Porphyromonas is a Gram-negative, asaccharolytic, anaerobic, bacteria belonging to the Porphyromonadaceae family 1. Porphyromonas gingivalis, is the species of the genus that has undergone the most in-depth study and is widely regarded as a significant pathogen connected to periodontal disease in humans 2. The ability to survive and transmit the infection are highly dependent on its numerous virulence factors, which include structural (lipopolysaccharide, fimbriae, heat shock proteins, etc.) and secretory components (gingipains and outer membrane vesicles) 3. The known risk factors for periodontitis are smoking and diabetes, which increase its prevalence and severity tenfold 4. Diabetes mellitus (DM) increases the severity, prevalence, and progression of periodontal disease. Not only does it affect the host immune response, but also modifies the oral environment, which may result in a different periodontal bacteria community than in non-diabetic condition. Increased gingival crevicular fluid glucose levels, in diabetic patients may alter the source of nutrition, influencing the proliferation of particular bacterial species. 5, 6, 7.

Smokers have been linked to having deeper tooth pockets, higher attachment loss, more noticeable radiographic indications of engagement with the furcation, and increased alveolar bone loss 8. Smoking is responsible for about 42% of periodontitis cases, and around 24% of the diabetics have been found to smoke, forming three high-risk categories for periodontal diseases: smokers, diabetics, and diabetic smokers 4, 9. Smoking causes peripheral vasoconstriction, reduced vascular permeability, hyperemia, decreased gingival crevicular fluid flow, and lower oxygen tension 4, 10, 11.

Thus, it is crucial to understand the function of P. gingivalis and its implications because DM and smoking are both large and critical risk factors for periodontitis 12. Therefore, this study was conducted to determine the prevalence of P. gingivalis and its association with diabetes mellitus and smoking in cases of periodontitis.

This was a cross-sectional study conducted in the department of Microbiology, Central lab, Sharda Hospital, Greater Noida, over a period of 3 months from February to April 2022.

Only those patients who had come to the outpatient department with periodontal plaques without any other oral pathology were included in the study. Plaque samples were then collected and immediately inoculated into Robertson's Cooked Meat (RCM) medium, which was sealed with paraffin oil to maintain anaerobiasis. The inoculated RCM was incubated for one week at 37°C and was checked for the turbidity. Once the turbidity was visible, a loopful of RCM was taken and subcultured on blood agar enriched with hemin and vitamin K. To maintain anaerobic conditions, the culture plates were incubated at 37°C for 1 week in a Gas-pak (HiMedia, Mumbai) jar system along with culture plate inoculated with Pseudomonas aeruginosa serving as a negative control for anaerobic culture. The sachet in the Gas Pak jar contained sodium bicarbonate and sodium borohydride, which reacted chemically in the presence of water to form hydrogen and CO₂ gas, and hence eliminated oxygen effectively.

The plates were examined for bacterial growth after one week. The anaerobic conditions were found to be effective as the plates inoculated with P. aeruginosa did not show any growth. The colonies showing black colour on the culture media were screened as Porphyromonas gingivalis. Presumptive diagnosis was made on the basis of Gram’s staining (Gram negative bacilli), catalase test (negative) and motility (non -motile) testing. Confirmation of the isolates was done by automated VITEK-2 Compact system.

The other anaerobic bacteria isolated during the study included Peptostreptococcus species and Bacteroides species, which were identified on the basis of Gram stain only. Considering these to be oral commensal flora, further identification by Vitek 2 Compact was not done. 

A total of 38 plaque samples were cultured out of which 27 showed growth of P. gingivalis (Table 1). So, the prevalence of P. gingivalis in dental caries was found to be 71.05%.

Out of 38 samples, 26 (68.4%) were from males and 12 (31.5%) from females. Twenty (52.6%) samples were from patients in the age group between 15–30 years, 31.5% (12) between 31–50 years and 15.7% (6) were over >50 years of age. Smokers accounted for 63.1% (24), while non-smokers for 36.8% (14) of the cases. Diabetic patients comprised 57.8% (22) of the total samples, whereas non-diabetics were found to be 42.1% (16) as shown in Table 2.

The association between P. gingivalis and the demographic characteristics of the patients has been depicted in Table 3. The growth was not found to be significantly associated with gender, age, and smoking. However, it was statistically significant with diabetics as well as with diabetic smokers.

The present study was conducted to determine the prevalence of an important oral pathogen Porphyromonas gingivalis in dental caries and to determine its association with smoking and diabetes.

We discovered that the prevalence rate of P. gingivalis was 71% over the course of our investigation, which was concordant with the studies conducted by Ingalagi P et al. in 2022 in India 13 and Ardila Carlos M et al. in 2011 in Columbia 14, with prevalence rates of 89.5% and 67.1%, respectively. This suggests that P. gingivalis is a major periodontic pathogen found in dental plaques and may be the primary etiological agent in the development of periodontal disease. 

Tobacco smoke boosts the production of the collagenase matrix metalloproteases, nicotine causes the degradation of periodontal collagen, which leads to the separation of the periodontal ligaments exposing the tooth to bacterial infections. 15 The prevalence of P. gingivalis in smokers was found to be 70% in our study, which is comparable with the study conducted by Abreu MGL et al. in 2019 in Brazil 16 showing 66.7% prevalence of the bacteria in smokers. The probable reason could be due to a weakened immune response and/or increased bacterial pathogenicity 8. Although the prevalence of P. gingivalis in smokers was high, the two were not significantly associated (p > 0.05). Our findings are supported by a study done by Zambon and Kinane's 17, 15 on the prevalence of P. gingivalis in smokers, which showed that there was no statistically significant difference between smokers and non-smokers.

Our study showed a prevalence of 70.3% for the growth of P. gingivalis in diabetics, which was comparable to the study done by Montevecchi M et al. in 2021 in Korea 18, having a positivity rate of 56% and to the study conducted by Mohamed HG et al. in 2016 in Norway 19 showing 59.3% of the prevalence in diabetic individuals. So, it can be concluded that low levels of defence cells and high glucose levels in DM patients promote the growth of periodontogenic flora. P. gingivalis being a predominant oral pathogen, predisposes diabetic patients to develop severe periodontal disease. 8

Given that there have been no prior studies assessing the factors of both smoking and diabetes to ascertain the prevalence of P. gingivalis in patients with periodontitis, we found that diabetic smokers contributed a statistically significant (p = 0.003) growth of P. gingivalis i.e., 66.6%.

It has been suggested that smoking impairs subcutaneous blood flow and decreases insulin absorption from subcutaneous tissues. Additionally, it promotes the development of pathogenic microorganisms which reduces the immunological host response and increases inflammatory mediator release. 20 Those inflammatory mediators that are linked to type 2 diabetes can be increased by P. gingivalis. Bacterial endotoxin or lipopolysaccharide (LPS) from infected periodontal areas enters the circulatory system easily and causes endotoxemia. Thus, bacterial endotoxins may drive inflammatory responses in distant tissues, resulting in elevated levels of systemic inflammatory mediators, which increase insulin resistance and promote diabetes development. 21, 22

According to research by Gupta et al. 10 and Orbak R et al. 20, smoking and diabetes are the main risk factors for periodontitis. However, there is currently no existing study that establishes a connection between P. gingivalis and both of these factors together. Therefore, further research is needed to investigate this relationship.

To the best of our knowledge, this is the first study to look at the prevalence of P. gingivalis in periodontitis patients in relation to both diabetes and smoking in Indian community. According to our study, P. gingivalis is one of the major pathogens responsible for periodontitis, taking into account smoking and diabetes as the key risk factors for periodontitis. Periodontal problems are more common among diabetic smokers. Hence, such people, besides having frequent monitoring of their blood sugar levels, should also be motivated to stop smoking, and pay frequent visits to their dentists so as to avoid oral and dental complications.