Scand. J. Dent. Res. 1977: 85: 114-121 (Key words: bacteria; microbiology^ oral;
periodontitis)
The predominant cultivable microflora of advanced periodontitis JORGEN SLOTS Department of Periodontology and Department of Microbiology, Royal Dental College, Copenhagen, Denmark ABSTRACT — A Study on the predominant cultivable microorganisms harbored in the base of deep periodontal pockets of eight patients aged 34—48 years was carried out using the roll tube culture technique. From a total of 475 isolates, 425 (89.5 %) were obligate anaerobes, 356 (74.9 %) were Gram-negative, and 441 (92.8%) were categorized as rods. Baeteroides melaninogenicus and Fusobacterium nucleatuni constituted the majority of the isolates in seven samples, but their mutual proportions differed considerably between the samples. One sample was dominated by Actinomyces species. Some peraodontopathic potentials of the predominating Gram-negative rods are summarised. (Accepted for publication 23 May 1976)
Several studies on the microflora as.sociated with chronic destructive periodontal disease have been made (reviewed by SocRANSKY 1970, KELSTROP & THEILADE 1974, CoBB & CoBB 1975). In many of these investigations selective and nonselective bacterial plate media were used in combination with conventional anaerobic jars. However, recent findings on isolation of dental plaque bacteria have revealed that a conventional anaerobic jar technique allowed toi grow only about half of the total colony counts recovered with the Hungate roll tube technique (SLOTS 1975). Therefore, the possibility exists that the nse of a strictly anaerobic culture technique might increase our knowledge of the oral microflora. The present .study attempted to describe and quantitate the predominant cultivable microorganisms inhabiting the base of
deep periodontal pockets in middle-aged subjects. The Hungate roll tube method as modified by HOLDEMAN & MOORE (1973) was. used for this purpose. Material and methods SUBJECTS
Eight patients, aged 34 to 48 years, with advanced periodontitis were selected for the study. Each patient fulfilled the following criteria described by DwYER & SocRANSKY (1968): (a) they had 16' or more teeth, (b) at least 10 teeth had pockets of 5 mm or more, (c) there was radiographic evidence of bone loss, (d) there was no history of scaling or dental treatment within the last 6 months, and (e) they did not have a history of certain systematic conditions such as diabetes, blood dyscrasias, nutritional deficiencies, etc., which might have contributed to the progress of the disease. Patients who had an obvious history of juvenile periodontitis were excluded. Table 1 summarizes the clinical data of the subjects.
MICROFLORA OF PERIODONTITIS
115
Table 1 Summary of clinical data of eight individuals with advanced periodontitis
Suhject
Age in
Sex
years B.S. V.C. J.D. D.K. B.O. A.J. E.J. T.S.
37 48 38 35 42 38 37 34
Sample site
F M M F F F M F
Pocket depth in mm at sample site
+ 6^ 2+2
6+2 3+* 4+2 6-2 +42
SAMPLE COLLECTION
The iiidividual specimens consisted of materials from the base of one lnterproximal pocket, and they were collected by a method described by SLOTS (1976). The sample was transferred to a test tube containing" 3.0 nil "Reduced Transport Fluid" (SYED & LoBSCHE 1972), The sampling and the subsequent micro bio logic aJ procedures took place under anaerobic conditions by flushing with a mixture of 85% N^, 10% Hg, and 5 % CO's-
SAMPLE DISPERSION AND INOCULATION
PROCEDUiRES
The samples were dispersed by sonication using a 100 W MSE® ultrasonic disintegrator equipped with a microprobe of a 3-mm end diameter. The sonication was performed for 20 sec with an amplitude of 8 ^.m. After sonication the samples were processed through serial 10-fold dilutions in tubes of the "Reduced Transport Fluid". Fractions of O.,l ml of a dilution (up to 10"^^) were added with Carlsberg pipettes to prereduced and melted agar tubes of the various media maintained at 45—50° G. Immediately after inoculation, roil tubes were made by rapid rotation and simultaneously cooling with cold water. In addition, solidified MM 10 roll tubes were inoculated for surface growth. Duplicate tubes were prepared from each dilution and medium. The roll tubes were inoculated at 37" C for 14 d. Plates with Sabouraud Maltose agar were streaked with aliquots on the surface and inoculated aerobically at 37° G for 4 d.
8 5 6
a
10 12 9 8
Total no. of teeth
No. of teeth with pociiets > 5' mm
No. of teeth with pockets > 7 mm
24 20 30 22 24 26 24 27
24 20 30 22 22 17 24 27
6 10 16 22 8 16 11 17,
GROWTH MEDIA
The following media were inoculated: 1) MM 10 (LoESCHE &. SYED 1973') for non-selective bacterial growth; 2) Mitis Salivarius agar (Difco); 3) Rogosa Selective Lactobacilius agar (Difco); 4) Rogosa Veillonella agar (Difco); 5) FM agar (Difco) for isolation of Fusobacterium; 6) Sabouraud Maltose agar (Difco) for selective growth of yeast. Heart Infusion agar medium supplemented with 10% blood (GORDOX, STUTMAN & LOE-
SCHE J971) prepared in roll tubes were inoculated with aliquots from four patients to compare the efficiency of this medium and of MM 10 with respect to recovery of Baeteroides melaninogenicus. The number of black pigmented colonies^ characteristic of Baeteroides melaninogenicus, was in each sample on MM 10 equal to or exceeded that on Heart Infusion - \0% blood agar medium. The total colony counts of all samples were higher on MM 10 in accordance with previous findings (SLOTS 1975). Preparation of all media took place according to the original descriptions and to the roll tube technique (except for Sabouraud Maltose agar) as described in. V.P.L Anaerobe laboratory manual (HoiDEMAN & MOORE 1973), COLONY COUNTS Colony counts of the 14-d incubated roll tubes with deep agar colonies were obtained by use of the dissecting microscope. The ratio of colony counts from selective media to the nonselective MM 10 was calculated for each sample and medium examined.
116
SLOTS Table 2 Distribution of pocket-organisms from eight patients with advanced periodontitis*
Subject B.S. V.G. J.D. D.K. B.O. A.J. E.J. T.S. Mean Median Range
B. nielaninogenicusx 100/ MM 10** 41.5 3.0
61,1 27.4 5.6 0
56.0 58.6 31.7 34.5 0-61.1
M.S. X 100/ MM 10** 11.8 62.1 33.1 2.9
40.4 33.1 4.0 3.4
23.9 22.5 2.9-62.1
S.L. X 100/ MM 10** 0.5 9.3 1.3 1.6 0.4 1.8 5.0 0 2.5 1.5
0-9.3
Veill. X 100/ MM 10** 6.1
FM X 100/ MM 10»*
6.5 8.9
1.3 2.6 1.1 1.6
15.2
14.5
7.1 4.5 0.4
0 4.0 0 3.1 1.5
68.8
14.7 6.8
0.4-68.8
0-14.5
*In none of the samples was yeast growth detected on Sabouraud Maltose agar. **Total colony counts on selective growtb media (Mitis Salivarius agar (M.S.)^ Selective Lactobacillus agar (S.L.), VeiUonella agar (Veill.), Fusobacterium agar (FM)) are expressed as percentage of the number of colonies grown on MM 10. Black pigmented Bacteroides melaninogenicus colonies are counted on MM 10 and expressed in percent of the total colony counts on MM 10.
ISOLATION AND PARTIAL CHARACTERIZATION OF MICROORGANISMS
Subcultivation took place after 7 d of incubation from one MM 10 roll tube containing wellseparated colonies. The colonies were picked regardless of appearance. Colonies first visible after 7—14 d of incubation were included from a corresponding MM 10 roll tube. The colonies present in this tube were marked by ink on the 7th day of incubation. After 14 d of incubation the non-tnarked colonies were subcultured. Every colony was streaked onto MM 10 roll tube and whenever possible, a portion of the colony was also pidced for Gram-staining. Efforts were made to minimize loss of those kinds of bacteria difficult to subcultivate. The coionies were picked a third time, if possible, and transferred to supplemented Brain Heart Infusion broth (HOLDEMAN & MOORE 1973). In
addition, representative colonies were picked and transferred directly to the supplemented Brain Heart Infusion broth and/or to other prereduced, anaerobicaliy sterilized broths. Each pure isolate was classified according to nnorphology. Gram reaction, aerobic growth, fermentation, and biochemical test following the outlines in V.P.I. Anaerobe 'laboratory manual
& MOORE 1973), and in Bergey's manual of determinative bacteriology (BUGHAKAK & GIBBONS 1974). Analysis of fernaentation products in glucose was made by use of a gas chromatograph tnounted with flame-ionization detector (Perkin-Elmer®, model 900). Column packing was 5 % FFAP on chroctiosorb-G treated with DMSG as recommended by HOLDEMAN & MooEE (1973). Results The data in Table 2 show the effect of different media upon the culture counts obtained on eight pocket samples from patients with advanced periodontitis. A notable intersample variation was found in the ratios of the total colony counts from the selective media to MM 10. The proportion of Bacteroides melaninogenicus also varied considerably between the samples, constituting from 0 to 61.1 % of the total colony counts on MM 10 (Table 2). The predominant cultivable pocket flora associated with advanced periodontitis are
MICROFLORA OF PERIODONTITIS
117
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listed in Table 3. From each individual, 42-98 bacterial strains were subcultured from MM 10 roll tubes. A total of 475 bacterial strains were examined from the eight samples: 425 isolates (89.5%) were obligate anaerobes, 356 (74.9%) were Gram-negative, and 441 (92.8%) were categorized as rods. Gram-negative anaerobic rods were the most predominant organisms in seven of the eight samples and averaged 74.3 % of the cultivable microflora (Table 3). Many of the Gram-negative rods were difficult to maintain and were lost on subculture before full classification. Bacteroides melaninogenicus was found to be the predominant Gram-negative rod in samples. B.S., J.D., E.J., and T.S. (Table 2). The majority of the Bacteroides melaninogenicus strains were proteolytic, non-fermentative, and produced acetic, propionic, isobutyric, butyric, isovaleric, lactic, and succinic acids in glucose broth cultures, indicating that they might be strains of Bacteroides melaninogenicus ss. asaccharolyticus. A group of Bacteroides melaninogenicus strains were proteofermentative to non-fermentative and lacked propionic and butyric acids among their metabolic end products, characteristics typical of Bacteroides melaninogenicus ss. intermedius. Fusiform organisms whose major fermentation product was butyric acid with no or negligible amounts of isobutyric and isovaleric acids were isolated from all the sam.ples and were predominant in samples B.O. and A.J. The majority of the strains exhibited straight or slightly curved cellular forms with tapered or rounded ends. Strains dominated by long, definitely curved cells were also regularly isolated. A few strains contained distinctly pleomorphic cells, Most of these isolates fit the morphologic and biochemical descriptions of Fusobacterium nucleatum.
Some kinds of Gram-negative, anaerobic, non-sporulating rods that were difficult to speciate on the basis of the data available were encountered in the predominant flora. Among these isolates some had "bacteroides-like" surface colonies:
periodontitis)
The predominant cultivable microflora of advanced periodontitis JORGEN SLOTS Department of Periodontology and Department of Microbiology, Royal Dental College, Copenhagen, Denmark ABSTRACT — A Study on the predominant cultivable microorganisms harbored in the base of deep periodontal pockets of eight patients aged 34—48 years was carried out using the roll tube culture technique. From a total of 475 isolates, 425 (89.5 %) were obligate anaerobes, 356 (74.9 %) were Gram-negative, and 441 (92.8%) were categorized as rods. Baeteroides melaninogenicus and Fusobacterium nucleatuni constituted the majority of the isolates in seven samples, but their mutual proportions differed considerably between the samples. One sample was dominated by Actinomyces species. Some peraodontopathic potentials of the predominating Gram-negative rods are summarised. (Accepted for publication 23 May 1976)
Several studies on the microflora as.sociated with chronic destructive periodontal disease have been made (reviewed by SocRANSKY 1970, KELSTROP & THEILADE 1974, CoBB & CoBB 1975). In many of these investigations selective and nonselective bacterial plate media were used in combination with conventional anaerobic jars. However, recent findings on isolation of dental plaque bacteria have revealed that a conventional anaerobic jar technique allowed toi grow only about half of the total colony counts recovered with the Hungate roll tube technique (SLOTS 1975). Therefore, the possibility exists that the nse of a strictly anaerobic culture technique might increase our knowledge of the oral microflora. The present .study attempted to describe and quantitate the predominant cultivable microorganisms inhabiting the base of
deep periodontal pockets in middle-aged subjects. The Hungate roll tube method as modified by HOLDEMAN & MOORE (1973) was. used for this purpose. Material and methods SUBJECTS
Eight patients, aged 34 to 48 years, with advanced periodontitis were selected for the study. Each patient fulfilled the following criteria described by DwYER & SocRANSKY (1968): (a) they had 16' or more teeth, (b) at least 10 teeth had pockets of 5 mm or more, (c) there was radiographic evidence of bone loss, (d) there was no history of scaling or dental treatment within the last 6 months, and (e) they did not have a history of certain systematic conditions such as diabetes, blood dyscrasias, nutritional deficiencies, etc., which might have contributed to the progress of the disease. Patients who had an obvious history of juvenile periodontitis were excluded. Table 1 summarizes the clinical data of the subjects.
MICROFLORA OF PERIODONTITIS
115
Table 1 Summary of clinical data of eight individuals with advanced periodontitis
Suhject
Age in
Sex
years B.S. V.C. J.D. D.K. B.O. A.J. E.J. T.S.
37 48 38 35 42 38 37 34
Sample site
F M M F F F M F
Pocket depth in mm at sample site
+ 6^ 2+2
6+2 3+* 4+2 6-2 +42
SAMPLE COLLECTION
The iiidividual specimens consisted of materials from the base of one lnterproximal pocket, and they were collected by a method described by SLOTS (1976). The sample was transferred to a test tube containing" 3.0 nil "Reduced Transport Fluid" (SYED & LoBSCHE 1972), The sampling and the subsequent micro bio logic aJ procedures took place under anaerobic conditions by flushing with a mixture of 85% N^, 10% Hg, and 5 % CO's-
SAMPLE DISPERSION AND INOCULATION
PROCEDUiRES
The samples were dispersed by sonication using a 100 W MSE® ultrasonic disintegrator equipped with a microprobe of a 3-mm end diameter. The sonication was performed for 20 sec with an amplitude of 8 ^.m. After sonication the samples were processed through serial 10-fold dilutions in tubes of the "Reduced Transport Fluid". Fractions of O.,l ml of a dilution (up to 10"^^) were added with Carlsberg pipettes to prereduced and melted agar tubes of the various media maintained at 45—50° G. Immediately after inoculation, roil tubes were made by rapid rotation and simultaneously cooling with cold water. In addition, solidified MM 10 roll tubes were inoculated for surface growth. Duplicate tubes were prepared from each dilution and medium. The roll tubes were inoculated at 37" C for 14 d. Plates with Sabouraud Maltose agar were streaked with aliquots on the surface and inoculated aerobically at 37° G for 4 d.
8 5 6
a
10 12 9 8
Total no. of teeth
No. of teeth with pociiets > 5' mm
No. of teeth with pockets > 7 mm
24 20 30 22 24 26 24 27
24 20 30 22 22 17 24 27
6 10 16 22 8 16 11 17,
GROWTH MEDIA
The following media were inoculated: 1) MM 10 (LoESCHE &. SYED 1973') for non-selective bacterial growth; 2) Mitis Salivarius agar (Difco); 3) Rogosa Selective Lactobacilius agar (Difco); 4) Rogosa Veillonella agar (Difco); 5) FM agar (Difco) for isolation of Fusobacterium; 6) Sabouraud Maltose agar (Difco) for selective growth of yeast. Heart Infusion agar medium supplemented with 10% blood (GORDOX, STUTMAN & LOE-
SCHE J971) prepared in roll tubes were inoculated with aliquots from four patients to compare the efficiency of this medium and of MM 10 with respect to recovery of Baeteroides melaninogenicus. The number of black pigmented colonies^ characteristic of Baeteroides melaninogenicus, was in each sample on MM 10 equal to or exceeded that on Heart Infusion - \0% blood agar medium. The total colony counts of all samples were higher on MM 10 in accordance with previous findings (SLOTS 1975). Preparation of all media took place according to the original descriptions and to the roll tube technique (except for Sabouraud Maltose agar) as described in. V.P.L Anaerobe laboratory manual (HoiDEMAN & MOORE 1973), COLONY COUNTS Colony counts of the 14-d incubated roll tubes with deep agar colonies were obtained by use of the dissecting microscope. The ratio of colony counts from selective media to the nonselective MM 10 was calculated for each sample and medium examined.
116
SLOTS Table 2 Distribution of pocket-organisms from eight patients with advanced periodontitis*
Subject B.S. V.G. J.D. D.K. B.O. A.J. E.J. T.S. Mean Median Range
B. nielaninogenicusx 100/ MM 10** 41.5 3.0
61,1 27.4 5.6 0
56.0 58.6 31.7 34.5 0-61.1
M.S. X 100/ MM 10** 11.8 62.1 33.1 2.9
40.4 33.1 4.0 3.4
23.9 22.5 2.9-62.1
S.L. X 100/ MM 10** 0.5 9.3 1.3 1.6 0.4 1.8 5.0 0 2.5 1.5
0-9.3
Veill. X 100/ MM 10** 6.1
FM X 100/ MM 10»*
6.5 8.9
1.3 2.6 1.1 1.6
15.2
14.5
7.1 4.5 0.4
0 4.0 0 3.1 1.5
68.8
14.7 6.8
0.4-68.8
0-14.5
*In none of the samples was yeast growth detected on Sabouraud Maltose agar. **Total colony counts on selective growtb media (Mitis Salivarius agar (M.S.)^ Selective Lactobacillus agar (S.L.), VeiUonella agar (Veill.), Fusobacterium agar (FM)) are expressed as percentage of the number of colonies grown on MM 10. Black pigmented Bacteroides melaninogenicus colonies are counted on MM 10 and expressed in percent of the total colony counts on MM 10.
ISOLATION AND PARTIAL CHARACTERIZATION OF MICROORGANISMS
Subcultivation took place after 7 d of incubation from one MM 10 roll tube containing wellseparated colonies. The colonies were picked regardless of appearance. Colonies first visible after 7—14 d of incubation were included from a corresponding MM 10 roll tube. The colonies present in this tube were marked by ink on the 7th day of incubation. After 14 d of incubation the non-tnarked colonies were subcultured. Every colony was streaked onto MM 10 roll tube and whenever possible, a portion of the colony was also pidced for Gram-staining. Efforts were made to minimize loss of those kinds of bacteria difficult to subcultivate. The coionies were picked a third time, if possible, and transferred to supplemented Brain Heart Infusion broth (HOLDEMAN & MOORE 1973). In
addition, representative colonies were picked and transferred directly to the supplemented Brain Heart Infusion broth and/or to other prereduced, anaerobicaliy sterilized broths. Each pure isolate was classified according to nnorphology. Gram reaction, aerobic growth, fermentation, and biochemical test following the outlines in V.P.I. Anaerobe 'laboratory manual
& MOORE 1973), and in Bergey's manual of determinative bacteriology (BUGHAKAK & GIBBONS 1974). Analysis of fernaentation products in glucose was made by use of a gas chromatograph tnounted with flame-ionization detector (Perkin-Elmer®, model 900). Column packing was 5 % FFAP on chroctiosorb-G treated with DMSG as recommended by HOLDEMAN & MooEE (1973). Results The data in Table 2 show the effect of different media upon the culture counts obtained on eight pocket samples from patients with advanced periodontitis. A notable intersample variation was found in the ratios of the total colony counts from the selective media to MM 10. The proportion of Bacteroides melaninogenicus also varied considerably between the samples, constituting from 0 to 61.1 % of the total colony counts on MM 10 (Table 2). The predominant cultivable pocket flora associated with advanced periodontitis are
MICROFLORA OF PERIODONTITIS
117
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listed in Table 3. From each individual, 42-98 bacterial strains were subcultured from MM 10 roll tubes. A total of 475 bacterial strains were examined from the eight samples: 425 isolates (89.5%) were obligate anaerobes, 356 (74.9%) were Gram-negative, and 441 (92.8%) were categorized as rods. Gram-negative anaerobic rods were the most predominant organisms in seven of the eight samples and averaged 74.3 % of the cultivable microflora (Table 3). Many of the Gram-negative rods were difficult to maintain and were lost on subculture before full classification. Bacteroides melaninogenicus was found to be the predominant Gram-negative rod in samples. B.S., J.D., E.J., and T.S. (Table 2). The majority of the Bacteroides melaninogenicus strains were proteolytic, non-fermentative, and produced acetic, propionic, isobutyric, butyric, isovaleric, lactic, and succinic acids in glucose broth cultures, indicating that they might be strains of Bacteroides melaninogenicus ss. asaccharolyticus. A group of Bacteroides melaninogenicus strains were proteofermentative to non-fermentative and lacked propionic and butyric acids among their metabolic end products, characteristics typical of Bacteroides melaninogenicus ss. intermedius. Fusiform organisms whose major fermentation product was butyric acid with no or negligible amounts of isobutyric and isovaleric acids were isolated from all the sam.ples and were predominant in samples B.O. and A.J. The majority of the strains exhibited straight or slightly curved cellular forms with tapered or rounded ends. Strains dominated by long, definitely curved cells were also regularly isolated. A few strains contained distinctly pleomorphic cells, Most of these isolates fit the morphologic and biochemical descriptions of Fusobacterium nucleatum.
Some kinds of Gram-negative, anaerobic, non-sporulating rods that were difficult to speciate on the basis of the data available were encountered in the predominant flora. Among these isolates some had "bacteroides-like" surface colonies:
