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Streptococci isolated from post-extraction bacteraemias
BritishJournal of&al surgery
STREPTOCOCCI
(1975),
13,
91-94
ISOLATED FROM POST-EXTRACTION BACTERAEMIAS
J. M. SYMINGTON, M.Sc., F.D.S.R.C.S. Department
of Oral Surgey,
Turner Dental School, Manchester
Summary. One hundred and fifty streptococci were isolated from blood cultures taken following the extraction of teeth. The cultural, biochemical and serological characteristics of the organisms are presented and their antibiotic sensitivities discussed. It is shown that a detailed assessment of these organisms can be made rather than considering them all as Streptococcus oiridans. INTRODUCTION THE role of streptococci in the causation of subacute bacterial endocarditis has been known for many years (Horder, agog; Libman, 1913) and as early as 1927 Smith and Brumfield suggested oral sepsis as a likely avenue to the blood stream. Okell and Elliot (1935) proved the existence of a post-extraction bacteraemia and this work has subsequently been confirmed on many occasions. Surprisingly, the literature on the organisms isolated contains few detailed bacteriological investigations, especially with reference to the streptococci. Discussion and subdivision has been largely on the basis of the appearance of the haemolysis on the blood agar plates, grouping being by a haemolysis, fl haemolysis and y or non-haemolytic The viridans (a haemolytic) streptococci were cited as causal orappearance. ganisms of subacute bacterial endocarditis in 1914 by Moorhead and Stokes, the name having been first given to this group of bacteria by Schottmuller (1903). This rather inexact concept of the streptococci has persisted despite the opportunity which exists for developing a more precise knowledge and terminology. Several workers have attempted this greater precision, Green (1939) used GrilIiths typing to compare streptococci from the blood and from the heart valves, while Lazansky et al. (1950) used sugar fermentation to identify organisms in the blood following oral surgical procedures. In a further investigation into the relationship of the flora of the oral cavity to the bacteria reported as aetiological agents in the production of subacute bacterial endocarditis, Farmer (1953) studied the streptococci from the mouths of healthy subjects. Serological studies showed that 37 per cent were able to be grouped by Lancefield’s method. Groups H and K were most frequently found, Groups F and 0 were less commonly isolated. This paper presents the biochemical and serological reactions of streptococci isolated from post-extraction bacteraemias and the use of this information to attempt more precise classification of the organisms found. Clinical Methods and Material. Eighty adult patients who required dental extractions under general anaesthesia were the subjects chosen for study. Following preparation of the skin with I per cent iodine in spirit, venepuncture was performed before and after tooth extraction. Ten millilitres of blood was Received
18.2.74.
Accepted 91
29.4.74.
92
BRITISH JOURNAL
OF ORAL
SURGERY
taken prior to surgery, 5 ml being inoculated into IOOml of glucose broth and 5 ml into IOO ml of Brewer’s medium. Fifteen millilitres of blood was withdrawn after the extraction and divided into three parts; two were treated as for the pre-extraction blood, but in addition, 5 ml of the sample was inoculated into O&i’s medium. Laboratory Methods. The blood cultures were incubated for 24 hours. Blood agar plates were then inoculated, those from the glucose broth being incubated aerobically and those from the Brewer’s medium anaerobically. This procedure was repeated each day for 14 days or until growth was seen on the blood agar plates. On&i’s medium was only subcultured when visible growth occurred. Subculturing on blood agar plates was continued until a pure culture was obtained. Tolerance to aerobic culture was then carried out and the subsequent investigations carried out under the appropriate conditions. A 24-hour broth culture was prepared, where necessary supporting growth with added horse serum. This broth culture was used for microscopy, serology and antibiotic sensitivity. Microscopy. Gram staining and a cell wall stain (Hales, 1953) were used routinely on preparations derived both from liquid and solid media. The cell wall stain was especially useful in distinguishing beading on filamentous organisms from true streptococci. Biochemical Reactions. Eight sugar media were used: glucose, lactose, sucrose, raflinose, trehalose, mannitol, sorbitol and salicin. In addition, reactions were tested to aesculin, sodium hippurate, arginine, methylene blue milk and litmus milk. Growth on blood agar plates containing either salt, potassium tellurite or bile salts and on salivarius mitis agar was also recorded. Serological Tests. The technique used was Shattock’s modification of Lancefield’s method. The antiserum was commercially prepared. The test was performed using capillary tubing, the second layer being added with a fine drawn pipette. The occurrence of an opaque ring at the interface was taken as a positive result. Antibiotic Sensitivity. This was determined with the use of an Oxoid Multidisc on a blood agar plate seeded with a 24-hour broth culture. The disc carriedpenicillin 1-5units, streptomycin IO pg, sulphafurazole IOO pg, chloramphenicol IO pg, erythromycin IO pg and tetracycline IO pg. RESULTS One hundred and teristics of streptococci reactions on serological divided on the basis of
fifty organisms which showed the morphological characwere isolated. Fifty-three (35.3 per cent) gave positive testing (Table I). The remaining organisms were subbiochemical tests and could be defined as follows:
(a) Four Streptococcus saliva&s -by
the characteristic growth on salivariusl mitis agar. (b) Eighty Streptococcus mitis- where the reactions exhibited most of the characteristics of this group as defined by Bergey (1957).
STREPTOCOCCI
ISOLATED
FROM
POST-EXTRACTION
TABLE
BACTERAEMIAS
93
I
Types of streptococci
isolated No. isolated
Group
21 FI
I3
I
f 2
Tsi
I3
0 Str. saliva&s Str. mitis
8:
Not identified
I3
/ (c)
Thirteen
not identified.
Only M.and y haemolytic streptoCultural and Biochemical Reactions. tc haemolysis accounted for 105 of the organisms and y cocci were isolated. haemolysis for 45. No consistant pattern was encountered either within or between the groups. When the ability of the various organisms to ferment the sugar media is studied, few conclusions can be drawn. As might have been expected, glucose Organisms were placed and lactose were commonly utilised and acid produced. as Streptococcus mitis by the ability to ferment glucose, lactose and sucrose, a positive reaction with salicin but not sorbitol being taken as confirmatory evidence. The production of ammonia from arginine was variable; 19 of the organisms with positive Lancefield grouping gave a negative reaction to this test while 64 Streptococcus mitis and IO unidentified streptococci also gave negative results. The use of salivarius/mitis agar was useful in detecting the large mucoid colonies produced by Streptococcus salivarius, of which four were isolated. The brokenglass appearance said to be significant in the identification of colonies of Streptococcus mitis was not, however, found of value during this investigation. Antibiotic Sensitivity. 54.7 per cent of the organisms isolated showed some form of antibiotic resistance to the tests applied (Table II). As can be seen, the results show the not unexpected finding that resistance was largely to streptomycin and sulphafurazole. When the Lancefield groups are compared with the antibiotic sensitivities no significant correlation was found. DISCUSSION Those organisms which could begrouped by the modified Lancefield’s method numbered 35.3 percent, a figure very close to that given by Farmer (1953) for streptococci isolated from the mouth. However, in contradistinction to his findings, the greatest numbers were in Groups F, H and 0, rather than Groups H and K. Since 19 organisms which gave a positive reaction with the antisera used did not produce ammonia from arginine, a negative result from this test does not mean that the organism will not group.
BRITISH
94
JOURNAL
OF ORAL SURGERY
TABLE
II
Antibiotic resistance in organisms isolated AntiBiotics
No. of organisms resistant
Penicillin Streptomycin Tetracycline Erythromycin Chloramphenicol Sulphafurazole
7: I 0
3:
Although 80 organisms were placed in the group Streptococcus m&isit would seem very likely that further subdivision could be made in this group using specific antisera as performed, for example, by Soloway (1942). Comparison of the fermentation reactions of this group with those reacting with Lancefield antisera shows that while Group H is more reactive, little difference can be detected between the remainder. The unidentified streptococci as a group proved unable to ferment more than a very few of the sugar media. Colonial morphology and the type of haemolysis on blood agar plates was of no value in the subdivision of the streptococci isolated. Organisms with the same Lancefield group could exhibit either a or y haemolysis. The fkniings in relation to the antibiotic sensitivities were not altogether unexpected, but would highlight the inadequacy of streptomycin and sulphonamides for therapeutic use against oral streptococci.
ACKNOWLEDGEMENTS I would like to thank Professor G. L. Slack and Mr G. Bowden of the Dental Institute, The London Hospital, and Professor J. R. Moore of The Turner Dental School, Manchester, for their kind assistance and encouragement.
REFERENCES BERGEY, D. A. (1957). Manual
Smith.
of Determinative Bacteriology, 7th ed.
Edited by N. A.
FARMER, E. D. (1953). Proceedings of the Royal Society of Medicine, 46, 201. GREEN,C. A. (1939). Annals of Rheumatic Diseases, I, 86. HALES,C. M. F. (1953). Laboratory Practice, 2, 11s. Quarterly Journal of Medicine, 2, 289. HORDER, T. J. (1909). LAZANSKY,J. l?., ROBINSON,L. & RODOFSKY, L. (1950). Journal of Dental Research, 28,533. LIBMAN, E. (1913). American Journal of Medicine Science, 146, 635. MOoRHEAD, T. G. & STOKES,A. (1914). Practitioner, 92, 407. OKEZL, C. C. & ELLIOT, S. D. (1935). Lancet, 2, 869. ONISI, M. (1959). Journal of Dental Research, 38, 311. SCHOTTMULLER, H. VON (1903). Munch. Med. Woch., 50, 849. SMITH, F. J. & BRUMFIELD, D. M. (1927). Michigan State Medical Society Journal, 26,245. SOLOWAY, M. (1942). $urnal of Experimental Medicine, 76, 109.
STREPTOCOCCI
(1975),
13,
91-94
ISOLATED FROM POST-EXTRACTION BACTERAEMIAS
J. M. SYMINGTON, M.Sc., F.D.S.R.C.S. Department
of Oral Surgey,
Turner Dental School, Manchester
Summary. One hundred and fifty streptococci were isolated from blood cultures taken following the extraction of teeth. The cultural, biochemical and serological characteristics of the organisms are presented and their antibiotic sensitivities discussed. It is shown that a detailed assessment of these organisms can be made rather than considering them all as Streptococcus oiridans. INTRODUCTION THE role of streptococci in the causation of subacute bacterial endocarditis has been known for many years (Horder, agog; Libman, 1913) and as early as 1927 Smith and Brumfield suggested oral sepsis as a likely avenue to the blood stream. Okell and Elliot (1935) proved the existence of a post-extraction bacteraemia and this work has subsequently been confirmed on many occasions. Surprisingly, the literature on the organisms isolated contains few detailed bacteriological investigations, especially with reference to the streptococci. Discussion and subdivision has been largely on the basis of the appearance of the haemolysis on the blood agar plates, grouping being by a haemolysis, fl haemolysis and y or non-haemolytic The viridans (a haemolytic) streptococci were cited as causal orappearance. ganisms of subacute bacterial endocarditis in 1914 by Moorhead and Stokes, the name having been first given to this group of bacteria by Schottmuller (1903). This rather inexact concept of the streptococci has persisted despite the opportunity which exists for developing a more precise knowledge and terminology. Several workers have attempted this greater precision, Green (1939) used GrilIiths typing to compare streptococci from the blood and from the heart valves, while Lazansky et al. (1950) used sugar fermentation to identify organisms in the blood following oral surgical procedures. In a further investigation into the relationship of the flora of the oral cavity to the bacteria reported as aetiological agents in the production of subacute bacterial endocarditis, Farmer (1953) studied the streptococci from the mouths of healthy subjects. Serological studies showed that 37 per cent were able to be grouped by Lancefield’s method. Groups H and K were most frequently found, Groups F and 0 were less commonly isolated. This paper presents the biochemical and serological reactions of streptococci isolated from post-extraction bacteraemias and the use of this information to attempt more precise classification of the organisms found. Clinical Methods and Material. Eighty adult patients who required dental extractions under general anaesthesia were the subjects chosen for study. Following preparation of the skin with I per cent iodine in spirit, venepuncture was performed before and after tooth extraction. Ten millilitres of blood was Received
18.2.74.
Accepted 91
29.4.74.
92
BRITISH JOURNAL
OF ORAL
SURGERY
taken prior to surgery, 5 ml being inoculated into IOOml of glucose broth and 5 ml into IOO ml of Brewer’s medium. Fifteen millilitres of blood was withdrawn after the extraction and divided into three parts; two were treated as for the pre-extraction blood, but in addition, 5 ml of the sample was inoculated into O&i’s medium. Laboratory Methods. The blood cultures were incubated for 24 hours. Blood agar plates were then inoculated, those from the glucose broth being incubated aerobically and those from the Brewer’s medium anaerobically. This procedure was repeated each day for 14 days or until growth was seen on the blood agar plates. On&i’s medium was only subcultured when visible growth occurred. Subculturing on blood agar plates was continued until a pure culture was obtained. Tolerance to aerobic culture was then carried out and the subsequent investigations carried out under the appropriate conditions. A 24-hour broth culture was prepared, where necessary supporting growth with added horse serum. This broth culture was used for microscopy, serology and antibiotic sensitivity. Microscopy. Gram staining and a cell wall stain (Hales, 1953) were used routinely on preparations derived both from liquid and solid media. The cell wall stain was especially useful in distinguishing beading on filamentous organisms from true streptococci. Biochemical Reactions. Eight sugar media were used: glucose, lactose, sucrose, raflinose, trehalose, mannitol, sorbitol and salicin. In addition, reactions were tested to aesculin, sodium hippurate, arginine, methylene blue milk and litmus milk. Growth on blood agar plates containing either salt, potassium tellurite or bile salts and on salivarius mitis agar was also recorded. Serological Tests. The technique used was Shattock’s modification of Lancefield’s method. The antiserum was commercially prepared. The test was performed using capillary tubing, the second layer being added with a fine drawn pipette. The occurrence of an opaque ring at the interface was taken as a positive result. Antibiotic Sensitivity. This was determined with the use of an Oxoid Multidisc on a blood agar plate seeded with a 24-hour broth culture. The disc carriedpenicillin 1-5units, streptomycin IO pg, sulphafurazole IOO pg, chloramphenicol IO pg, erythromycin IO pg and tetracycline IO pg. RESULTS One hundred and teristics of streptococci reactions on serological divided on the basis of
fifty organisms which showed the morphological characwere isolated. Fifty-three (35.3 per cent) gave positive testing (Table I). The remaining organisms were subbiochemical tests and could be defined as follows:
(a) Four Streptococcus saliva&s -by
the characteristic growth on salivariusl mitis agar. (b) Eighty Streptococcus mitis- where the reactions exhibited most of the characteristics of this group as defined by Bergey (1957).
STREPTOCOCCI
ISOLATED
FROM
POST-EXTRACTION
TABLE
BACTERAEMIAS
93
I
Types of streptococci
isolated No. isolated
Group
21 FI
I3
I
f 2
Tsi
I3
0 Str. saliva&s Str. mitis
8:
Not identified
I3
/ (c)
Thirteen
not identified.
Only M.and y haemolytic streptoCultural and Biochemical Reactions. tc haemolysis accounted for 105 of the organisms and y cocci were isolated. haemolysis for 45. No consistant pattern was encountered either within or between the groups. When the ability of the various organisms to ferment the sugar media is studied, few conclusions can be drawn. As might have been expected, glucose Organisms were placed and lactose were commonly utilised and acid produced. as Streptococcus mitis by the ability to ferment glucose, lactose and sucrose, a positive reaction with salicin but not sorbitol being taken as confirmatory evidence. The production of ammonia from arginine was variable; 19 of the organisms with positive Lancefield grouping gave a negative reaction to this test while 64 Streptococcus mitis and IO unidentified streptococci also gave negative results. The use of salivarius/mitis agar was useful in detecting the large mucoid colonies produced by Streptococcus salivarius, of which four were isolated. The brokenglass appearance said to be significant in the identification of colonies of Streptococcus mitis was not, however, found of value during this investigation. Antibiotic Sensitivity. 54.7 per cent of the organisms isolated showed some form of antibiotic resistance to the tests applied (Table II). As can be seen, the results show the not unexpected finding that resistance was largely to streptomycin and sulphafurazole. When the Lancefield groups are compared with the antibiotic sensitivities no significant correlation was found. DISCUSSION Those organisms which could begrouped by the modified Lancefield’s method numbered 35.3 percent, a figure very close to that given by Farmer (1953) for streptococci isolated from the mouth. However, in contradistinction to his findings, the greatest numbers were in Groups F, H and 0, rather than Groups H and K. Since 19 organisms which gave a positive reaction with the antisera used did not produce ammonia from arginine, a negative result from this test does not mean that the organism will not group.
BRITISH
94
JOURNAL
OF ORAL SURGERY
TABLE
II
Antibiotic resistance in organisms isolated AntiBiotics
No. of organisms resistant
Penicillin Streptomycin Tetracycline Erythromycin Chloramphenicol Sulphafurazole
7: I 0
3:
Although 80 organisms were placed in the group Streptococcus m&isit would seem very likely that further subdivision could be made in this group using specific antisera as performed, for example, by Soloway (1942). Comparison of the fermentation reactions of this group with those reacting with Lancefield antisera shows that while Group H is more reactive, little difference can be detected between the remainder. The unidentified streptococci as a group proved unable to ferment more than a very few of the sugar media. Colonial morphology and the type of haemolysis on blood agar plates was of no value in the subdivision of the streptococci isolated. Organisms with the same Lancefield group could exhibit either a or y haemolysis. The fkniings in relation to the antibiotic sensitivities were not altogether unexpected, but would highlight the inadequacy of streptomycin and sulphonamides for therapeutic use against oral streptococci.
ACKNOWLEDGEMENTS I would like to thank Professor G. L. Slack and Mr G. Bowden of the Dental Institute, The London Hospital, and Professor J. R. Moore of The Turner Dental School, Manchester, for their kind assistance and encouragement.
REFERENCES BERGEY, D. A. (1957). Manual
Smith.
of Determinative Bacteriology, 7th ed.
Edited by N. A.
FARMER, E. D. (1953). Proceedings of the Royal Society of Medicine, 46, 201. GREEN,C. A. (1939). Annals of Rheumatic Diseases, I, 86. HALES,C. M. F. (1953). Laboratory Practice, 2, 11s. Quarterly Journal of Medicine, 2, 289. HORDER, T. J. (1909). LAZANSKY,J. l?., ROBINSON,L. & RODOFSKY, L. (1950). Journal of Dental Research, 28,533. LIBMAN, E. (1913). American Journal of Medicine Science, 146, 635. MOoRHEAD, T. G. & STOKES,A. (1914). Practitioner, 92, 407. OKEZL, C. C. & ELLIOT, S. D. (1935). Lancet, 2, 869. ONISI, M. (1959). Journal of Dental Research, 38, 311. SCHOTTMULLER, H. VON (1903). Munch. Med. Woch., 50, 849. SMITH, F. J. & BRUMFIELD, D. M. (1927). Michigan State Medical Society Journal, 26,245. SOLOWAY, M. (1942). $urnal of Experimental Medicine, 76, 109.