Bacterial study of pyogenic infections of dental origin Charles B. Sabiston, Jr., B.S., D.D.S., Ph.D.,” William R. Grigsby, B.A., D.D.S., Ph.D.,** and Nancy Segerstrom, M.T.,**# Iowa City, Iowa UNIVERSITY
OF IOWA,
COLLEJGE
OF DENTISTRY
Fluctuant abscesses of dental origin were sampled by syringe aspiration. These samples were cultured by means of techniques suitable for isolation and growth of obligate anaerobes. Oral abscesses are usually mixed infections that include a number of different species. More than 65 per cent of the species isolated were obligate anaerobes, which indicates the need for anaerobic methods in studying these infections. Anaerobic gram-negative rods and facultative streptococci were the groups isolated most frequently.
0
ur study of anaerobic bacteria in oral infections, reported in 1974,* described bacteria isolated from eight fluctuant abscessesof dental origin. We have now studied exudates from a total of sixty-five dental abscesses.The cases selected for this report were limited to abscessesof dental origin which had extended beyond the alveolus to cause obvious erythematous fluctuant swelling. Differentiation between abscessesof periodontal origin and those of pulpal origin was not made and, indeed, frequently could not be made since both pulpal and periodontal lesions were associated with the same tooth. There are inherent difficulties in obtaining and processing exudates from oral abscesses.Sampling in an area with a substantial normal flora is likely to result in an inoculum which includes that normal flora. Any delay in plating the specimen directly onto a suitable medium or in incubating in an anaerobic environment reduces anaerobic recovery and distorts the determination of relative numbers of species of bacteria present in the abscess.Our methods of This study was supported by Grant D.E. 03662 from the United States Public Health Service, National Institute of Dental Research. *Associate Professor of Periodontology and Director, Collegiate Microbiology Research Laboratories. **Associate Professor of Periodontology. ***Research Assistant.
430
Volume 41 Number 4
Pyogenic
i~~fecti4m of dental origin
431
study have been designed to minimize certain inherent difficulties in isolating, propagating, and identifying bacteria. IJnique aspects include sampling by syringe aspiration of pus from the abscess in an attempt to minimize contamination of the specimen with normal flora; immediate culturing of all specimens in order to minimize loss of oxygen-sensit,ive anaerobes and other distortions of relative proportions of species contained in the specimen; use of a modified Hungate roll tube technique in addition to the anaerobe jar technique; use of multiple media and conditions of incubation. The literature provides little information on the species of bacteria found in abscesses of dental origin. Feldmann and Large1 reported a series in which a cautery was used to minimize contamination of the specimen. A recent study by Turner and associates5 relied on techniques which might minimize recovery of obligate anaerobes. METHODS
The first eight specimens were taken from patients being treated at Medical College of Virginia Hospital, Outpatient Clinic, Richmond, Virginia. The remainder were taken from patients in the clinics of the College of Dentistry, University of Iowa. Abscesses were isolated and dried with sterile gauze, and the exudate was obtained by aspiration with the use of a sterile disposable syringe with a No. 16 gauge needle. The exudate was expressed directly into a tube under oxygen-free gas, within 5 minutes for specimens collected at the University of Iowa and within 30 minutes for the eight specimens collected in Richmond. Some abscesses were sampled by sterile paper point after the tooth had been opened for endodontic therapy. In these cases, at chairside, the paper point was dropped into a gassed-out tube which had been opened under oxygenfree CO,. This sampling procedure used a mobile anaerobe cart similar to that described by Fulghum.2 Our techniques of culture and identification are those developed by the staff of the Anaerobe Laboratory at Virginia Polytechnic Institute and State University. Methods are described in detail in the manual published by the Anaerobe Laboratory .3 Each specimen was examined grossly, a wet preparation was made for phase-contrast microscopy, a Gram preparation was made, and the following media were inoculated: a brain-heart infusion (prereduced) agar roll tube (supplemented with heme, vitamin K, and yeast extract) ; a blood agar plate freshly poured from prereduced medium and reduced in an anaerobe jar; and a stock blood agar plate incubated in a CO, incubator. All media were incubated at 37’ C. Representative colonies were picked in 3 to 5 days. The blood media were further incubated for 10 days in order to detect Bactewides melaniwogenicus. Methods of identification were those of Holdeman and Moore3 and relied on morphology, Gram reaction, a battery of biochemical tests, and gas chromatographic analysis of fatty acids and alcohols produced in culture medium. RESULTS
Sixty-five specimens were sampled. Seven of these specimens were culturally negative. From the remaining fifty-eight specimens, we isolated from one to
Grigsby,
Sabiston,
Table
I. Frequency of occurrence of groups in specimens
Group
Anaerobic gram-positive rods Anaerobic gram-negative rods Anaerobic gram-positive cocci Anaerobic gram-negative cocci Facultative gram-positive rods Facultative gram-negative rods Facultative gram-positive cocci Facultative gram-negative cocci
and
Oral April,
432
LSegerstrom
Number of specimens containing the group
4’: 28 10 10 4: 5
Surg. 1976
Percentage of positive specimens containing the group
41.4 69.0 48.3 17.2 17.2 5.2 12.4 8.6
twelve species. The average number of distinct species in each culturally positive specimen was 3.8. Obligate anaerobes numbered 147, or 65.9 per cent of the total species isolated. Spirochetes were observed in phase preparations of seven specimens, all of which were culturally positive for other species as well. A single organism in pure culture was isolated from seven specimens, one of which was an obligate anaerobe (six facultative streptococci; one Veillonelln alcalescens). Table I lists the number of specimens which contained a given group, and the percentage of all positive specimens (fifty-eight) which that number represents. Table II lists the genus and species designation of strains isolated and the number of specimens in which that species, genus, or group occurred. The frequency of occurrence of each is also given and is expressed as a percentage of the total number of specimens studied (sixty-five). It should be noted that species of the genera Lactobacillus and Actinomyces appear under two categories since both facultative and anaerobic species commonly occur in both genera. No attempt was made to identify facultative isolants at the species level; accordingly, all facultative streptococcal strains were considered as a single species in each of the specimens in which they were present. Organisms are listed as unidentified if they were lost before identification, or if their characteristics did not closely fit those of recognized species. The species designated as Bacteroides ruminicola S.S. brevis has the characteristics described in the Virginia Polytechnic Institute Avlaerobe Laboratory Mawa1,3 but since it has not been directly and extensively compared with reference strains, it may not be identical to the rumen organism. These data represent the presence of a given organism without indicating its quantitative importance. Precise quantitation is not feasible using clinical methods, although such data could be important. We made estimations of relative proportions of morphotypes present when the primary plating media were picked, but we do not think that these estimations are sufficiently precise to report in detail. In general, the numbers of colonies observed on media incubated anaerobically were several orders of magnitude higher than those on plates incubated aerobically or under CO,. This observation is particularly significant
Volume 41 Number 4
Pyogenic
Table II. Identity
infections of
dental
origin
433
of strains isolated
Species, genus or group
Anaerobic gram-positive rods unidentified
Number of specimens from which tiolated
1
Eubacterium lentum Eubacterium alactoiyticum &bacterium species Lactobacillus species Actinomyces species Bifidobacterium species
1.1 ::: 1%
17:2 10.3 25.9 5.2 13.8 13.8 3.4
Anaerobic gram-negative rods unidentified Bacteroides oralis B. ruminicola S.S.brevis B. melaninogenicus S.S.intermedius B. melaninogenicus S.S.melaninogenicus Bacteroides species Fusobacterium nucleatum Fusobacterium species
2:.: 1:7 13.8
Anaerobic gram-positive cocci unidentified Peptococcus magnus Streptococcus morbillorum Peptostreptococcus anaerobius Peptostreptococcus micros Streptococcus constellatus Streptocuccus intermedius
ii 15:5 22.4 ii:;1
Anaerobic gram-negative cocci Veillonella parvula
10.3 6.9 6.9 10.3 5.2 5.2
Veillonella alcalescens
Facultative gram-positive rods unidentified Lactobacillus species Actinomyces species Facultative gram-negative rods unidentified Facultative gram-positive cocci streptococci-catalase neg., unspeciated
10.7
1.7 13.8
Streptococcuspyogenes Staphylococcus epidermidis Staphylococcus aureus
Facultative gram-negative cocci unidentified Other unidentified bacterium Yeast (not Capdida albicans) . _ Spirochetes (phase-contrast microscopy)
Percentage ofpositive cultures from which isolated
it 1:7 /
1:::
in the case of facultative streptococci isolated only from plates incubated aerobically with or without some COz in twelve of the forty-one cases in which they were isolated. In six additional instances they constituted less than 20 per cent of colonies present on media incuba,ted anaerobically. Five additional specimens from which they were isolated yielded very few total colonies on any media, so that the total number of specimens from which facultative streptococci were isolated in numbers above 20 per cent of the bacterial population was eighteen. The data tables, indicating a high frequency of occurrence of facultative gram-positive cocci, most of which were facultative streptococci, should be interpreted with these observations in mind. CONCLUSIONS Pyogenic oral infections contain a complex mixture of species of bacteria. Facultative streptococci occurred with greater frequency than other groups ;
434
Sabiston, Grigsby,
and Negerstrom
Oral Surg. -April, 1976
however, they were detected from some specimens only on plates incubated under aerobic conditions, which would tend to select for them. This ma; intjicate a diminutive numerical significance in some abscesses. Anaerobic gram-positive and gram-negative rods and gram-positive cocci, along with facultative streptococci, dominated the bacteria present in our series of abscesses. Facultative gramnegative rods were rare, and especially so considering their frequency in pyogenic infections elsewhere in the body. This observation may indicate a local source of the bacteria causing oral pyogenic infections. Facultative and anaerobic gram-negative cocci and facultative gram-positive rods were intermediate in occurrence. The species identified are essentially those found in the mouth in the absence of infection.G These observations support the proposition that oral abscesses are caused by opportunistic pathogens whose presence, under other circumstances, can be consistent with clinical health. The mixed anaerobic nature of the infections is of interest. The data may indicate that most of the species isolated a,re incapable of producing infections alone. With one exception, abscesses from which a single species was isolated were all apparently caused by facultative streptococci. No single species, group, or combination could be identified as essential to the production of oral abscesses. It is evident from our data that anaerobic techniques arc essential to the study of pyogenic oral infections and that protocols without such techniques are likely to give a distorted picture because of the inherent selectivity of aerobic incubation. Our data should be interpreted with some reserve since culturing remains a technically imperfect art. The organism responsible for the infection may not invariably be present in cultivable condition in the pus. In addition, the presence in pus of a given bacterium does not, with certainty, indicate that it plays the role of pathogen. Further study into the role played in a mixed infection by a given isolant is needed. The capability for identification of organisms in the original specimen by fluorescent antibody techniques needs to be developed and the clinical course of infections should be carefully correlated with the species or combinations isolated. The initiation of antibiotic therapy in oral infections, where indicated, remains essentially empirical because of the time required for sensitivity testing of some slow-growing anaerobes. A knowledge of the bacteria usually encountered in these infections should be of assistance in managing these infections. REFERENCES
Flora of Submucous Abscesses Originating 1. Feldmann, G., and Large, 0.: The Bacterial From Chronic Exacerbating Osteitis, Acta Odontol. &and. 24: 129-145, 1966. 2. Fulghum, R. S.: Mobile Anaerobe Laboratory, Appl. Microbial. 21: 769-770, 1971. Manual, Blacksburg, Va., 3. Holdeman, L. V., and Moore, W. E. C.: Anaerobe Laboratory 1972, Virginia Polytechnic Institute Anaerobe Laboratory. 4. Sabiston, C. B., Jr., and Gold, W. A.: Anaerobic Bacteria in Oral Infections, ORAL SURG. 38: X37-192, 1974. 5. Turner, J. E., Moore, D. W., and Shaw, B. S.: Prevalence and Antibiotic Susceptibility
Volume 41 Number 4
Pyogenic
infections
of dental
origin
435
of Organisms Isolated From Acute Soft-tissue Abscesses Secondary to Dental Caries, ORAL SURG. 39: 848-857, 1975.
6. Wittgow, W. C. and Sabiston, C. B., Jr.: Microorganisms From Pulpal Chambers of Intact Teeth With Necrotic Pulps, J. Endodontics 1: 168-171, 1975. Reprint requests to: Dr. C. B. Sabiston, Jr. Department of Periodontology College of Dentistry University of Iowa Iowa City, Iowa 52242
OF IOWA,
COLLEJGE
OF DENTISTRY
Fluctuant abscesses of dental origin were sampled by syringe aspiration. These samples were cultured by means of techniques suitable for isolation and growth of obligate anaerobes. Oral abscesses are usually mixed infections that include a number of different species. More than 65 per cent of the species isolated were obligate anaerobes, which indicates the need for anaerobic methods in studying these infections. Anaerobic gram-negative rods and facultative streptococci were the groups isolated most frequently.
0
ur study of anaerobic bacteria in oral infections, reported in 1974,* described bacteria isolated from eight fluctuant abscessesof dental origin. We have now studied exudates from a total of sixty-five dental abscesses.The cases selected for this report were limited to abscessesof dental origin which had extended beyond the alveolus to cause obvious erythematous fluctuant swelling. Differentiation between abscessesof periodontal origin and those of pulpal origin was not made and, indeed, frequently could not be made since both pulpal and periodontal lesions were associated with the same tooth. There are inherent difficulties in obtaining and processing exudates from oral abscesses.Sampling in an area with a substantial normal flora is likely to result in an inoculum which includes that normal flora. Any delay in plating the specimen directly onto a suitable medium or in incubating in an anaerobic environment reduces anaerobic recovery and distorts the determination of relative numbers of species of bacteria present in the abscess.Our methods of This study was supported by Grant D.E. 03662 from the United States Public Health Service, National Institute of Dental Research. *Associate Professor of Periodontology and Director, Collegiate Microbiology Research Laboratories. **Associate Professor of Periodontology. ***Research Assistant.
430
Volume 41 Number 4
Pyogenic
i~~fecti4m of dental origin
431
study have been designed to minimize certain inherent difficulties in isolating, propagating, and identifying bacteria. IJnique aspects include sampling by syringe aspiration of pus from the abscess in an attempt to minimize contamination of the specimen with normal flora; immediate culturing of all specimens in order to minimize loss of oxygen-sensit,ive anaerobes and other distortions of relative proportions of species contained in the specimen; use of a modified Hungate roll tube technique in addition to the anaerobe jar technique; use of multiple media and conditions of incubation. The literature provides little information on the species of bacteria found in abscesses of dental origin. Feldmann and Large1 reported a series in which a cautery was used to minimize contamination of the specimen. A recent study by Turner and associates5 relied on techniques which might minimize recovery of obligate anaerobes. METHODS
The first eight specimens were taken from patients being treated at Medical College of Virginia Hospital, Outpatient Clinic, Richmond, Virginia. The remainder were taken from patients in the clinics of the College of Dentistry, University of Iowa. Abscesses were isolated and dried with sterile gauze, and the exudate was obtained by aspiration with the use of a sterile disposable syringe with a No. 16 gauge needle. The exudate was expressed directly into a tube under oxygen-free gas, within 5 minutes for specimens collected at the University of Iowa and within 30 minutes for the eight specimens collected in Richmond. Some abscesses were sampled by sterile paper point after the tooth had been opened for endodontic therapy. In these cases, at chairside, the paper point was dropped into a gassed-out tube which had been opened under oxygenfree CO,. This sampling procedure used a mobile anaerobe cart similar to that described by Fulghum.2 Our techniques of culture and identification are those developed by the staff of the Anaerobe Laboratory at Virginia Polytechnic Institute and State University. Methods are described in detail in the manual published by the Anaerobe Laboratory .3 Each specimen was examined grossly, a wet preparation was made for phase-contrast microscopy, a Gram preparation was made, and the following media were inoculated: a brain-heart infusion (prereduced) agar roll tube (supplemented with heme, vitamin K, and yeast extract) ; a blood agar plate freshly poured from prereduced medium and reduced in an anaerobe jar; and a stock blood agar plate incubated in a CO, incubator. All media were incubated at 37’ C. Representative colonies were picked in 3 to 5 days. The blood media were further incubated for 10 days in order to detect Bactewides melaniwogenicus. Methods of identification were those of Holdeman and Moore3 and relied on morphology, Gram reaction, a battery of biochemical tests, and gas chromatographic analysis of fatty acids and alcohols produced in culture medium. RESULTS
Sixty-five specimens were sampled. Seven of these specimens were culturally negative. From the remaining fifty-eight specimens, we isolated from one to
Grigsby,
Sabiston,
Table
I. Frequency of occurrence of groups in specimens
Group
Anaerobic gram-positive rods Anaerobic gram-negative rods Anaerobic gram-positive cocci Anaerobic gram-negative cocci Facultative gram-positive rods Facultative gram-negative rods Facultative gram-positive cocci Facultative gram-negative cocci
and
Oral April,
432
LSegerstrom
Number of specimens containing the group
4’: 28 10 10 4: 5
Surg. 1976
Percentage of positive specimens containing the group
41.4 69.0 48.3 17.2 17.2 5.2 12.4 8.6
twelve species. The average number of distinct species in each culturally positive specimen was 3.8. Obligate anaerobes numbered 147, or 65.9 per cent of the total species isolated. Spirochetes were observed in phase preparations of seven specimens, all of which were culturally positive for other species as well. A single organism in pure culture was isolated from seven specimens, one of which was an obligate anaerobe (six facultative streptococci; one Veillonelln alcalescens). Table I lists the number of specimens which contained a given group, and the percentage of all positive specimens (fifty-eight) which that number represents. Table II lists the genus and species designation of strains isolated and the number of specimens in which that species, genus, or group occurred. The frequency of occurrence of each is also given and is expressed as a percentage of the total number of specimens studied (sixty-five). It should be noted that species of the genera Lactobacillus and Actinomyces appear under two categories since both facultative and anaerobic species commonly occur in both genera. No attempt was made to identify facultative isolants at the species level; accordingly, all facultative streptococcal strains were considered as a single species in each of the specimens in which they were present. Organisms are listed as unidentified if they were lost before identification, or if their characteristics did not closely fit those of recognized species. The species designated as Bacteroides ruminicola S.S. brevis has the characteristics described in the Virginia Polytechnic Institute Avlaerobe Laboratory Mawa1,3 but since it has not been directly and extensively compared with reference strains, it may not be identical to the rumen organism. These data represent the presence of a given organism without indicating its quantitative importance. Precise quantitation is not feasible using clinical methods, although such data could be important. We made estimations of relative proportions of morphotypes present when the primary plating media were picked, but we do not think that these estimations are sufficiently precise to report in detail. In general, the numbers of colonies observed on media incubated anaerobically were several orders of magnitude higher than those on plates incubated aerobically or under CO,. This observation is particularly significant
Volume 41 Number 4
Pyogenic
Table II. Identity
infections of
dental
origin
433
of strains isolated
Species, genus or group
Anaerobic gram-positive rods unidentified
Number of specimens from which tiolated
1
Eubacterium lentum Eubacterium alactoiyticum &bacterium species Lactobacillus species Actinomyces species Bifidobacterium species
1.1 ::: 1%
17:2 10.3 25.9 5.2 13.8 13.8 3.4
Anaerobic gram-negative rods unidentified Bacteroides oralis B. ruminicola S.S.brevis B. melaninogenicus S.S.intermedius B. melaninogenicus S.S.melaninogenicus Bacteroides species Fusobacterium nucleatum Fusobacterium species
2:.: 1:7 13.8
Anaerobic gram-positive cocci unidentified Peptococcus magnus Streptococcus morbillorum Peptostreptococcus anaerobius Peptostreptococcus micros Streptococcus constellatus Streptocuccus intermedius
ii 15:5 22.4 ii:;1
Anaerobic gram-negative cocci Veillonella parvula
10.3 6.9 6.9 10.3 5.2 5.2
Veillonella alcalescens
Facultative gram-positive rods unidentified Lactobacillus species Actinomyces species Facultative gram-negative rods unidentified Facultative gram-positive cocci streptococci-catalase neg., unspeciated
10.7
1.7 13.8
Streptococcuspyogenes Staphylococcus epidermidis Staphylococcus aureus
Facultative gram-negative cocci unidentified Other unidentified bacterium Yeast (not Capdida albicans) . _ Spirochetes (phase-contrast microscopy)
Percentage ofpositive cultures from which isolated
it 1:7 /
1:::
in the case of facultative streptococci isolated only from plates incubated aerobically with or without some COz in twelve of the forty-one cases in which they were isolated. In six additional instances they constituted less than 20 per cent of colonies present on media incuba,ted anaerobically. Five additional specimens from which they were isolated yielded very few total colonies on any media, so that the total number of specimens from which facultative streptococci were isolated in numbers above 20 per cent of the bacterial population was eighteen. The data tables, indicating a high frequency of occurrence of facultative gram-positive cocci, most of which were facultative streptococci, should be interpreted with these observations in mind. CONCLUSIONS Pyogenic oral infections contain a complex mixture of species of bacteria. Facultative streptococci occurred with greater frequency than other groups ;
434
Sabiston, Grigsby,
and Negerstrom
Oral Surg. -April, 1976
however, they were detected from some specimens only on plates incubated under aerobic conditions, which would tend to select for them. This ma; intjicate a diminutive numerical significance in some abscesses. Anaerobic gram-positive and gram-negative rods and gram-positive cocci, along with facultative streptococci, dominated the bacteria present in our series of abscesses. Facultative gramnegative rods were rare, and especially so considering their frequency in pyogenic infections elsewhere in the body. This observation may indicate a local source of the bacteria causing oral pyogenic infections. Facultative and anaerobic gram-negative cocci and facultative gram-positive rods were intermediate in occurrence. The species identified are essentially those found in the mouth in the absence of infection.G These observations support the proposition that oral abscesses are caused by opportunistic pathogens whose presence, under other circumstances, can be consistent with clinical health. The mixed anaerobic nature of the infections is of interest. The data may indicate that most of the species isolated a,re incapable of producing infections alone. With one exception, abscesses from which a single species was isolated were all apparently caused by facultative streptococci. No single species, group, or combination could be identified as essential to the production of oral abscesses. It is evident from our data that anaerobic techniques arc essential to the study of pyogenic oral infections and that protocols without such techniques are likely to give a distorted picture because of the inherent selectivity of aerobic incubation. Our data should be interpreted with some reserve since culturing remains a technically imperfect art. The organism responsible for the infection may not invariably be present in cultivable condition in the pus. In addition, the presence in pus of a given bacterium does not, with certainty, indicate that it plays the role of pathogen. Further study into the role played in a mixed infection by a given isolant is needed. The capability for identification of organisms in the original specimen by fluorescent antibody techniques needs to be developed and the clinical course of infections should be carefully correlated with the species or combinations isolated. The initiation of antibiotic therapy in oral infections, where indicated, remains essentially empirical because of the time required for sensitivity testing of some slow-growing anaerobes. A knowledge of the bacteria usually encountered in these infections should be of assistance in managing these infections. REFERENCES
Flora of Submucous Abscesses Originating 1. Feldmann, G., and Large, 0.: The Bacterial From Chronic Exacerbating Osteitis, Acta Odontol. &and. 24: 129-145, 1966. 2. Fulghum, R. S.: Mobile Anaerobe Laboratory, Appl. Microbial. 21: 769-770, 1971. Manual, Blacksburg, Va., 3. Holdeman, L. V., and Moore, W. E. C.: Anaerobe Laboratory 1972, Virginia Polytechnic Institute Anaerobe Laboratory. 4. Sabiston, C. B., Jr., and Gold, W. A.: Anaerobic Bacteria in Oral Infections, ORAL SURG. 38: X37-192, 1974. 5. Turner, J. E., Moore, D. W., and Shaw, B. S.: Prevalence and Antibiotic Susceptibility
Volume 41 Number 4
Pyogenic
infections
of dental
origin
435
of Organisms Isolated From Acute Soft-tissue Abscesses Secondary to Dental Caries, ORAL SURG. 39: 848-857, 1975.
6. Wittgow, W. C. and Sabiston, C. B., Jr.: Microorganisms From Pulpal Chambers of Intact Teeth With Necrotic Pulps, J. Endodontics 1: 168-171, 1975. Reprint requests to: Dr. C. B. Sabiston, Jr. Department of Periodontology College of Dentistry University of Iowa Iowa City, Iowa 52242
