Bacteremia following scaling and root planing: A clinico-microbiological study.

BACKGROUND: Bacteremia frequently occurs after treatment procedures such as extractions, scaling, root planing, periodontal surgery. There is currently significant interest in the possibility that bacteremia with oral bacteria may play role in pathogenesis of atherosclerosis. There are well-conducted studies that have determined the frequency of passage of periodontal microorganisms to the bloodstream after periodontal treatment. There is scarce information related to the incidence of periodontopathic microorganisms during bacteremia induced by this procedure. AIM: The aim of this study was to establish the frequency of passage of periodontopathic microorganisms in peripheric blood after scaling and root planing in patients with periodontitis.
MATERIALS AND METHODS: Forty subjects with chronic periodontitis were included in the study. Blood samples were drawn from each patient at following intervals pre-treatment i.e., before SRP (P1), immediately after SRP (P2), and 30 minutes after SRP (P3). Following SRP, blood samples were analyzed for following microorganisms: Porphyromonasgingivalis, Tannerella. forysthus, Eikenellanella. corrodens, Campylobacter species, Micromonas. micros, and Prevotella. intermedia. STATISTICAL ANALYSIS USED: Chi-square test.
RESULTS: Bacteremia was found in 70% (28/40) immediately after SRP and after 30 min, it was reduced to 25% (10/40) and 7.5% (3/40) presented bacteremia before SRP.
CONCLUSIONS: It was concluded that bacteremia frequently occurs immediately after SRP with P. gingivalis showing the highest frequency in blood.

INTRODUCTION

Bacteremia occurs when bacteria enters the blood stream transiently and can be detected by laboratory blood culture techniques. Though bacteremia are transient, it has long been recognized that oral bacteria may cause distant infections.[1] There is currently significant interest in the possibility that bacteremia with oral bacteria may also play role in pathogenesis of atherosclerosis.[2]

There is evidence that components of these causative bacteria of oral infections, particularly lipopolysaccharide, may promote atherosclerosis, affect blood coagulation, the function of platelets and prostaglandin synthesis.[3] Such functions are important in thrombus formation, which may lead to cerebral and myocardial infarction. Hence, cardiovascular disease may be detrimentally influenced by repeated entry of bacteria into the blood stream.[4]

Epidemiologic studies have reported a risk association between periodontal disease and acute myocardial infarction and stroke.[5] Local infection and transient bacteremia due to periodontopathic bacteria can be related to the pathogenesis of the association between periodontal disease and cardiovascular disease by different mechanisms.[6] The micro-organisms more frequently associated with periodontitis are Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tanerella forysthus, Echinella corrodens, Campylobacter rectus, Micromonas micros, Treponema denticola, Fusobacterium Nucleatum, and Prevotella intermedia.[7]

Bacteremia frequently occurs after treatment procedures such as extractions,[8] scaling,[9] scaling and root planing,[10] periodontal probing,[11] periodontal surgery,[12] suture removal,[13] orthodontic treatment,[14] restorative dentistry,[15] non-surgical root canal treatment.[16] However, not only professional treatment, but also chewing,[17] subgingival irrigation,[18] and oral hygiene procedures such as tooth brushing[19] and flossing[20] have been reported to give rise to bacteremia. However, the results showed considerable variability due to the techniques used, timing of blood sample collection, and periodontal status and identification methods for the isolation of microorganisms.

There are well-conducted studies that have determined the frequency of passage of periodontal microorganisms to the bloodstream after periodontal treatment. They have evaluated this process only after scaling. Root planing in addition to scaling is the procedure most used in periodontal therapy and in the maintenance of patients with periodontitis and may be a risk factor for bacteremia. However, there is scarce information related to the incidence of periodontopathic microorganisms during bacteremia induced by this procedure. The aim of this study was to establish the frequency of passage of periodontopathic microorganisms in peripheric blood after scaling and root planing in patients with periodontitis.

MATERIALS AND METHODS

The patients for the study were selected from department of periodontics. Approval for the study was obtained from the ethical committee. Forty volunteers were invited to participate in the study after giving written informed consent.

Patients who exhibited previously untreated chronic periodontitis with at least 10 sites having pocket depth >5 mm were included in the study. Exclusion criteria were subjects with abnormal hematological profile, those at the risk of infective endocarditis, subjects who had a significant medical or dental condition (such as diabetes, malignancy, or desquamative oral lesions, etc.), those taking any medication, those with a history of infectious disease (e.g., hepatitis, HIV) drug abuse or allergy to dental products or previous problems with venepuncture were excluded.

Three blood samples were drawn from each patient at the following interval: Pre-treatment before the SRP (P1), immediately after SRP (P2), and 30 minutes after SRP (P3).

On the day of the blood sample collection, patients were advised not to use any plaque control measures and to consume only liquids in the morning. Five ml of blood was drawn before the treatment procedure (P1). In order to standardize the time at all the 10 sites, SRP [Figure 1] was conducted for 1 min and immediately blood was drawn (P2) and another blood sample was collected 30 min after treatment (P3).

Figure 1

Scaling procedure

Blood was obtained from veins in the antecubital fossa. Prior to each sampling, the site was wiped with isopropyl alcohol to minimize the number of potential skin contaminants, cannulation of the cubital vein was performed, and 5 ml of blood was drawn. Blood samples were drawn through the needle into the bottle [Figure 2] containing brain heart infusion biphasic medium consisting of one solid and one liquid phase (Himedia company) [Figure 3]. Blood culture bottles were incubated at 37° C and continuously maintained over 14 days for the presence of microorganisms. Samples that indicated positive bacteria were then further analyzed. Samples from culture–positive bottles were inoculated by streaking method on blood agar plates [Figure 4]. These plates were incubated in an anaerobic chamber [Figure 5] for 48 hours and examined for the presence of bacterial growth. Preliminary identification of pure bacterial cultures was based on colony morphology and gram-staining characteristics done by microbial analysis [Figure 6], and specific biochemical tests [Figure 7] were performed for particular organism.

Figure 2

Blood collection

Figure 3

Material used: Anaerobic gas pack sachet, blood agar plates. BI hasic brain heart infusion medium

Figure 4

Black pigmented colonies on blood agar plates

Figure 5

Anaerobic gas pack jar

Figure 6

Microscopic analysis

Figure 7

Biochemical tests for confirmation of bacteria

RESULTS

Frequency of microorganisms in the blood cultures after SRP [Table 1, Graph 1]. Bacteremia was found in 7.5% (3/40) before SRP (P1), 70% (28/40) immediately after SRP (P2) and 30 min after SRP (P3), it was reduced to 25 % (10/40). The frequency of periodontopathic microorganisms in peripheric blood was as follows P. gingivalis 37.5% (15/40), M. micros 22.5% (9/40), P. intermedia 15% (6/40), T. forysthus 12.5% (5/40), E. corrodens 12.5% (5/40), and Campylobacter spp. 7.5% (3/40) [Table 2, Graph 2].

Table 1

Frequency of micro-organisms in blood

Graph 1

Frequency of micro-organisms at different time intervals

Table 2

Periodontopathic micro-organisms isolated from generalized chronic periodontitis patients in blood cultures at different time interval

Graph 2

Frequency of bacteremia in blood amongst the study population

The matching data was cross-classified according to presence or absence of micro-organisms.

Contingency table [Table 3] showing the presence/absence of microbes in patients before and immediately after SRP reveals that organisms were present in 2 patients before surgery and continued showing presence immediately after SRP, while 1 patient showed presence before and then absence after SRP. The organisms were absent in 26 patients before SRP but were present after SRP, while in 11 patients, the occurrence was not observed in either states.

Table 3

Contingency table showing the presence/absence of microbes in patients before and immediately after SRP

Here, we test the null hypothesis (H0) that the probability of transition from presence to absence of microbes in patient immediately after SRP is same as probability of transition from absence to presence. Since we are observing the change, the patients with microbes before and after SRP (2) and those without microbes before and after SRP (11) don’t supply any comparative information. Hence, we target only the discordant i.e., off-diagonal cells. Under null hypothesis, the probability of transition from presence to absence would be 0.5, and since there are 27 discordant pairs (26 + 1), the expected number of patients under H0 would be 27/2 = 13.5 of each type. The resulting Chi-square using the observed and expected counts of discordant cells is 23.148, indicating P < 0.001. Thus, we reject null hypothesis and conclude that there is highly significant difference in the transition likelihood of patient from presence - absence and absence - presence. In other words, the presence of microbes in patients is more likely immediately after SRP.

On similar lines, the microbial count data for 30 minutes after SRP was compared with that of before SRP. Table 4 shows the cross-classified table for the two instances.

Table 4

Contingency table showing the presence/absence of microbes in patients before and after 30 minutes of SRP

The same analysis was performed for 30 min after SRP, which resulted into a Chi-square value of 6.4 with a P value of 0.0114 (P < 0.05). This indicates that although the number of cases with presence of microbes after 30 minutes has reduced, the number still indicates higher likelihood of finding microbes in patients at this time point.

DISCUSSION

The oral microflora consists of more than 600 species, half of which have not yet been cultured.[21] Furthermore, as cultivable taxa of oral bacteria have a range of growth requirements, no single isolation medium satisfies all. Transient bacteremia is been directly related to dental disease and its treatment.[22] This transient bacteremia is associated with acute or chronic oral odontogenic infections, such as periodontal disease may represent a far greater risk for the development of endocarditis than occasional health-care procedures administered in a professional setting.[23]

After scaling and root planing, bacteremia has been analyzed in only aerobic gram-positive bacteria; the presence of anaerobic and facultative bacteria has not been reported, perhaps due to lack of accurate procedures for isolating and identifying this kind of micro-organisms. The objective of this study was to evaluate the passage of periodontal bacteria in the bloodstream after scaling and root planing in order to know the incidence of bacteremia induced for these micro-organisms in periodontitis patients. In the present study, only anaerobic bacteria were isolated; they represent only a fraction of the micro-organisms present in blood. Aerobic bacteria were not considered in this study.

Messini et al. conducted a study to investigate the initial time and duration of bacteremia in disabled patients after SRP under general anesthesia[24] and found 83% aerobic and anaerobic bacteria in the patient's blood. In 2005, a study which analyzed patients with periodontitis, reported a frequency of 23% by polymerase chain reaction (PCR) (using universal bacterial primers that target the 16S ribosomal RNA gene) and 13% by culture method aerobic and anaerobic bacteria after full-mouth ultrasonic scaling.[25] A similar study was conducted in 2006 and found 75% aerobic and anaerobic bacteria in periodontitis patients after scaling.[2627]

In the present study, P. gingivalis, M. micros were more frequently isolated followed by P. intermedia, Campylobacter spp, E. corrodens, T. forsythensis. Several species such as P. gingivalis, P. intermedia have the ability to invade cells and tissues in vitro.[28] P. gingivalis showed highest frequency of isolation in peripheric blood after scaling and root planing, suggesting that these patients may be at risk of cell or tissue invasion. This microorganism has also been isolated frequently from atheromas, which may represent an additional risk for the development of vascular lesions.[29] A study conducted in animals showed that recurrent P. gingivalis bacteremia induces aortic and coronary lesions consistent with atherosclerosis in normocholesterolemic pigs and increases aortic and coronary atherosclerosis in hypercholesterolemic pigs.[30]

This study evaluated the presence of bacteria in peripheric blood before, immediately after SRP and 30 min after SRP. The highest incidence was observed immediately after treatment (70%) and decreased 30 min. after treatment (25%). P. gingivalis and M. micros were found in the blood 30 min. after procedure. Animal studies have shown that peak bacteremia occurs quickly (within the first minute) when human oral microorganisms are injected into the blood stream.[31] The reduction in bacteremia over several minutes after dental instrumentation is due to effectiveness of the host defense system in rapidly clearing micro-organisms from the blood.[32] The capability of neutralizing the microorganism in blood varies among patients and may represent an additional risk factor for developing remote infections. Moreover, degradation of gram-negative bacteria by the immune system can promote the expression of lipopolysaccharides in the peripheral blood initializing cell activation and the subsequent production of pro-inflammatory cytoquines.[33] SRP can also elevate significantly the levels of pro-inflammatory cytokines in a short time.[3435]

In this study, 3 patients presented bacteremia before treatment. A possible explanation could be that the patient did not follow the recommendations, although all the factors in the methodology were kept optimal for the patients such as brushing, oral hygiene aids etc., Tooth brushing and chewing have demonstrated induction of bacteremia in periodontitis patients.[2526] In some cases, detection of microorganisms was not possible immediately after treatment (P2) or 30 min after treatment (P3). This result suggests that although blood cultures are considered the gold standard, evaluation of bacteremia by this method can lead to false-negative results and cannot detect bacteria degraded by the immune system. This indicates the need of evaluating other methods for the detection of periodontopathic microorganisms in bacteremia studies using molecular or immunologic techniques, especially when investigating the association between periodontal and cardiovascular disease.

This study supports the evidence that bacteremia is highly associated with periodontopathic microorganisms after SRP in patients with chronic periodontitis. It also supports the relationship between periodontal disease associated bacteria and its distant effects in the human body in general, and the interaction between periodontal disease and cardiovascular disease, in particular. Because of high prevalence of bacteremia in periodontitis, patients at risk of infective endocarditis should receive antibiotic prophylaxis. The policy for antibiotic prophylaxis during dental treatment may demand reconsideration as further epidemiological data becomes available.[36]