observed in periodontitis patients. In such preparation during enzyme-linked immunosorbent assay (ELISA), binding of antibodies to disease-associated antigens may be overshadowed by binding of antibodies to antigens that are unrelated to periodontal disease. It is therefore interesting to examine the level of antibodies to isolated fraction of bacterial antigen like lipopolysaccharide (LPS) of P. gingivalis, which is the important virulence factor of this organism.
The aims of the present study were to compare P. gingivalis in subgingival plaque samples and IgA antibodies against LPS of P. gingivalis in saliva of individuals with periodontal health and disease.
| Materials and Methods|| |
Totally thirty patients with chronic periodontitis and thirty periodontally healthy subjects were included for the study based on clinical examination. Study protocol was approved by the ethical committee of the institution. Study protocols were explained to the patients, and written informed consent was taken from all the subjects.
Inclusion criteria for chronic periodontitis group were presence of generalized clinical signs of gingival inflammation, generalized probing depth ≥5 mm (millimeter), and generalized clinical attachment loss ≥3 mm. Inclusion criteria for healthy group were absence of clinical signs of gingival inflammation, no increased probing depth, and no clinical attachment loss.
Exclusion criteria were subjects below 30 years of age, subjects who have received periodontal therapy or antimicrobial therapy within the last 3 months, subjects with history of any systemic disease/condition, smokers, and pregnant and lactating women.
Plaque index (Silness and Loe, 1964), gingival index (Loe and Silness, 1963), probing depths, and clinical attachment loss were recorded in all the subjects. All the recordings were done by a single examiner. Probing depths and clinical attachment loss for each tooth at six sites were measured and recorded to the nearest millimeter mark, using the University of North Carolina-15 periodontal probe.
Unstimulated saliva was collected from the study subjects. Spitting method was used to collect the saliva. Four to five milliliters (ml) of saliva was collected from each study subject. Saliva sample was then stored at −80°C in the department of molecular biology and immunology.
Pooled subgingival plaque sample was collected from each subject. Randomly selected sites were isolated with sterile cotton rolls. In chronic periodontitis subjects, after supragingival plaque and calculus were removed carefully, a sterile Gracey's curette was inserted into the selected site and the subgingival plaque sample was obtained. In periodontally healthy subjects, subgingival plaque samples were collected from the deepest sites of the gingival sulcus. Plaque samples were immediately transferred in a reduced transport fluid and sent to the department of molecular biology and immunology. Plaque samples were cultured in blood agar (enriched medium) and Kanamycin blood agar (selective medium for P. gingivalis). After completion of incubation, the plates were removed and the colony characters of P. gingivalis were checked. The number of colony-forming units (CFUs)/ml was calculated for each sample from Kanamycin blood agar. In case of no growth on Kanamycin blood agar, CFUs were counted from blood agar.
Extraction and purification of LPS were done from the standard strain of P. gingivalis (ATCC 33277). LPS was extracted by the hot phenol-water method as described previously with some modifications suggested by Westphal in 1965.
Polystyrene microtiter plate was used for ELISA. LPS i.e. Lipopolysaccharide was extracted from outer cell membrane of P.gingivalis. This LPS was used as an antigen in ELISA test to detect antibodies against it (LPS antigen).50 micro-litres of LPS were mixed with 5 ml of coating solution. This solution was then used to coat the wells of micro-titre plate used in ELISA. One hundred microliters of coating solution was added to each well.
Frozen saliva samples were thawed. All saliva samples were diluted with assay buffer, and 25-μl diluted saliva sample was added to individual well. Then, 100 μl of conjugate diluted using assay buffer was added to each well. Then, the plate was incubated for 1 h in dark. Then, wells were washed thrice with washing solution and 100 μl of substrate was then added to each well. Plate was incubated for 15 min in dark after which color change was observed. One hundred microliters of stop solution was then added to stop the reaction. Then, the color change was read by putting the microtiter plate into LISA plus microplate reader. Reader provided μl/ml of salivary IgA antibodies in each salivary sample.
Software SPSS 9 (SPSS Inc., Chicago, USA) was used for data analysis. Mann–Whitney U-test was used to compare number of CFU/ml of P. gingivalis and to compare the levels of salivary IgA antibodies against LPS of P. gingivalis in periodontal health and disease. Spearman's rank correlation coefficient test was used to correlate CFUs/ml of P. gingivalis with level of salivary IgA antibodies against LPS of P. gingivalis in the healthy group as well as the chronic periodontitis group.
| Results|| |
30 subjects each were included in periodontally healthy and chronic periodontitis group. In healthy group 19 were males and 11 were females. In chronic periodontitis group 23 were males and 7 were females [Table 1]. Mean age of subjects, in healthy group was 37.43 ± 8.20, in chronic periodontitis group was 42.00 ± 9.85 and in total sample was 39.72 ± 9.28 [Table 2].
|Table 1: Distribution of males and females in healthy and chronic periodontitis groups|
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|Table 2: Mean and standard deviation age of males and females in healthy and chronic periodontitis groups|
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[Table 3] shows comparison of healthy and chronic periodontitis groups with respect to CFUs/ml of P. gingivalis. Mean ± SD CFUs/ml of P. gingivalis (103) in healthy group was 22.00 ± 29.55 and in chronic periodontitis group was 30.07 ± 48.78. Standard error (SE) values were 5.40 and 8.91 in healthy and chronic periodontitis groups respectively. Though mean ± SD CFUs/ml count was more in chronic periodontitis group compared to healthy group, the difference did not reach statistical significance as p value was 0.4779.
|Table 3: Comparison of healthy and chronic periodontitis groups with respect to colony-forming units/ml of Porphyromonas gingivalis (103) by Mann–Whitney U-test|
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[Table 4] shows comparison of healthy and chronic periodontitis groups with respect to salivary IgA. The mean ± SD salivary IgA concentration in healthy group was 22.53 ± 12.66 μg/ml and in chronic periodontitis group was 43.86 ± 20.67 μg/ml. Standard error (SE) values were 2.31 and 3.77 in healthy and chronic periodontitis groups respectively. Salivary IgA levels were significantly higher in chronic periodontitis group compared to healthy group as p value was 0.00001.
|Table 4: Comparison of healthy and chronic periodontitis groups with respect to salivary immunoglobulin A (ug/ml) by Mann–Whitney U-test|
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[Table 5] and [Table 6] shows correlation between CFUs/ml of P. gingivalis (103) and salivary IgA (μg/ml) in healthy group and Chronic periodontitis group respectively. No correlation was observed between CFUs/ml of P. gingivalis and salivary IgA levels in both the groups.
|Table 5: Correlation between colony-forming units/ml of Porphyromonas gingivalis (103) and salivary immunoglobulin A ug/ml in healthy group|
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|Table 6: Correlation between colony-forming units/ml of Porphyromonas gingivalis (103) and salivary immunoglobulin A ug/ml in chronic periodontitis group|
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| Discussion|| |
Chronic periodontitis is an inflammatory disease of supporting tissues of teeth, leading to periodontal destruction. Complex bacterial species interact with host tissues in chronic periodontitis, causing release of a broad array of immune and inflammatory mediators.
P. gingivalis is implicated as one of the periodontal pathogens. P. gingivalis have several different virulence factors. It creates dysbiosis in host oral microorganisms, and this may represent one mechanism by which periodontitis can be initiated. Therefore, Darveau et al. (2012) termed P. gingivalis as a keystone pathogen.
Bacterial culturing has been the reference diagnostic technique for many years. In the present study, P. gingivalis CFUs were detected using culture method and salivary IgA levels were assessed using ELISA technique.
Many studies have relied upon dental professionals such as hygienists and dental students as a healthy or control group. However, Christopher et al. (1996) have shown that the prevalence and levels of P. gingivalis are atypically low in this group, probably due to unusually thorough oral hygiene. Therefore, healthy subjects selected for the present study were taken from patient pool to provide a more representative control group.
P. gingivalis was present in 46.66% (14 out of 30) samples in the healthy group and 66.66% samples in the chronic periodontitis group. In a study by Griffen et al. (1998), a sensitive polymerase chain reaction (PCR) assay was used to detect P. gingivalis in health and disease. P. gingivalis was detected in 25% (46 of 181) of the healthy subjects and was detected in 79% (103 of 130) subjects of the periodontitis group. Griffen et al studied prevalence of P. gingivalis and correlated with periodontal health status. They observed that P. gingivalis was significantly higher in patients having periodontal disease. They used polymerase chain reaction (PCR) technique to detect P. gingivalis during this study. Yang et al. in 2004 used indirect immunofluorescent assay using species-specific antisera to detect P. gingivalis in health and disease. They found P. gingivalis in 85.7% of diseased subjects and only 23.1% of healthy subjects. These results can be attributed to high sensitivity of immunofluorescent assay.
Mean ± standard deviation CFUs/ml of P. gingivalis (×103) count was more in the chronic periodontitis group compared to the healthy group [Table 3], but the difference was not statistically significant. This finding is similar to culture a study by van Winkelhoff et al. (2002) who found that periodontitis patients had on average a higher mean percentage of P. gingivalis, but in their study also, the difference did not reach the level of significance.
LPS is the constituent of the cell wall in Gram-negative bacteria and possesses several biological activities. Although the LPS of P. gingivalis has antigenic qualities, it displays low endotoxic activity as measured by the Shwartzman reaction. Because of low endotoxic activity, P. gingivalis induces weak host reaction and escapes host immune response.
In many studies, elevated levels of antibodies against whole cells or crude extracts of P. gingivalis were observed in periodontitis patients. Such preparations include a number of antigens, some of which may be associated with periodontitis and others that are unrelated to the disease. Thus, in ELISA, the binding of antibodies to disease-associated antigens may be overshadowed by the binding to unrelated antigens. The majority of previous studies have used P. gingivalis whole cells or sonicated P. gingivalis whole cells as antigens to determine antibody level and avidity.
In one study, antibody levels against P. gingivalis LPS highly correlated with that against P. gingivalis whole cells, indicating that LPS is a major immunodominant antigen of P. gingivalis. Therefore, in the present study, the levels of antibodies to isolated fraction of the bacterial antigen and LPS of outer membrane of P. gingivalis were assessed and compared in health and periodontal disease.
In the chronic periodontitis group, salivary IgA was detected in all salivary samples. 83.33% (25 out of 30) samples in the healthy group were positive for salivary IgA antibodies. However, the healthy group consistently showed low levels of antibodies against LPS. Low levels of antibodies in healthy subjects may represent a natural protective response in these periodontally healthy subjects.
In experimental gingivitis studies, it was observed that, in subjects who stopped using oral hygiene, the development of gingivitis was delayed when high levels of IgA antibodies against Actinobacillus actinomycetemcomitans, P. gingivalis, and Streptococcus mutans were present in parotid saliva, suggesting a protective role of IgA antibodies. Raised antibody levels against LPS can protect against its toxic activities, and modulate infections with Gram-negative microorganisms. Evans et al. suggested that antibody to LPS might act as a limiting factor in the progression of periodontal diseases.
In the present study, the salivary IgA levels were significantly higher in the chronic periodontitis group compared to the healthy group [Table 4]. In a study by Schenck in 1985, ELISA was used to assess antibodies in serum samples from subjects with clinically healthy gingiva and patients with chronic periodontitis. They found that patients with periodontitis had significantly higher levels of specific IgG and IgA antibodies than patients with healthy gingiva. In the present study, similar finding is observed in saliva samples.
Doty et al. (1982) studied serum antibodies against various oral microorganisms. They did not observe any significant difference between healthy and diseased subjects with respect to serum IgA levels against Bacteroides gingivalis. Their result is in contrast to the results of the present study, though in the present study, specific IgA antibodies in saliva were detected.
In the present study, no correlation was observed between number of CFUs/ml of P. gingivalis (103) and salivary IgA (ug/ml) in both the healthy and chronic periodontitis groups [Tables 5 and 6]. Salivary IgA is derived from two sources: secretary IgA which is secreted from salivary glands and serum IgA present in the serum and released through gingival sulcus into the saliva. Secretary IgA mainly affects microorganisms in supragingival plaque, whereas IgA of serum origin affects subgingival plaque. In the present study, total IgA (serum + salivary origin) antibodies were detected and that could be the reason for observed lack of correlation between total salivary IgA levels and CFUs of P. gingivalis in subgingival plaque.
In the chronic periodontitis group, salivary IgA antibodies against LPS of P. gingivalis were detected in all the salivary samples, but P. gingivalis was not detectable in all the subgingival plaque samples. Humoral immune response against P. gingivalis LPS may have cleared this organism from periodontal pockets in these patients. Another explanation could be that, in these patients, source of P. gingivalis for IgA secretion could be from nonsampled area.
These also could be the reasons for lack of correlation between salivary IgA levels and CFUs/ml of P. gingivalis in subgingival plaque.
The increased release of serum IgA due to an increased permeability of the crevicular epithelium during gingival inflammation may contribute to the rise in IgA levels in saliva during inflammatory periodontal disease.
Higher secretory IgA levels have also been observed in parotid saliva of subjects with gingival inflammation.,
These studies suggested that an increased antigenic load from dental plaque induces secretory IgA response. In contrast to the abovementioned studies, no direct correlation was found between the concentration of total salivary IgA and plaque accumulation,, and which supports the finding of the present study.
Wide inter-subject variation of antibody production was observed in both the healthy and chronic periodontitis groups in the present study. The extent and duration of P. gingivalis infection could be the possible factors in this variability. Wide inter-individual variability of humoral immune response against P. gingivalis infection was observed even in an experimental study using nonhuman primates in which P. gingivalis was equally infected among the experimental population. Hence, difference in the individual host immune response could be another important factor responsible for observed lack of correlation between number of CFUs/ml of P. gingivalis (103) and salivary IgA levels (ug/ml).
Eick and Pfister (2002) suggested that nucleic acid techniques should replace culture methods as gold standard in microbial diagnosis of progressive periodontitis. Use of less sensitive culture method to detect and quantitate P. gingivalis is one of the limitations of the present study. Furthermore, this could be a reason for observed lack of correlation between number of CFUs/ml of P. gingivalis (103) and salivary IgA levels (ug/ml). However, the use of PCR is still hampered by the high cost and the equipment necessary for the processing. In contrast, culture techniques still hold some inherent capabilities, which makes this diagnostic tool the current reference standard in periodontal microbiology.
Further intensive study on the antibodies against the LPS of other strains of P. gingivalis and other periodontal bacteria would give us useful information about illness typing and management of periodontitis.
| Conclusions|| |
Although mean CFUs/ml of P. gingivalis was more in the chronic periodontitis group compared to the healthy group, the difference was not statistically significant. No correlation was observed between CFUs/ml of P. gingivalis and salivary IgA levels in both the healthy and chronic periodontitis groups.
The significant difference in salivary antibody levels between the healthy and the chronic periodontitis groups implies that salivary IgA against LPS of P. gingivalis is a promising indicator in the serological diagnosis of chronic periodontitis. Chronic periodontitis can be diagnosed by measuring the antibody titers in a patient's saliva against the LPS of P. gingivalis.
Ethical clearance was obtained from ethical clearance board of Maratha Mandal's Nathajirao G Halgekar institute of dental sciences and research centre, Belgaum.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]