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Antibiotic prophylaxis recommendations for the prevention of infective endocarditis are based in part on studies of bacteremia from dental procedures, but tooth brushing may pose a greater threat. The purpose of this study was to compare the incidence, duration, nature and magnitude of endocarditis-related bacteremia from single tooth extraction and tooth brushing, and to determine the impact of amoxicillin prophylaxis on single tooth extraction.
In this double-blind, placebo-controlled study, 290 subjects were randomized to 1) tooth brushing, 2) single tooth extraction with amoxicillin prophylaxis, or 3) single tooth extraction with identical placebo. Blood was drawn for bacterial culturing and identification at six time points before, during and after these interventions. The focus of our analysis was on bacterial species reported to cause infective endocarditis. We identified 98 bacterial species, 32 of which are reported to cause endocarditis. Cumulative incidence of endocarditis-related bacteria from all 6 blood draws was 23%, 33% and 60% for the tooth brushing, extraction-amoxicillin and extraction-placebo groups, respectively (p<0.0001). Significant difference were identified among the three groups at draws 2, 3, 4 and 5 (all p<0.05). Amoxicillin resulted in a significant decrease in positive cultures (p<0.0001).
Although amoxicillin has a significant impact on bacteremia from a single tooth extraction, given the greater frequency for oral hygiene, tooth brushing may be a greater threat for individuals at risk for infective endocarditis.
Historically, much of the emphasis on prevention of infective endocarditis (IE) has focused on the risk from dental and other non-dental procedures.1 The American Heart Association (AHA) recommendations for antimicrobial prophylaxis for IE are controversial due to the lack of definitive evidence for efficacy, and because they are based largely on studies utilizing surrogate measures for risk.2-12 Studies of incidence, duration, nature (species) and magnitude of bacteremia from dental procedures are often in conflict due to variability in study design. Although there are many reports that address bacteremia from dental extractions – considered the most invasive of dental office procedures – there are few such data on routine daily activities such as tooth brushing. We identified 7 papers with bacteremia from tooth brushing alone as an outcome, 5 of which were published between 1954 and 1977.13-17 The other 2 papers were published following patient enrollment for the present study.18, 19 These 7 papers had a range in the number of subjects from 20-30 and they had a wide range of bacteremia incidence. Only one of these papers measured bacteremia beyond 20 minutes from brushing.19 All of these studies used non-molecular microbiology methods with their inherent limitations. Incidence figures for bacteremia in adults range from 0% to 100% for single tooth extractions,5, 7, 20-22 and 0% to 57% for tooth brushing.13-15, 17, 19 There is no large prospective comparison study of the incidence, duration, nature and magnitude of bacteremia from procedures with these perceived extremes of invasiveness. The literature suggests that the detection of bacteria following a tooth extraction drops off sharply after 10 minutes, and isolated reports indicate that positive blood cultures can be detected for as long as 30 min post dental procedure.8, 12, 20, 23 Finally, the extent to which systemic antibiotics reduce the incidence, duration, nature and magnitude of bacteremia from dental procedures is controversial as well.5, 24
Our review of the literature identified over 170 species of bacteria that have been isolated from blood following dental procedures, and over 275 species of bacteria that have been reported to cause IE. However, there are no studies that focus on the subset of bacteria that are common to both of these groups. The purpose of this study was to compare the incidence, duration, nature and magnitude of bacteremia from endocarditis-related bacteria during and following brushing or a single tooth extraction, and to determine the impact of AHA-recommended amoxicillin prophylaxis on the incidence, duration, nature and magnitude of bacteremia following a single tooth extraction.
Patients presented to our urgent care service with the need for extraction of at least one erupted tooth. Exclusion criteria included: less than ten teeth; use of systemic antibiotics within the previous 2 weeks; need for antibiotic prophylaxis based on current practice guidelines; active viral disease; immunocompromise; poorly controlled systemic disease; history of penicillin allergy; temperature greater than 100.5 degrees Fahrenheit; facial cellulitis; and manipulation of the gingival tissues (e.g., chewing, tooth brushing) within 1 hr prior to the study. Patients who met the inclusion/exclusion criteria were informed of the study and IRB-approved consent was obtained.
Patients were randomly assigned by a computer-generated list, with a block size of 12, to one of three interventions: 1) tooth brushing, 2) single tooth extraction with amoxicillin prophylaxis according to the AHA recommendations (extraction-amoxicillin), or 3) single tooth extraction with an identical placebo (extraction-placebo). Study drug and identical placebo capsules were placed in sealed, opaque envelopes identified only by study identification number. When a patient was enrolled, the next envelope in sequence was opened and the treatment assignment implemented. Baseline data included demographics, medical history, and a thorough clinical and radiographic examination of the teeth and periodontium. Dental and periodontal disease parameters included: mean periodontal pocket depths for all remaining teeth; calculus scores (range 0-3), gingival erythema scores (range 0-3), and plaque scores (range 0-3) (Table 1). For all three arms of the study, a specific protocol was followed (Fig. 1). The puncture site was scrubbed and a large bore (18-22 g) angiocath needle was placed in the usual manor and attached to a bag of normal saline. The baseline blood sample (20 mL) was then drawn and 7-8 mL was inoculated directly into both aerobic and anaerobic BACTEC® bottles for bacterial culturing. Patients in the extraction arms were anesthetized with 1.8 mL of 2% lidocaine with 1:100,000 epinephrine approximately 15 min before surgery. Carbocaine 3% without vasoconstrictor was used if further local anesthesia was necessary. Following AHA recommendations, the extraction began 1 hr following ingestion of the amoxicillin or placebo. For the brushing arm of the study, subjects brushed all surfaces of the teeth adjacent to the gingiva with a new toothbrush (without toothpaste) for 2 min, timed as 30 seconds for each of the maxillary and mandibular quadrants of teeth. Subsequent blood draws of 20 mL were taken at 1.5 min and at 5 min after the initiation of surgery or brushing. Additional blood samples (20 mL) were drawn 20, 40, and 60 min following the end of the procedure. Two mL of blood were drawn into a new syringe and discarded before each of the 6 blood draws, and the catheter was flushed with 2 mL of saline from a new syringe following each blood draw. Patients randomized to the brushing group had their dental extraction accomplished at the end of the study period, after the last blood draw, or on a subsequent visit.
Blood samples were cultured in BACTEC Plus Aerobic/F and LYTIC/10 Anaerobic/F (Becton, Dickinson, Sparks, MD). Bacterial colonies were isolated on both selective and non-selective media such as blood agar, Chocolate agar and MacConkey II agar (BD & Co., Sparks, MD) for aerobes, and on anaerobic blood agar, Columbia CNA agar and blood agar plates supplemented with Phenylethyl Alcohol (PEA) and Kanamycin/Vancomycin (KV) for anaerobes. All false-positive bottles (i.e., bottles that were signaled positive but the subculture was negative) were further incubated for the total of 2 weeks. Bottles with positive cultures were also kept for two weeks and subcultured periodically to ensure recovery of additional species. The 16S rRNA sequencing method was used for bacterial identification. Bacterial lysates were used as templates in PCR using 16S rRNA universal primers following standard protocols.25 PCR products were sequenced using an ABI 3100 DNA sequencer according to manufacturer's instructions (PE Applied Biosystem, Foster City, CA). Identification of strains was based on comparisons of the first 500 bases using Database Project (RDP) (http//rrna.uia.ac.be/rrna/ssu/forms/index) and GenBank by BLAST (http://www.ncbi.nih.nlm.gov). For those strains that were potentially new species (i.e., less than 98% similarity to their closest relatives), full 1,500 base pair sequences were obtained.25, 26 Investigators involved in bacterial culturing and identification were blinded as to subject randomization.
Sensitive, real-time, quantitative polymerase chain reaction (RT qPCR) was used to quantify bacteria. Bacterial DNA was isolated from patient blood draws and from blood seeded with known quantities of several common oral pathogens. Patients were selected who had positive cultures following tooth brushing (N=16), single tooth extraction with amoxicillin (N=20), or with placebo (N=23). Additional patient samples were drawn from a prior pilot study of multiple tooth extractions (N=5). A modification of QIAGEN DNA Blood Mini Kit (QIAGEN, Valencia, CA) procedure was used for optimal recovery of bacterial DNA from blood. For RT qPCR, Taqman technology and probes (Biosearch Technologies, Novaro, CA) and universal 16S rRNA primers (Integrated DNA Technologies, Coralville, Iowa) conserved among oral pathogens were employed using SmartCycler (Cepheid, Sunnyvale, CA). We established standard curves for the seeded pathogens and calculated the levels of bacteria in subject blood cultures. The sensitivity of the method was 25 colony forming units (CFUs) per PCR reaction, corresponding to 103-104 CFU/mL of blood.
Our comprehensive search of the literature provided a list of 275 species of bacteria reported to cause IE; which we compared with the list of bacterial species identified in this study. The bacterial species common to both lists was used in our analysis.
Demographic and baseline clinical characteristics of participants are reported as means and standard deviations, or frequencies and percents. For the analysis of incidence, each patient was assessed at each blood draw, and coded as positive for any bacteria that was common to the list of 397 bacterial species reported to cause IE. Comparisons by study arm at each blood draw, and a summary comparison by study arm combining all draws, were made with chi-square tests. Duration of bacteremia was defined as the number of blood draws at which any target organism was cultured. Intercurrent negative findings were rare (N=2), were judged to be spurious, and were considered positive for analysis. Duration to specific intervals by study arm was compared with chi-square tests. Statistical significance (α) of 0.05 was used in all cases.
Calculation of the required sample size was based on comparison of the rates of incidence in the hygiene and extraction-with-placebo study arms. Our prior work suggested that the incidence of bacteremia from single tooth extraction would be between 70 and 100 percent.7 There was no consensus opinion available on the incidence of bacteremia following tooth brushing in adults – estimates ranged from 30 percent to over 60 percent. Assuming a significance level of 0.05, we estimated that 80 participants per study arm would yield nominal power of 90% to detect a difference in cumulative incidences of at least 20%.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
During the 3-year study period, we screened 600 patients and subsequently randomized 290 patients to one of three groups (Fig. 2). The mean age was 40 years, 58% were men, and 73% were African-American. Baseline characteristics were well balanced between the study groups with the exception of a higher percentage of complex extractions in the extraction-placebo group compared to the extraction-amoxicillin group (Table 1). However, no meaningful differences were found in extraction times or in the incidence of bacteremia between those with simple and complex extractions in the relevant study arms.
The overall incidence of bacteremia at any of the 6 draws was 32%, 56% and 80% for the brushing, amoxicillin and placebo groups respectively (Chi-Square p<0.0001). The highest incidence occurred at the time of the procedures in the placebo group (79%), followed by the extraction-amoxicillin (56%) and brushing (28%) groups. All baseline blood cultures were negative, with the exception of 3 instances, likely from skin contamination (e.g., Staphylococcus epidermidis).27, 28
Two percent of the placebo group (N=2) and 9% (N=9) of the brushing group were still bacteremic at 60 minutes post procedure. Two subjects (2%) in the extraction-amoxicillin group were positive at 40 minutes.
We identified 98 different bacterial species, the most common of which belonged to the genera Streptococcus (49%), Prevotella (9%), Actinomyces (5%), and Fusobacterium (5%).
All analyzed samples were below the detection threshold of 104 CFUs per milliliter of blood.
Of the 98 bacterial species identified, 32 species overlapped with our list of 275 species reported to cause IE, and the following results focus on these 32 oral bacterial species.
All baseline blood cultures were negative, with the exception of one patient (with 2 species) in the brushing group (Fig. 3 & Table 2). The cumulative incidence of bacteremia from all 6 blood draws was 23%, 33% and 60% for the brushing, extraction-amoxicillin and extraction-placebo groups, respectively (p<0.0001). There was a significant difference in the incidence of positive cultures among the three groups at draws 2, 3, 4 and 5 (all p<0.05). The highest incidence of positive cultures occurred in the first 5 minutes of the procedures (combining draws 2 and 3), with incidence figures of 19%, 33%, and 58% for brushing, extraction-amoxicillin and extraction-placebo groups, respectively. The extraction-placebo group had a significantly greater number of positive cultures at 20 minutes (10%) as compared to the extraction-amoxicillin (1%) and tooth brushing (1%) groups (p=0.001). This pattern persisted to 40 minutes.
The vast majority of bacteremic subjects (93%) had a brief duration time (<20 min.) (Fig. 3). There was a significant drop in the incidence of positive cultures at 20 minutes in all 3 groups (all p<0.0001), and this continued at 40 and 60 minutes, with little difference between the brushing and extraction-amoxicillin groups at draws 4-6. Five percent of the extraction-placebo group and 2% of the brushing group were still bacteremic at 60 minutes.
Ten (43%) of the 32 IE-associated oral bacterial species are (viridans) streptococci (Table 2). Thirteen of 27 (48%) positive cultures in the brushing group were (viridans) streptococci, by comparison with the extraction-amoxicillin (23/47, 49%) and extraction-placebo (106/151, 70%) groups. The majority of the non-streptococcal species occurred in the extraction groups. With the exception of one subject in the placebo group, polymicrobial blood cultures occurred only within the first 5 minutes of the procedure, albeit at a low rate in the brushing (2%) and extraction-amoxicillin (6%) groups, by comparison with the extraction-placebo (29%) group.
As noted above, all analyzed samples were below the detection threshold of 104 CFUs per milliliter of blood.
Amoxicillin resulted in a significant reduction in the incidence of positive cultures at draws 2, 3, and 4 (p<0.0001), and it reduced the incidence of positive cultures from all species by 69% (from 151 to 47) and by 78% (106 to 23) for (viridans) streptococci (Table 2).
Although there is a strong emphasis on prevention of bacteremia in the dental office setting, the relative risk for IE from dental procedures versus routine daily events such as tooth brushing is unknown. Bacteria commonly gain entrance to the circulation through ulcerated gingival crevicular tissue surrounding the teeth.23 While dental extractions are among the most likely of dental procedures to cause bacteremia, tooth brushing may disrupt a far larger surface area of gingival crevicular tissue. While brushing does not appear to have the same incidence, duration, nature, and therefore magnitude of bacteremia as a dental extraction, we found a substantial incidence of bacteremia (23%) of IE-causing species from this common daily oral hygiene activity. In addition, the brushing group had a larger percentage of positive cultures at 60 minutes (9% vs. 2% respectively). This suggests that brushing poses a risk for bacteremia similar to a dental extraction, given professional guidelines that recommend tooth brushing at least twice per day. Therefore, there is the potential for bacteremia from tooth brushing alone to occur over two hundred times per year, by comparison with an average of less than two dental office visits per year per person in the United States.29 Although amoxicillin has a significant impact on bacteremia from a dental extraction, a notable number the extraction patients who received prophylaxis in this study nonetheless showed evidence of bacteremia, including IE-related species. This lack of 100% efficacy alters the per-dose risk-benefit ratio, increasing the number needed to treat to avert a distant site infection.
The duration of bacteremia likely reflects the nature and number of bacteria that enter the circulation, as well as multiple other host factors such as immune responses. Although it is not clear what role duration has on the risk for bacterial seeding of cardiac valves, our data demonstrate that bacteria are cleared rapidly, particularly in the presence of amoxicillin. However, some pathogenic species persisted for at least 60 min. following brushing and extraction without antibiotic.
The human oral cavity is colonized by a larger variety of bacterial flora than any other anatomical area. Over 700 species of bacteria have already been identified, 400 of which were found in the periodontal pocket adjacent to teeth.30 Streptococci represent a significant proportion of the flora around the teeth, especially in the supragingival plaque, and they are frequently associated with IE. An extensive search of the literature yields a common list of 126 individual bacteria reported in blood cultures following extractions (N=131) or tooth brushing (N=26), all identified by conventional clinical laboratory methods. However, data on the incidence, duration, nature and magnitude of bacteremia from non-IE-associated species, and bacteria identified by non-molecular means, are of little or no help to clinicians or policy makers.31 We focused on the 32 bacterial species identified in the present study that were also on the list of 275 bacteria reported to cause IE, 11 (32%) of which have not been reported previously in studies of brushing or extractions. Finally, we identified 71 species and subspecies of bacteria not previously reported in blood cultures following extractions or tooth brushing, 30 of which are novel.
It is difficult to quantitate the magnitude of bacteria that initially gain entrance to the circulation following dental procedures due to factors such as heart rate, blood volume, proximity of the blood collection site to the source of the bacteremia, and the rapid bacterial clearance by the reticuloendothelial system. Although animal model data have established that the rate of infection of damaged heart valves is dependent upon the inoculum size of the bacterial challenge, with larger inocula yielding higher infection rates, there are no data indicating the range of inocula which result in endocarditis in vulnerable patients. Although we were able to reliably detect and therefore quantitate a bacteria in blood at or above a concentration between 103 and 104 CFU/mL, the magnitude from extractions and tooth brushing was below this level in all samples examined. Therefore, all we can say is that the magnitude of bacteria in the blood cultures was below 104 CFU/ml.
We have over twice the number of complex extractions in the extraction – placebo group (19 vs. 9) which, although not statistically significant, suggests that 20% of the placebo group vs. 9% of the amoxicillin group had a more invasive procedure. This might contribute to the increased incidence and duration of bacteremia in the placebo group, and this would likely explain the (non-significant) increased extraction time for the placebo group. If this is the case, there is less of an impact from Amoxicillin than Figure 3 suggests.
There are potential limitations to this study. First, since all subjects were seen at a hospital-based clinic, and all needed an extraction, they may have been demographically distinct from, and had a greater burden of dental disease than, the general population. Our population was similar to the US population in gender distribution (56% male in the study vs. 49% male nationally), but was different in racial/ethnic break-down (69% African –American in the study vs. 12% nationally and 28% locally) [US Census Bureau, American Community Survey, 2006].32 We are not aware of any differences between racial groups in terms of oral bacterial flora in disease or health, and we therefore feel that these data are easily extrapolated to other racial and ethnic groups. Second, although the number of bacterial CFU per ml of blood was always below 10,000, this does not exclude the possibility of significant differences in CFU/ml below this threshold between the three groups, which might be important in terms of risk of heart valve colonization. Our data suggest that brushing and single tooth extraction, generally thought to be at different ends of the spectrum of invasiveness, are similar from the standpoint of magnitude.
There is ongoing debate concerning the health risks, cost-effectiveness, and practicality of the routine use of prophylactic antibiotics.3, 4, 33-35 The lack of efficacy data for this practice has to be weighed against risk factors (e.g., drug reactions), potential for resistant strains and various economic costs to society from the routine use of antibiotics for common dental procedures. Although the 2007 AHA recommendations call for far fewer people to receive antibiotic prophylaxis than earlier guidelines, these recommendations have been adopted for over 20 groups of noncardiac patients as well.36 The incidence, duration, nature and magnitude and daily occurrence of bacteremia from tooth brushing and other routine daily events (e.g., chewing food) calls into question the appropriateness and emphasis on prophylaxis for periodic dental procedures. Given the unfeasible concept of advocating antibiotic coverage for tooth brushing, we suggest that a controlled clinical trial is indicated to resolve this longstanding issue. In the meantime, there should be a greater focus on avoidance of dental disease in patients at risk for distant site infection in general, and IE in particular.
The authors wish to thank Jenene Noll, RN and Louise Kent, RN for their dedicated effort with subject enrollment and data collection; Shirley Coleman, MS and Jignya Ashar, MS for their contribution to bacterial isolation; Tainika Williams for her skills with manuscript preparation; Bridget Loven, MLIS for her skills with biomedical information; and Anne Olson for her Adobe Illustrator skills. We are also grateful to Larry Baddour, MD, Stanford Shulman, MD, Brian Strom, MD, MPH, and Kathryn Taubert, PhD for their helpful comments on this manuscript.
Funding Source: This study was supported by NIDCR/NIH Grant # R01 DE13559-01.
Conflict of Interest Disclosures: None.
Trial Registration: ClinicalTrials.gov
Identification Number: NCT00454285