Journal of Clinical Periodontology: 1977: 4: 92-99 Key words: Bacieiaeinia - hiimati experimenial'on - periodonial health flatus. Accepted for publication: July 16, 1976.
Experimental transient bacteraemias in human subjects with varying degrees of plaque accumulation and gingival inflammation JOHN G . SILVER, ALEXIS W . MARTIN AND BARRY C . MCBRIDE
Departments of Oral Medicine and MicrobioJogy, University of British Columbia, Vancouver, British Columbia, Canada Abstraci. Ninety-six subjects were assigned to one of four groups according to severity of gingiva] inflamimation and bacterial plaque accumulation on the teeth. Following a standardized toothbrushing procedure, blood specimens from a vein in the antecubital fossa were cultured under aerobic and stringent anaerobic conditions. The percentage of positive cultures increased significantly with increasing severity of gingival inflammation, as did the number of species of organisms isolated. Thirty different microbial species indigenous to the oral cavity, including many strict anaerobes, were recovered. The study has implications for standards of oral health which might be considered necessary in patients with congenital or acquired endocardial defects or cardiovascular prostheses.
Over forty years ago it was found that dental extractions produced a transitory bacteraemia and it was suggested that this phenomenon had a bearing on the aetiology of infective endocarditis in patients with rheumatic and congenital heart disease (Okell & Elliott 1935). It is known now that most dental manipulations including extractions (Jones et al. 1970), scaling (Bandt et al. 1964, Conner et al. 1967), surgical periodontal procedures (Korn & Schaffer 1962, Bender et al. 1963), filling of teeth (Harvey & Capone 1961) and root canal treatment (Farrington 1973) introduce bacteria into the blood stream as do such patient performed procedures as chewing (Cobe 1954), oral irrigation (Romans & App 1971, Berger et al. 1974) and tooth-
brushing (Cobe 1954, Rise et al. 1969, Sconyers et al. 1973, Madsen 1974). There is thus sufficient circumstantial evidence to recommend certain precautions be taken during oral manipulative procedures in patients with congenital and acquired heart defects (Cooke 1970) and specific recommendation for antibiotic cover has been made (American Heart Association 1965). Patients who have succumbed to one attack of infective endocarditis have been advised to have their remaining teeth extracted to prevetit further occurrence (Hobsen & Juel-Jensen 1956, Beeley 1969). It has been pointed out, however, that further information is needed before dental clearance can be advised (Croxson et al. 1971) and the current un-
BACTERAEMIAS AND VARYING PERIODONTAL HEALTH certainty of the dental assessment of patients with valvular defects and the need for further research also has been stated (Editorial, Lancet 1971). Bearing in mind tbe above points, the present study of experimental bacteraemias was undertaken. The study differed from previous investigations by utilizing a sample size suitable for statistical analysis and by employing strict epidemiological criteria for categorization of subjects. The method chosen for attempted creation of the bacteraemia was toothbrushing, the rationale for this method being its everyday occurrence in tbe hves of many persons and the relative paucity of previous research using a timed, specified technique of brushing. Additionally, correlations between bacteraemias and specified parameters of gingival health had not been proven (Cobe 1954, Rise et al. 1969, Tamini et al. 1969, Sconyers et al. 1973, Madsen 1974).
Methods
A total of 96 subjects (44 male, 52 female) aged 17-61 years were assigned to one of four groups according to modified criteria for the Gingival and Plaque Indices (Loe & Silness 1963, Silness & Loe 1964). The modifications involved omission of instrumentation within the gingival sulcus, thus reducing the possibility of inadvertent pre-experimental bacteraemias. Bleeding on brusbing resulted in tbe assignation of a score of 2 for the Gingival Index. All subjects were in good general health with no history of any antibiotic therapy within the preceding month. Each subject's mouth was divided into three segments for both mandibular and maxillary teeth. The gingival sulci of each segment were brushed by one of the investigators using an electric toothbrush (Touch-Tronic,®, Teledyne Aqua Tec, Ltd., Rexdale, Ont.) which started automatic
vibration by the application of pressure to the brush head. All brush heads, which were pre-sterilized in ethylene oxide, were of the same soft nylon texture and the pressure applied to the brush was just sufficient to start the automatic action. The buccal and lingual gingival sulci of each segment were brushed for 10 sec restilting in a total brushing time of 2 min for each subject. Immediately prior to brushing, 5 ml of blood were withdrawn from a vein in the antecubital fossa of one arm and, during the last 30-60 sec of brushing, 10 ml were withdrawn from a similar vein in the apposite arm. (Previous studies on dogs had shown that peak bacteraemia occurred 30-60 sec after introduction of bacteria into the circulation (Silver et al. 1975)). Prior to obtaining the blood samples, the skin of the antecubital fossae was cleansed thoroughly using, in succession, several swabs soaked in 70 % ethyl alcohol. The preoperative blood sample was divided equally and placed into 50 ml of prereduced anaerobically sterile (PRAS) "B" medium (McMinn & Crawford 1970) contained in an anaerobic flask, and 50 ml of aerobic trypticase soy broth. Three ml of the postoperative blood sample were placed into 100 ml of trypticase soy hrotb and 1 ml was mixed with 15 ml of premelted trypticase soy agar for aerobic incubation. The remaining 6 ml of blood was divided equally and placed into anaerobic containers holding respectively 100 ml of PRAS "E" broth; 50 ml of brain heart infusion broth supplemented with 0.3 % yeast extract, 1.0 % trypticase, 0.0005 % haemin, 0.05 % cysteine hydrochloride, and 25 ml of PRAS "E" broth supplemented with 2 % agar. Blood cultures were incubated at 37°C and inspected at intervals for growth. Aerobic cultures were maintained for a minimum of 5 days and anaerobic cultures
94
SILVER, MARTIN AND McBRlDE
Table 1. Relationship of positive blood cultures to groupings of the Ginglval Index Verhdltnis positlver Blutkulturen zu verschiedenen Gruppen des Gingival-Jndexes Relation des cultures positives de sang et des groupes d'index gingival Gingival Index Subjects No. in group No. with positive blood cultures Per cent with positive biood cultures
0-0.75
0.76-1.50
1.51-2.25
2.26-3.00
25
24
25
22
4
8
14
15
56%
68%
16%
33%
No. in group (Anzahl Probanden in der Gruppe, nombre dans le groupe). No. with positive blood cultures (Anzahl Probanden mit positiven Blutkulturen, nombre avec culture de sang positive). Per cent witb positive blood cultures (Anzahl Probanden mit positiven Blutkulturen in Prolent, potircentage de cultures de sang positives)
for 21 days. Samples were subciiltured onto blood agar plates, anaerobic cultures being plated and incubated in an anaerobic chamber (Coy Mfg. Co., Ann Arbor, Michigan). Identification of microorganisms was in accordance with criteria specified in Buchanan & Gibbons (1974) and Holdeman & Moore (1975). Results were analyzed and significant differences determined by utilization of a critical ratio test.
^^" ^ The relationships between numbers and percentages of subjects with positive blood cultures following toothbrushing and Gingival and Plaque Indices are shown in Tables 1 and 2. As can be seen, the percentage of subjects demonstrating bacteraemia increased with increasing severity of gingival inflammation. Significant differences were found between those groupings of the Gingival Index shown in Table 3,
Table 2. Relationship of positive blood cultures to groupings of the Plaque Index Verhdltnis positiver Blutkulturen zu verschiedenen Gruppen des Plaque-Indexes Relation des cultures positives de sang et des groupes d'index de plaque Plaque Index Subjects No. in group No. with positive blood cultures Per cent with positive blood cultures
0-0.75
0.76-1.50
1.51-2.25
2.26-3.00
40
26
16
14
14
9
10
.8
35%
35%
63%
57%
Subjects (Probanden, Sujets), No. in group (Anzahl Probanden in der Gruppe, nonibre dans le groupe). No. with positive blood cultures (Anzahl Probanden mit positiven Blutkulturen, nombre avec culture de sang positive). Percent positive blood cultures (Anzahl Probanden mit positiven Blutkulturen in Prozent, pourcentage de cultures de sang positives).
BACTERAEMIAS AND VARYING PERIODONTAL HEALTH Table 3. Groupings of the Gingival Index showing significant differences in occurrence of bacteraemias utilizing a Critical Ratio Test Gingival'Index-Gruppen. Gezeigt werden s'lgnifikante Unterschiede betr. Vorkommen von Bakteriaemie, beslimmt miltels Crtlical-RalioTest Groiipes de rindex gingival montrant des differences significalivcs de frequence de bacteriemies uiiiisanf un test de proportion critique Significance
Groupings compared 0-0.75 < 1.51-2.25 0-0.75 < 2.26-3.00 0.76-1.50 < 2.26-3.00
0.001
Experimental transient bacteraemias in human subjects with varying degrees of plaque accumulation and gingival inflammation JOHN G . SILVER, ALEXIS W . MARTIN AND BARRY C . MCBRIDE
Departments of Oral Medicine and MicrobioJogy, University of British Columbia, Vancouver, British Columbia, Canada Abstraci. Ninety-six subjects were assigned to one of four groups according to severity of gingiva] inflamimation and bacterial plaque accumulation on the teeth. Following a standardized toothbrushing procedure, blood specimens from a vein in the antecubital fossa were cultured under aerobic and stringent anaerobic conditions. The percentage of positive cultures increased significantly with increasing severity of gingival inflammation, as did the number of species of organisms isolated. Thirty different microbial species indigenous to the oral cavity, including many strict anaerobes, were recovered. The study has implications for standards of oral health which might be considered necessary in patients with congenital or acquired endocardial defects or cardiovascular prostheses.
Over forty years ago it was found that dental extractions produced a transitory bacteraemia and it was suggested that this phenomenon had a bearing on the aetiology of infective endocarditis in patients with rheumatic and congenital heart disease (Okell & Elliott 1935). It is known now that most dental manipulations including extractions (Jones et al. 1970), scaling (Bandt et al. 1964, Conner et al. 1967), surgical periodontal procedures (Korn & Schaffer 1962, Bender et al. 1963), filling of teeth (Harvey & Capone 1961) and root canal treatment (Farrington 1973) introduce bacteria into the blood stream as do such patient performed procedures as chewing (Cobe 1954), oral irrigation (Romans & App 1971, Berger et al. 1974) and tooth-
brushing (Cobe 1954, Rise et al. 1969, Sconyers et al. 1973, Madsen 1974). There is thus sufficient circumstantial evidence to recommend certain precautions be taken during oral manipulative procedures in patients with congenital and acquired heart defects (Cooke 1970) and specific recommendation for antibiotic cover has been made (American Heart Association 1965). Patients who have succumbed to one attack of infective endocarditis have been advised to have their remaining teeth extracted to prevetit further occurrence (Hobsen & Juel-Jensen 1956, Beeley 1969). It has been pointed out, however, that further information is needed before dental clearance can be advised (Croxson et al. 1971) and the current un-
BACTERAEMIAS AND VARYING PERIODONTAL HEALTH certainty of the dental assessment of patients with valvular defects and the need for further research also has been stated (Editorial, Lancet 1971). Bearing in mind tbe above points, the present study of experimental bacteraemias was undertaken. The study differed from previous investigations by utilizing a sample size suitable for statistical analysis and by employing strict epidemiological criteria for categorization of subjects. The method chosen for attempted creation of the bacteraemia was toothbrushing, the rationale for this method being its everyday occurrence in tbe hves of many persons and the relative paucity of previous research using a timed, specified technique of brushing. Additionally, correlations between bacteraemias and specified parameters of gingival health had not been proven (Cobe 1954, Rise et al. 1969, Tamini et al. 1969, Sconyers et al. 1973, Madsen 1974).
Methods
A total of 96 subjects (44 male, 52 female) aged 17-61 years were assigned to one of four groups according to modified criteria for the Gingival and Plaque Indices (Loe & Silness 1963, Silness & Loe 1964). The modifications involved omission of instrumentation within the gingival sulcus, thus reducing the possibility of inadvertent pre-experimental bacteraemias. Bleeding on brusbing resulted in tbe assignation of a score of 2 for the Gingival Index. All subjects were in good general health with no history of any antibiotic therapy within the preceding month. Each subject's mouth was divided into three segments for both mandibular and maxillary teeth. The gingival sulci of each segment were brushed by one of the investigators using an electric toothbrush (Touch-Tronic,®, Teledyne Aqua Tec, Ltd., Rexdale, Ont.) which started automatic
vibration by the application of pressure to the brush head. All brush heads, which were pre-sterilized in ethylene oxide, were of the same soft nylon texture and the pressure applied to the brush was just sufficient to start the automatic action. The buccal and lingual gingival sulci of each segment were brushed for 10 sec restilting in a total brushing time of 2 min for each subject. Immediately prior to brushing, 5 ml of blood were withdrawn from a vein in the antecubital fossa of one arm and, during the last 30-60 sec of brushing, 10 ml were withdrawn from a similar vein in the apposite arm. (Previous studies on dogs had shown that peak bacteraemia occurred 30-60 sec after introduction of bacteria into the circulation (Silver et al. 1975)). Prior to obtaining the blood samples, the skin of the antecubital fossae was cleansed thoroughly using, in succession, several swabs soaked in 70 % ethyl alcohol. The preoperative blood sample was divided equally and placed into 50 ml of prereduced anaerobically sterile (PRAS) "B" medium (McMinn & Crawford 1970) contained in an anaerobic flask, and 50 ml of aerobic trypticase soy broth. Three ml of the postoperative blood sample were placed into 100 ml of trypticase soy hrotb and 1 ml was mixed with 15 ml of premelted trypticase soy agar for aerobic incubation. The remaining 6 ml of blood was divided equally and placed into anaerobic containers holding respectively 100 ml of PRAS "E" broth; 50 ml of brain heart infusion broth supplemented with 0.3 % yeast extract, 1.0 % trypticase, 0.0005 % haemin, 0.05 % cysteine hydrochloride, and 25 ml of PRAS "E" broth supplemented with 2 % agar. Blood cultures were incubated at 37°C and inspected at intervals for growth. Aerobic cultures were maintained for a minimum of 5 days and anaerobic cultures
94
SILVER, MARTIN AND McBRlDE
Table 1. Relationship of positive blood cultures to groupings of the Ginglval Index Verhdltnis positlver Blutkulturen zu verschiedenen Gruppen des Gingival-Jndexes Relation des cultures positives de sang et des groupes d'index gingival Gingival Index Subjects No. in group No. with positive blood cultures Per cent with positive biood cultures
0-0.75
0.76-1.50
1.51-2.25
2.26-3.00
25
24
25
22
4
8
14
15
56%
68%
16%
33%
No. in group (Anzahl Probanden in der Gruppe, nombre dans le groupe). No. with positive blood cultures (Anzahl Probanden mit positiven Blutkulturen, nombre avec culture de sang positive). Per cent witb positive blood cultures (Anzahl Probanden mit positiven Blutkulturen in Prolent, potircentage de cultures de sang positives)
for 21 days. Samples were subciiltured onto blood agar plates, anaerobic cultures being plated and incubated in an anaerobic chamber (Coy Mfg. Co., Ann Arbor, Michigan). Identification of microorganisms was in accordance with criteria specified in Buchanan & Gibbons (1974) and Holdeman & Moore (1975). Results were analyzed and significant differences determined by utilization of a critical ratio test.
^^" ^ The relationships between numbers and percentages of subjects with positive blood cultures following toothbrushing and Gingival and Plaque Indices are shown in Tables 1 and 2. As can be seen, the percentage of subjects demonstrating bacteraemia increased with increasing severity of gingival inflammation. Significant differences were found between those groupings of the Gingival Index shown in Table 3,
Table 2. Relationship of positive blood cultures to groupings of the Plaque Index Verhdltnis positiver Blutkulturen zu verschiedenen Gruppen des Plaque-Indexes Relation des cultures positives de sang et des groupes d'index de plaque Plaque Index Subjects No. in group No. with positive blood cultures Per cent with positive blood cultures
0-0.75
0.76-1.50
1.51-2.25
2.26-3.00
40
26
16
14
14
9
10
.8
35%
35%
63%
57%
Subjects (Probanden, Sujets), No. in group (Anzahl Probanden in der Gruppe, nonibre dans le groupe). No. with positive blood cultures (Anzahl Probanden mit positiven Blutkulturen, nombre avec culture de sang positive). Percent positive blood cultures (Anzahl Probanden mit positiven Blutkulturen in Prozent, pourcentage de cultures de sang positives).
BACTERAEMIAS AND VARYING PERIODONTAL HEALTH Table 3. Groupings of the Gingival Index showing significant differences in occurrence of bacteraemias utilizing a Critical Ratio Test Gingival'Index-Gruppen. Gezeigt werden s'lgnifikante Unterschiede betr. Vorkommen von Bakteriaemie, beslimmt miltels Crtlical-RalioTest Groiipes de rindex gingival montrant des differences significalivcs de frequence de bacteriemies uiiiisanf un test de proportion critique Significance
Groupings compared 0-0.75 < 1.51-2.25 0-0.75 < 2.26-3.00 0.76-1.50 < 2.26-3.00
0.001
