Oral Microbiol Immunol 1991: 6; 177-181
Antineoplastic agents inhibit the growth of Streptococcus mutans and Streptococcus sanguis in vitro
Jukka H. Meurman\ Heini Torkko\ Seppo Pyrhonen^ 'Department of Cariology, University of Helsinki, ^Department of Radiotfierapy and Oncology, Llniversity Central Hospital, Helsinki, Finland
Meurman JH, Torkko H, Pyrhonen S. Antitieoplastie agents inhibit the growth of Streptococcus mutans attd Streptococcus sanguis in vitro. Oral Mierobiol Itnmunol 1991: 6: 177-181. . • The effect of methotrexate (MTX) and doxorubicin on the growth, metabolism and ultrastructure of Streptococcus mutatis and Streptococcus sanguis was studied '« vitro. Both anticancer drugs exerted an inhibitory effect on the oral streptococci. I^TX was more inhibitory than doxorubicin. The minimum inhibitory concentrations (MICs) of MTX to S. mutans were 0.25-2.5 /ig/ml and that of doxorubicin 0-2 mg/ml. The MICs of MTX and doxorubicin to 5. sanguis were 0.025 pgjmX Key words: cytostatic drug; cancer cfiemoand 2.0-0.02 mg/ml, respecdvely. When saliva samples of patients with rnalignant tfierapy; oral complication; oral microtumors receiving various doses of MTX were analyzed, MTX was found to be biology; Streptococcus mutans; Streptococcus sanguis secreted into the oral cavity at concentrations ranging from 0.014 to 4.486 pgl ml. The saliva of these patients was also found to inhibit the growth of S. mutans, Dr. J. H. Meurman, tjniversity of Helsinki, Department of Cariology, Mannerheimintie 172, and the inhibidon zones were in accordance with the MIC values observed. The SF-00300 Helsinki, Finland results suggest that anticancer therapy must be taken into account when the Accepted for publication September 10, 1990 salivary microbiological findings of cancer patients are interpreted.
inimunosuppression caused by cytostadc drugs in cancer chemotherapy niay alter the host response, which results in increased liability to infection. Approximately 40% of patients who receive cytostatic drugs develop oral comPlicadons (13). The oral cavity itself is a potendal focus for general infections °f myelosuppressed patients (1). '^urther, because modern treatment of "malignant diseases has a curative intent, Padents' preventive dental care in the 'ong term needs to be considered. The established antibacterial activity °i cytostatic drugs has been suggested to modify the pattern of infections in cancer patients with a selective supPression of normal bacterial flora (7). Methotrexate (MTX), a much used and effective chemotherapeutic agent in the treatment of leukemias, lymphomas and '^any other solid tumors, inhibits intracellular dihydrofolate reductase and henee DNA synthesis. It also binds to the cytoplasmic membrane of both eukaryotic and microbial cells (4). Doxorub^^'n, an anthracycline antibiotic which ^as originally purified from a Strepto"^yces species (6), tnay also interact with •Membrane funcdons (5). Our clinical experience with lymphpatients has shown a marked and
consistent decrease in salivary acidogenic bacteria, the mutans streptococci and lactobacilli, immediately after the onset of andcancer therapy (11). The levels of these bacteria also remained low throughout the anticancer treatment of 8 weeks. Our patients received a combinadon of cytostatic drugs in which the cornerstone of therapy was based on MTX and doxorubicin, or its parent compound 4-epidoxorubicin. Nevertheless, the study by Bodet et al. (2) has shown that these drugs should have only a minimal activity on Streptococcus and Lactobacillus species. This study investigated the in vitro effect of MTX and doxorubicin on the growth, metabolism, and ultrastructure oi Streptococcus mutans and Streptococcus sanguis. These 2 bacterial species are associated with the initiadon of dental caries (3). The mutans streptococci, in particular, should be monitored during caries therapy of all patients who belong to the so-called risk groups (10, 14), as do patients with a malignant disease. Thus, it was considered important to know whether the anticancer agents should be taken into account when interpreting the clinical oral microbiological findings of patients with a malignant disease.
Material and methods Bacterial strains and cuiture methods
Stock cultures of 5. tnutans ATCC 27351 and S. sanguis ATCC 10556 were grown in thioglycollate broth (NIH thioglycollate with Bacto dextrose (Difco Laboratories, Detroit, MI). In addition, 4 fresh chnical isolates of each of the 2 bacterial species were studied. The strains of S. tnutatis (128B, 013B, 132A and 121 A) had been isolated from patients suffering from hematological tnalignancies, the strains of S. sanguis (4063 and 1272 of serotype I, 2827 and 1089 of serotype II) from patients suffering from bacterial endocarditis. After incubadon for 18 h at 37°C, the cells were harvested by centrifugation (9000 g, 15 min), washed 3 dmes with 0.1 M phosphate-buffered saline (PBS) and suspended in 0.1 M PBS to a turbidity of 175 in a Klett-Summerson colorimeter (filter 62, range 590-660 nm). This corresponded to 3.4-6.1 x 10** cells/ ml for S. tnutans and 6.0 x lO^'-l.O x 10' cells/ml for S. sanguis. Antineoplastic agents
Doxorubicin (Adriamycin 2 mg/ml; Farmitalia, Milano, Italy) and metho-
178
Meurtnan et al.
trexate (Trexan 25 mg/ml; Laakefarmos, Turku, Finland) were used in the experiments. Doxorubicin was dissolved in sterile 0.9% NaCl as recommended by the manufacturer and used within 12 h. The cytotoxin dilutions were prepared in 0.1 M PBS.
diludon) were combined and the initial pH was measured. The test vials were placed in a 37°C water bath (no agitadon) and the pH was measured every 15 min for 2 h. The difference from initial pH to end pH after 2 h incubation was calculated. The experiment was carried out twice.
Assessment of viable counts after incubation with antineopiastic agents
Aliquots of 1 or 2 ml of antineoplasdc agents or their dilutions were added to bacterial suspensions (Klett 175), mixed thoroughly and incubated in a 37°C water bath for I h with gentle agitadon. The cells were washed 3 times (5000 g, 15 min) with 0.1 M PBS and suspended in the buffer in a volume equivalent to the aboye. Appropriate diludons were plated on blood agar (2 parallels; Brucella blood agar, BBL Microbiology Systems, Cockeysville, MD). After 48 h incubadon at 37°C, viable count was determined as colony-forming units (CFU)/ml. The experiment was carried out 3 dmes. Susceptibility testing
Bacterial suspensions (0.2 ml) were spread evenly on Mueller-Hinton blood agar plates (Orion Yhtyma Oy, Orion Diagnostica, Espoo, Finland). Nonimpregnated filter discs (d = 6 mm) were placed aseptically on the agar surface. The cytotoxins were serially diluted in sterile saline. Ten microliters of each cytotoxin dilution was absorbed onto the filter discs. The plates were incubated for 24 h at 37°C, after which the inhibition zones around the discs were measured. The minimal inhibitory concentradon (MIC) represented the lowest concentration of antimicrobial agent at which inhibition occurred. The experiment was carried out 6 dmes. The antibiotic effect of saliva collected from patients being treated with methotrexate was also tested using the above described method. Centrifuged, undiluted saliva was used and the inhibition zones were measured after 24 h incubadon. The collecdon of saliva is described below. Fermentation tests
Bacteria were grown and washed as described above, and suspended in 10% sucrose (in 0.9% NaCl) to a turbidity of Klett 175. Equal volumes of bacterial suspension and cytostatic drug (or its
Determination of methotrexate in patient saiiva
The secretion of antineoplastic agent into saliva was studied in 9 patients with lymphomas or other malignant tumors treated with intravenous infusions of methotrexate at the Department of Radiotherapy and Oncology, University Central Hospital of Helsinki. Three patients received fairly small daily doses of methotrexate (150-250 mg); 6 patients received high methotrexate doses (4.3-15.5 g). The administradon of the drug took at most 6 h. Paraffin-wax stimulated whole saliva was collected for 5 min. The saliva samples were taken 20 min after onset of therapy, 20 min after termination and 24 h after onset of therapy. The saliva was centrifuged for 15 min at 5000 g to remove solid particles. The concentration of methotrexate in the samples was assayed using afiuorescencepolarization immunoassay method (8). The level of detecdon was 0.009 pglm\. The antibiodc suscepdbility of the saliva samples was tested on blood agar plates, as described above.
Preparation of specimens for ultrastructure study
Equal volumes of bacterial suspensions and antineoplastic agents were mixed and incubated in a water bath for 1 h at 37°C with gentle agitation. Suspensions were centrifuged (15 min, 5000 g) and bacterial pellets were prefixed with 2.8% glutaraldehyde for 50 min, washed 3 dmes with 0.1 M PBS and postfixed with 1 % osmium tetroxide in the phosphate buffer for 2 h. The specimens were dehydrated in graded series of ethanol, embedded in Epon 812 and thin-sectioned in an LKB ultramicrotome (LKB, Stockholm, Sweden). The thin-sections were stained with uranyl acetate and lead citrate and studied in a Jeol 100 CX transmission electron microscope operadng at 60 kV (JEOL, Japan Electron Opdcs, Tokyo, Japan).
Table 1. Inhibition of growth of ATCC strains and clinical isolates of S. tnutans and S. .sanguis by methotrexate and doxorubicin after 24 h incubation. Restilts given as minimum inhibitory concentrations (MIC) Bacterial strain
Methotrexate Doxorubicin //g/ml mg/ml
S. mutans ATCC 27351 128B 013B I32A 121A
0.25 0.25 0.25 2.5 0.25
0.2 0.2 0.2 0.2 0.2
S. sanguis ATCC 10556 4063 1272 2827 1089
_* 0.025 0.025 0.025 0.025
0.2 0.2 0.02 2.0
* hazy inhibition zones at all concentrations down to 0.25
Statistical methods
Statistical analyses tested the effect of the 2 drugs and bacterial strains separately on the results, and the effect of time and MTX concentration on the patients' salivary findings. ANOVA statistics were used together with the Fisher test when applicable. Results
The MIC values for MTX and doxorubicin are given in Table 1. The ATCC strain of S. sanguis showed hazy growth at the inhibition zones in all test series with MTX, and this strain did not appear to be sensitive to doxorubicin. Thus, exact MIC values are not given to that strain. The clinical isolates, however, were disdnctly sensitive also to doxorubicin. The results of the inhibition of growth in both the S. mutans and S. sanguis bacteria are given in Table 2. As Table 2. Growth of S. mutans and S. satiguis after incubation with methotrexate and doxorubicin. Results given as CFU/ml Concentration of antineoplastic agent S. mutans S. .sanguis Methotrexate 12.5 mg/ml Methotrexate 2.5 mg/ml Doxorubicin I mg/ml Doxorubicin 0.2 tng/ml
6.1x10"
8.5x10*
1.5x10"
1.9x10*
2.9x10'
3.1x10*
1.2x10'
2.6x10'
1.8x10'
2.1x10'
Antineoplastic agents atid oral sireptoeoeei 179 Table 3. Effect of cytostatic drugs on the fermentation of sucrose by S. mutatis and S. satiguis. Fermentation is expressed as mean decrease in pH after 2 h incubation. 0.1 M PBS was used as control Anticancer drug (mg/ml)
^ g- I. Mueller-Hinton plates of S. mutatis showing: a) a distinct growth inhibition caused by xorubicin (arrow heads), and b) a not clearly detected effect by methotrexate, where only 'izy inhibidon zones could be seen in this case.
shown, the study drugs did not much atfeet the growth of 5. .satiguis, although statistically the observed differences l^ere significant compared with the conJ-ols in both the cytostatic series with tnis bacterium (P< 0.05). The growth of ''•mutans, however, showed a 10-fold fedution when incubated with doxorua highly significant difference n compared with the control ^^
Antineoplastic agents inhibit the growth of Streptococcus mutans and Streptococcus sanguis in vitro
Jukka H. Meurman\ Heini Torkko\ Seppo Pyrhonen^ 'Department of Cariology, University of Helsinki, ^Department of Radiotfierapy and Oncology, Llniversity Central Hospital, Helsinki, Finland
Meurman JH, Torkko H, Pyrhonen S. Antitieoplastie agents inhibit the growth of Streptococcus mutans attd Streptococcus sanguis in vitro. Oral Mierobiol Itnmunol 1991: 6: 177-181. . • The effect of methotrexate (MTX) and doxorubicin on the growth, metabolism and ultrastructure of Streptococcus mutatis and Streptococcus sanguis was studied '« vitro. Both anticancer drugs exerted an inhibitory effect on the oral streptococci. I^TX was more inhibitory than doxorubicin. The minimum inhibitory concentrations (MICs) of MTX to S. mutans were 0.25-2.5 /ig/ml and that of doxorubicin 0-2 mg/ml. The MICs of MTX and doxorubicin to 5. sanguis were 0.025 pgjmX Key words: cytostatic drug; cancer cfiemoand 2.0-0.02 mg/ml, respecdvely. When saliva samples of patients with rnalignant tfierapy; oral complication; oral microtumors receiving various doses of MTX were analyzed, MTX was found to be biology; Streptococcus mutans; Streptococcus sanguis secreted into the oral cavity at concentrations ranging from 0.014 to 4.486 pgl ml. The saliva of these patients was also found to inhibit the growth of S. mutans, Dr. J. H. Meurman, tjniversity of Helsinki, Department of Cariology, Mannerheimintie 172, and the inhibidon zones were in accordance with the MIC values observed. The SF-00300 Helsinki, Finland results suggest that anticancer therapy must be taken into account when the Accepted for publication September 10, 1990 salivary microbiological findings of cancer patients are interpreted.
inimunosuppression caused by cytostadc drugs in cancer chemotherapy niay alter the host response, which results in increased liability to infection. Approximately 40% of patients who receive cytostatic drugs develop oral comPlicadons (13). The oral cavity itself is a potendal focus for general infections °f myelosuppressed patients (1). '^urther, because modern treatment of "malignant diseases has a curative intent, Padents' preventive dental care in the 'ong term needs to be considered. The established antibacterial activity °i cytostatic drugs has been suggested to modify the pattern of infections in cancer patients with a selective supPression of normal bacterial flora (7). Methotrexate (MTX), a much used and effective chemotherapeutic agent in the treatment of leukemias, lymphomas and '^any other solid tumors, inhibits intracellular dihydrofolate reductase and henee DNA synthesis. It also binds to the cytoplasmic membrane of both eukaryotic and microbial cells (4). Doxorub^^'n, an anthracycline antibiotic which ^as originally purified from a Strepto"^yces species (6), tnay also interact with •Membrane funcdons (5). Our clinical experience with lymphpatients has shown a marked and
consistent decrease in salivary acidogenic bacteria, the mutans streptococci and lactobacilli, immediately after the onset of andcancer therapy (11). The levels of these bacteria also remained low throughout the anticancer treatment of 8 weeks. Our patients received a combinadon of cytostatic drugs in which the cornerstone of therapy was based on MTX and doxorubicin, or its parent compound 4-epidoxorubicin. Nevertheless, the study by Bodet et al. (2) has shown that these drugs should have only a minimal activity on Streptococcus and Lactobacillus species. This study investigated the in vitro effect of MTX and doxorubicin on the growth, metabolism, and ultrastructure oi Streptococcus mutans and Streptococcus sanguis. These 2 bacterial species are associated with the initiadon of dental caries (3). The mutans streptococci, in particular, should be monitored during caries therapy of all patients who belong to the so-called risk groups (10, 14), as do patients with a malignant disease. Thus, it was considered important to know whether the anticancer agents should be taken into account when interpreting the clinical oral microbiological findings of patients with a malignant disease.
Material and methods Bacterial strains and cuiture methods
Stock cultures of 5. tnutans ATCC 27351 and S. sanguis ATCC 10556 were grown in thioglycollate broth (NIH thioglycollate with Bacto dextrose (Difco Laboratories, Detroit, MI). In addition, 4 fresh chnical isolates of each of the 2 bacterial species were studied. The strains of S. tnutatis (128B, 013B, 132A and 121 A) had been isolated from patients suffering from hematological tnalignancies, the strains of S. sanguis (4063 and 1272 of serotype I, 2827 and 1089 of serotype II) from patients suffering from bacterial endocarditis. After incubadon for 18 h at 37°C, the cells were harvested by centrifugation (9000 g, 15 min), washed 3 dmes with 0.1 M phosphate-buffered saline (PBS) and suspended in 0.1 M PBS to a turbidity of 175 in a Klett-Summerson colorimeter (filter 62, range 590-660 nm). This corresponded to 3.4-6.1 x 10** cells/ ml for S. tnutans and 6.0 x lO^'-l.O x 10' cells/ml for S. sanguis. Antineoplastic agents
Doxorubicin (Adriamycin 2 mg/ml; Farmitalia, Milano, Italy) and metho-
178
Meurtnan et al.
trexate (Trexan 25 mg/ml; Laakefarmos, Turku, Finland) were used in the experiments. Doxorubicin was dissolved in sterile 0.9% NaCl as recommended by the manufacturer and used within 12 h. The cytotoxin dilutions were prepared in 0.1 M PBS.
diludon) were combined and the initial pH was measured. The test vials were placed in a 37°C water bath (no agitadon) and the pH was measured every 15 min for 2 h. The difference from initial pH to end pH after 2 h incubation was calculated. The experiment was carried out twice.
Assessment of viable counts after incubation with antineopiastic agents
Aliquots of 1 or 2 ml of antineoplasdc agents or their dilutions were added to bacterial suspensions (Klett 175), mixed thoroughly and incubated in a 37°C water bath for I h with gentle agitadon. The cells were washed 3 times (5000 g, 15 min) with 0.1 M PBS and suspended in the buffer in a volume equivalent to the aboye. Appropriate diludons were plated on blood agar (2 parallels; Brucella blood agar, BBL Microbiology Systems, Cockeysville, MD). After 48 h incubadon at 37°C, viable count was determined as colony-forming units (CFU)/ml. The experiment was carried out 3 dmes. Susceptibility testing
Bacterial suspensions (0.2 ml) were spread evenly on Mueller-Hinton blood agar plates (Orion Yhtyma Oy, Orion Diagnostica, Espoo, Finland). Nonimpregnated filter discs (d = 6 mm) were placed aseptically on the agar surface. The cytotoxins were serially diluted in sterile saline. Ten microliters of each cytotoxin dilution was absorbed onto the filter discs. The plates were incubated for 24 h at 37°C, after which the inhibition zones around the discs were measured. The minimal inhibitory concentradon (MIC) represented the lowest concentration of antimicrobial agent at which inhibition occurred. The experiment was carried out 6 dmes. The antibiotic effect of saliva collected from patients being treated with methotrexate was also tested using the above described method. Centrifuged, undiluted saliva was used and the inhibition zones were measured after 24 h incubadon. The collecdon of saliva is described below. Fermentation tests
Bacteria were grown and washed as described above, and suspended in 10% sucrose (in 0.9% NaCl) to a turbidity of Klett 175. Equal volumes of bacterial suspension and cytostatic drug (or its
Determination of methotrexate in patient saiiva
The secretion of antineoplastic agent into saliva was studied in 9 patients with lymphomas or other malignant tumors treated with intravenous infusions of methotrexate at the Department of Radiotherapy and Oncology, University Central Hospital of Helsinki. Three patients received fairly small daily doses of methotrexate (150-250 mg); 6 patients received high methotrexate doses (4.3-15.5 g). The administradon of the drug took at most 6 h. Paraffin-wax stimulated whole saliva was collected for 5 min. The saliva samples were taken 20 min after onset of therapy, 20 min after termination and 24 h after onset of therapy. The saliva was centrifuged for 15 min at 5000 g to remove solid particles. The concentration of methotrexate in the samples was assayed using afiuorescencepolarization immunoassay method (8). The level of detecdon was 0.009 pglm\. The antibiodc suscepdbility of the saliva samples was tested on blood agar plates, as described above.
Preparation of specimens for ultrastructure study
Equal volumes of bacterial suspensions and antineoplastic agents were mixed and incubated in a water bath for 1 h at 37°C with gentle agitation. Suspensions were centrifuged (15 min, 5000 g) and bacterial pellets were prefixed with 2.8% glutaraldehyde for 50 min, washed 3 dmes with 0.1 M PBS and postfixed with 1 % osmium tetroxide in the phosphate buffer for 2 h. The specimens were dehydrated in graded series of ethanol, embedded in Epon 812 and thin-sectioned in an LKB ultramicrotome (LKB, Stockholm, Sweden). The thin-sections were stained with uranyl acetate and lead citrate and studied in a Jeol 100 CX transmission electron microscope operadng at 60 kV (JEOL, Japan Electron Opdcs, Tokyo, Japan).
Table 1. Inhibition of growth of ATCC strains and clinical isolates of S. tnutans and S. .sanguis by methotrexate and doxorubicin after 24 h incubation. Restilts given as minimum inhibitory concentrations (MIC) Bacterial strain
Methotrexate Doxorubicin //g/ml mg/ml
S. mutans ATCC 27351 128B 013B I32A 121A
0.25 0.25 0.25 2.5 0.25
0.2 0.2 0.2 0.2 0.2
S. sanguis ATCC 10556 4063 1272 2827 1089
_* 0.025 0.025 0.025 0.025
0.2 0.2 0.02 2.0
* hazy inhibition zones at all concentrations down to 0.25
Statistical methods
Statistical analyses tested the effect of the 2 drugs and bacterial strains separately on the results, and the effect of time and MTX concentration on the patients' salivary findings. ANOVA statistics were used together with the Fisher test when applicable. Results
The MIC values for MTX and doxorubicin are given in Table 1. The ATCC strain of S. sanguis showed hazy growth at the inhibition zones in all test series with MTX, and this strain did not appear to be sensitive to doxorubicin. Thus, exact MIC values are not given to that strain. The clinical isolates, however, were disdnctly sensitive also to doxorubicin. The results of the inhibition of growth in both the S. mutans and S. sanguis bacteria are given in Table 2. As Table 2. Growth of S. mutans and S. satiguis after incubation with methotrexate and doxorubicin. Results given as CFU/ml Concentration of antineoplastic agent S. mutans S. .sanguis Methotrexate 12.5 mg/ml Methotrexate 2.5 mg/ml Doxorubicin I mg/ml Doxorubicin 0.2 tng/ml
6.1x10"
8.5x10*
1.5x10"
1.9x10*
2.9x10'
3.1x10*
1.2x10'
2.6x10'
1.8x10'
2.1x10'
Antineoplastic agents atid oral sireptoeoeei 179 Table 3. Effect of cytostatic drugs on the fermentation of sucrose by S. mutatis and S. satiguis. Fermentation is expressed as mean decrease in pH after 2 h incubation. 0.1 M PBS was used as control Anticancer drug (mg/ml)
^ g- I. Mueller-Hinton plates of S. mutatis showing: a) a distinct growth inhibition caused by xorubicin (arrow heads), and b) a not clearly detected effect by methotrexate, where only 'izy inhibidon zones could be seen in this case.
shown, the study drugs did not much atfeet the growth of 5. .satiguis, although statistically the observed differences l^ere significant compared with the conJ-ols in both the cytostatic series with tnis bacterium (P< 0.05). The growth of ''•mutans, however, showed a 10-fold fedution when incubated with doxorua highly significant difference n compared with the control ^^
