A prospective clinico-microbial study to evaluate the adherence and transmission of periodontal pathogens during periodontal probing. (Preprint)

Abstract

Approximately 500 bacterial taxa inhabit periodontal pockets,1 which provide a moist, warm, nutritious and anaerobic environment for microbial colonization and multiplication. The abundance and diversity of periodontal pocket microorganisms depend upon several factors, including effectiveness of oral hygiene measures, pocket depth, degree of gingivitis, flow of gingival crevice fluid, type of interacting microbes and viruses, transmission rate of microbes from other individuals, and the antimicrobial efficacy of the host immune response.2 The oral cavity is an environment in itself which provides a nutritive medium for bacterial growth. Dental plaque, both supragingival and in the periodontal pocket, is a major source of these organisms. Cross-contamination and cross-infection can occur by direct contact with micro-organisms, indirect contact with contaminated objects, droplet transmission, and inhalation of airborne pathogens. These routes are also responsible for the intra-oral transmission of periodontal pathogens. The most evident way is the transport of bacteria from one site in the mouth to the other by means of the saliva. The finding that Streptococcus mutans can be transmitted intraorally with a dental explorer3 led to the speculation that a periodontal probe also has the potential to transmit bacteria from site to site. Periodontal probes have previously been shown to harbor several bacterial types or species after probing periodontally diseased pockets4. The periodontal probe is used during evaluation of the clinical status of pockets and can encounter sites with high proportions of periodontal pathogens. It was found that probes recovered high numbers of bacteria having a morphology that has been associated with a pathogenic flora.4 Depending on the ability of bacteria to attach to the probe, it is feasible that pathogenic bacteria could be transported from one site to another. The transmission of these bacteria to other periodontal sites may be avoided by disinfecting the probe tip between applications.5 Iodine and its compounds, such as polyvinylpyrrolidone-Iodine (PVP-I or Betadine) are a large group of antiseptics with a broad spectrum of effect used widely in different areas of medicine. Even a brief exposure of 15 seconds to Betadine provides effective in vitro killing of various periodontopathic bacteria including two of the major periodontal pathogens i.e. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.6,7 Also, povidone-iodine exhibits marked anticytomegalovirus activity8, a herpesvirus implicated in the pathogenesis of periodontitis.9 Hence the aim of the study was to evaluate the possibility and control of intra oral cross transmission of periodontal pathogens that might occur by probing clinically diseased and healthy sites. Other objectives of the study were to: 1) To study the adherence of the bacteria to the periodontal probe following insertion into deep pockets of >5 mm. 2) To examine the transmissibility of periodontal pathogens from infected periodontal pockets to healthy sites during routine dental examination. 3) To evaluate the effect of betadine irrigation in reducing this transmissibility.

1) To study the adherence of the bacteria to the periodontal probe following insertion into deep pockets of >5 mm. 2) To examine the transmissibility of periodontal pathogens from infected periodontal pockets to healthy sites during routine dental examination. 3) To evaluate the effect of betadine irrigation in reducing this transmissibility.

The study population was obtained from the outpatient department of Periodontology. Ethical clearance was obtained from institutional ethical committee (IEC) and the guidelines of declaration of Helsinki were strictly followed. After obtaining the ethical approval, a voluntary written informed consent was taken from all the patients for their participation in the study. A total of 16 sites were selected for the study. The sites were divided into 8 periodontal sites and 8 healthy sites. All the test sites exhibited periodontal pockets of 5 mm or greater (diseased sites), and the control sites had sulcus depths of 3 mm or less (healthy sites). None of the subjects had a history of systemic disease, or had received antibiotics or periodontal therapy in the previous 6 months. For the baseline Colony Forming Units (CFUs), a UNC 15 probe was inserted into diseased sites and into a healthy sites in the same arch separately. After individual probing the probe was then placed in 1 ml of saline and thoroughly agitated. This way it was possible to analyze the bacterial load at baseline in healthy and diseased sites. Subsequently in total, eight times the probing was done in diseased sites first and then in healthy sites. The study samples were divided into two groups: Group I (wherein the probe was transferred from diseased site to healthy site without disinfection) and Group II (wherein the probe was disinfected with betadine first before inserting in the healthy gingival sulci). Coin toss method was used for the randomization of the sites. For disinfecting the probe, the probe was held under a syringe filled with 5% concentrated betadine and irrigated with for approximately 5 seconds.10 The required dilutions were performed and the sample was cultured on a blood agar plate. The total number of Colony forming units (CFUs) were counted. After 3 weeks the changes in the bacterial load as a result of cross transmission at the then healthy sites was determined by counting the CFUs obtained from cultures of samples taken from probes inserted in the baseline healthy sites of both the groups. Statistical Analysis: Unpaired t-test was performed to determine the difference between the colony forming units at baseline and at 3 weeks. The value of p < 0.05 was considered significant.

At baseline, the CFUs recovered in the healthy sites were less (193) compared to the CFUs in the periodontal sites (226.75). At 3 weeks follow up, a minor increase in the bacterial count in the healthy sites was observed compared to baseline where the probe was not disinfected between probing sites (p =0.8819). In contrast, number of CFUs were reduced at 3 weeks follow up in the healthy sites when the probe was disinfected with betadine first (p=0.4231). (Figure 1 and 2) Figure 1: Graph showing the mean scores of the CFU at baseline and after 3 weeks. (a) (b) (c) (d) The mean score of the CFUs in the healthy sites of the betadine group at baseline was 260 and at the third week was 193. A mild change was observed in the CFUs at the baseline and at the third week as the colonies in the healthy sites were decreased. However, this difference was not significant as the P value was > 0.05. The mean scores of the CFUs in the healthy sites of the without betadine group at baseline and at three weeks was 138.75 and 226.75 respectively. The colony count increased after 3 weeks in the healthy site when the probe was inserted directly without the use of betadine. However, this change was not significant as the p value was > 0.05. (Table 1) Group I (with betadine) Group II (without betadine) At Baseline At 3 weeks At baseline At 3 weeks No of sites 4 4 4 4 Mean score 260 193 138.75 226.75 Standard deviation ±102.31 ±98.63 ±64.598 ±107.74 Standard error 51.153 49.082 32.299 53.872 P value 0.9474 0.4231 Table 1: Mean scores of the colony counts of both the groups and the healthy sites at baseline and at 3 week.

Discussion: Potentially pathogenic microorganisms may be transmitted between oral sites by seeding from other sites via the saliva, direct extension from adjacent sites or by manipulation as occurs in dental treatment. Dental instruments have the potential to transmit microbes from one site to another within the oral cavity. Loesche et al. 3 demonstrated that Streptococcus mutans can be transferred between teeth by a dental explorer. Other investigators4,5,11 using scanning electron microscopy confirmed that microbes adhered to periodontal probes and concluded that the instrument had the potential of spreading bacteria to other locations. Christersson et al.12,13 verified that probes could transmit A. actinomycetemcomitans between sites in patients with juvenile Periodontitis. They found that probe insertion into a pocket colonized with A. actinomycetemcomitans resulted in the probe becoming contaminated (13 out of 14 times) with at least 1,000 A. actinomycetemcomitans cells. Furthermore, when the probe was transferred from a lesion site to a healthy sulcus, A. actinomycetemcomitans was deposited within the healthy sulcus 28 out of 30 times, however, these bacteria did not persist. The findings in the study conducted by Barnett et al.4 indicate that subgingival organisms will adhere to a periodontal probe during a routine periodontal examination. They detected large numbers of gram-negative cocci, filaments and rods, flagellated filaments, and small and medium spirochetes. Their findings indicate that at least in some instances subgingival organisms can adhere to a probe, suggesting that they could be transmitted from an involved site to another site in the oral cavity. It has been shown that surface roughness plays a role in the initial bacterial adhesion to a surface, by facilitating the establishment of a firm attachment.14The results of the present study were also consistent with the above study as a large number of bacteria were recovered from periodontal sites. This suggests that periodontal probes may have the potential of transmitting pathogenic bacteria from involved sites to other sites in the mouth. The CFUs in the healthy sites were reduced at 3 weeks follow up in the study group, where the probe was disinfected with betadine first. This states that irrigating the probe with betadine has helped in reducing the cross transmission from the periodontal sites to the healthy sites. Similar results were proposed Hoang et al.15 that the use of PVP-I is associated with a 95% reduction in periodontal pathogens in 44% of the pockets with a depth of 6 mm or more, whereas this rate was only 12.5% in pockets irrigated with normal saline. This finding revealed the antimicrobial efficacy of PVP-I 10%. Whereas, the CFUs in the healthy sites were increased as compared to the baseline when the probe was directly transferred from the periodontal sites to the healthy sites. This states that the cross transmission had indeed taken place which led to the transfer of the bacteria. The large number of bacteria present in the recipient sites, and the persistence of the organisms for 3 weeks after transfer, suggests that bacterial replication and colonization had indeed taken place in the previously healthy sites. However, the results of this study indicate that the transfer of the bacterial load by the periodontal probe is reduced by irrigating the probe by betadine first prior to the transfer. Although the difference found was not statistically significant but still the increase in CFUs can be prevented by simple disinfection with betadine then it is worth the effort. The reason why there was not statistically significant increase in CFUs at healthy site could be seen because the receiving site was already occupied with a stable bacterial morphology with adequate immune response. This prevented the adhesion and extensive growth of newly entered micro-organisms. In case where the site is at inactive stage or has compromised immune response, the cross contamination might lead to drastic increase in the pathogenic microflora. However, currently there are no tests to predict that the pocket is in active stage or whether the local immune balance is compromised in a particular pocket. Hence, the prevention of cross- contamination of pathogenic microbiota should be undertaken even it is statistically non-significant. Further studies should be done using large number of sites and between different grades of disease progression. Conclusion: Although statistically non-significant, but still an increase in the bacterial load was seen in the periodontally healthy sites as a result of cross transmission. Use of betadine as an irrigant between probing sites helped to reduce the chances of cross transmission.