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Synergism between Clindamycin and Colloidal Bismuth Sub citrate against Helicobacter (Campylobacter) pylori in vitro
Zbl. Bakt. 274, 246-249 (1990) © Gustav Fischer Verlag, StuttgartlNew York
Synergism between Clindamycin and Colloidal Bismuth
Subcitrate against Helicobacter (Campylobacter) pylori in vitro KONST ANZE VOGT and HELMUT HAHN Institut fur Medizinische Mikrobiologie und Infektionsimmunologie der Freien Universitat Berlin, D-1000 Berlin 45 With 1 Figure· Received December 4, 1989 . Accepted in revised form May 31, 1990
Summary A combination of clindamycin and colloidal bismuth subcitrate was evaluated for synergistic inhibition of Helicobaeter pylori employing the agar dilution method. A total of 47 clinical isolates of Helieobaeter pylori were examined. Synergistic interaction was observed in 36%, additive behaviour in 64% of the strains. No antagonism could be detected. Zusammenfassung Die Kombination von Clindamycin und kolloidalem Wismutsubcitrat wurde mit der Agardilutionstechnik auf synergistische Wechselwirkungen bei 47 Isolaten von Helicobacter pylori untersucht. Bei 36% der Stamme wurde ein Synergismus beobachtet, bei 64% eine additive Interaktion. Ein Antagonismus zwischen den beiden Substanzen trat nicht auf. Introduction
Campylobaeter pylori - recently renamed Helieobaeter pylori (H. pylori) (1) - is nowadays accepted as a possible etiologic agent in chronic gastritis and peptic ulcer disease (2). Elimination of the bacteria with antibiotic substances leads to both clinical and histological improvement (7). The therapeutic regimen of choice is still unknown; antibiotic monotherapy, e.g. with ampicillin, results in short-term elimination rates of 68% immediately after treatment (7). Long-term studies, however, show a relapse rate for H. pylori infection of 67% after 1 year (7). A similar experience has been made with bismuth preparations (6). Combinations of antibiotics have resulted in lower long-term relapse rates. A combined therapy with ampicillin and colloidal bismuth subcitrate (CBS), for example, has resulted in a relapse rate of only 39% after one year (7). Similar results were reported for a CBS/metronidazole combination (12). In the present study, we have investigated the interaction of colloidal bismuth subcitrate and clindamycin against H. pylori in vitro. Clindamycin which is mostly
Clindamycin and CBS against H. pylori
247
employed for the treatment of coccal or anaerobic infections inhibits bacterial protein synthesis and acts specifically on the 50 S subunit of the bacterial ribosome (5). It is well absorbed after oral administration. Furthermore, the presence of food in the stomach does not impair its absorption (5). Its in vitro activity against H. pylori has been studied by Goodwin and coworkers; they found an MIC so of 0.5 mg/I (3). Materials and Methods
47 recent clinical isolates of H. pylori were tested. The bacteria were grown, identified and subcultured according to conventional methods described before (3). Clindamycin was supplied by Upjohn GmbH, Heppenheim, colloidal bismuth subcitrate by Byk Gulden, Konstanz. Synergy testing was performed with an agar dilution technique with Mueller-Hinton agar supplemented with 10% sheep blood and 1 mg/I amphotericin B. 1 ml clindamycin solution (0.0625 mg/l up to 4 mg/l) and 1 ml CBS solution (0.5 mg/l to 32 mg/l) were added to 18 ml agar according to the checkerboard scheme. For inoculation, the bacteria were resuspended in 3 ml brain heart broth supplemented with 5 ~g/l hemin to give a concentration of 107/ml. Plates were inoculated with a Steer multipoint inoculator (transfer volume 4 ~l), incubated at 3rC in a microaerophilic atmosphere (6% 02> 10% CO 2 ), and read after 72 h. The results obtained were recorded and the fractional inhibitory index (FIC index) was calculated employing the scheme of Krogstad and Moellering (4): FIC values of < 0.5 were considered synergistic; such between 0.51 and 1.0 were considered additive while FIC values of > 2 were thought to be antagonistic. Results Among the 47 H. pylori strains tested, synergistic action of clindamycin and CBS was found in 36% (17 strains) of the isolates under study. All other strains (64% = 30 strains) showed an additive behaviour. A typical result of synergistic action has been illustrated in Fig. 1: The MIC of clindamycin for H. pylori (4 mg/I) was reduced to 0.5 mg/I in the presence of 4 mg/I CBS.
4
2
1
0.5 0.25 ~.125 0,063
Clindamycin mg/l
32 16
0.56
+
+
8
0.31
+
+
+
+
+
4
0.38 0.25
2
0.56
+
+
+
+
+
1
0.53
+
+
+
+
+
+ i+
+
+
+
+
0.5
CBS mgll
Fig. 1. Checkerboard scheme with clindamycin and colloidal bismut subcitrate (CBS), H. pylori strain 169. For the zones of bacterial growth inhibition, the FIC index is calculated. The combination is highly synergistic at the concentrations for clindamycin of 0.5 mg/l and CBS of 4 mg/1. + = bacterial growth 17 ZbL Bakt. 274/2
248
K. Vogt and H. Hahn
Discussion Bismuth preparations are a well-proven therapeutic principle for chronic gastritis and peptic ulcer disease (9). Although they are not completeley soluble, significant levels are reached in gastric juice which exceed the MIC for H. pylori (10). Nevertheless, the bacteria often regrow in their sheltered niche underneath the gastric mucosal barrier which may lead to a relapse (7). A combined antibiotic therapy is apparently more effective, provided that both components reach the bacteria in their habitat. In establishing such combinations, clindamycin seems to be a suitable partner since it can be administered orally, is well distributed in body fluids and is acid-stable (5). The results of our study show a high rate of synergistic interaction of clindamycin and CBS in vitro. The FIC index calculation of Krogstad and Moellering employed (4), however, does not separate indifferent from additive interaction. Ullmann (11) has modified the respective definition by calculating an FIC index of 0.51-1.0 for addition and 1.1-1.9 for indifference. To find out whether the remaining 30 out of 47 strains were additive or indifferent, we used this more precise interpretation of results. It could be shown that the 30 strains were still attached in an additive mode by the two substances. Presumably, there is no antagonism between CBS and clindamycin. The molecular basis of the reported interaction is unknown. It is possible that the aggregation of CBS underneath the bacterial cell wall as described by Rauws and Tytgat (8) enhances the penetration of clindamycin into the bacteria. This might increase its bactericidal action on H. pylori. Clinical follow-up studies should be done to determine whether these promising invitro results can be applied to in-vivo conditions and whether H. pylori is just temporarily reduced by this combined treatment or in fact eliminated from the gastric mucosa.
Acknowledgement. We thank Petra Wiedersatz for technical assistance. References 1. Goodwin, C. S., J. A. Armstrong, T. Chi/vers, M. Peters, M. D. Collins, L. Sly, W. McConnell, and W. E. S. Harper: Transfer of Campylobaeter pylori and Campylobaeter mustelae to Helieobaeter gen. nov. as. Helieobaeter pylori comb. nov. and Helieobaeter mustelae comb. nov., respectively. Int. J. System. Bact. 39 (1989) 397-405 2. Goodwin, C. S., J. A. Armstrong, and B. J. Marshall: Campylobaeter pyloridis, gastritis, and peptic ulceration. J. Clin. Path. 39 (1986) 353-365 3. Goodwin, C. S., P. Blake, and E. Blineow: The minimum inhibitory and bactericidal concentrations of antibiotics and anti-ulcer agents against Campylobaeter pyloridis. J. Antimicrob. Chemother. 17 (1986) 309-314 4. Krogstad, D. and R. C. Moellering: Combination of antibiotics, mechanisms of interaction against bacteria. In: Antibiotics in Laboratory Medicine, 1st ed. (V. Lorian ed.), pp. 298-341. Williams and Wilkins, Baltimore-London (1980) 5. Kueers, A. and N. MeK. Bennett: Lincomycin and clindamycin. In: The Use of Antibiotics, 4th edition (A. Kueers and N. MeK. Bennett, eds.), pp. 819-850. William Heinemann Medical Books, London (1987) 6. Lee, F. 1., 1. M. Samloff, and M. Hardman: Comparison of tri-potassium di-citrate bismuthate tablets with ranitidine in healing and relapse of duodenal ulcers. Lancet I (1985) 1299-1301
Clindamycin and CBS against H. pylori
249
7. Rauws, E. A. j., W. Langenberg, H. J. Houthoff, H. C. Zanen, and C. N. j. Tytgat: Campylobaeter pyloridis-associated chronic active antral gastritis. A prospective study 8. 9. 10. 11. 12.
of its prevalence and the effects of antibacterial and antiulcer treatment. Gastroenterol. 94 (1988) 33-40 Rauws, E. A. j. und C. N. Tytgat: Elektronenmikroskopische Befunde wiihrend einer Behandlung Campylobaeter-pylori-positiver Gastritiden mit Wismutsalzen. Z. Gastroenterol. 25 (S4 (1987) 41-43 Rosch, W.: Therapie des peptischen Ulcus und der chronischen Gastritis mit Wismutsalzen. Z. Gastroenterol. 25 S4 (1987) 34-40 Skoglund, M.L. and K. Watters: Bismuth concentration at site of Campylobaeter pylori colonization. Gastroenterol. 94 (1988) A430 Ullmann, U.: Synergism between ciprofloxacin and fosfomycin in vitro. Infection 15 (1987) 264 Weil, j., C. D. Bell, P. Cant, J. Trowell, and C. Harrison: High success rate for eradication of Campylobaeter pylori infection using a colloidal bismuth sub citrate (CBS) metronidazole combination. Gut 30 (1989) A733-734
Dr. Konstanze Vogt, Institut fiir Medizinische Mikrobiologie der Freien Universitiit Berlin, Hindenburgdamm 27, D-lOOO Berlin 45
Synergism between Clindamycin and Colloidal Bismuth
Subcitrate against Helicobacter (Campylobacter) pylori in vitro KONST ANZE VOGT and HELMUT HAHN Institut fur Medizinische Mikrobiologie und Infektionsimmunologie der Freien Universitat Berlin, D-1000 Berlin 45 With 1 Figure· Received December 4, 1989 . Accepted in revised form May 31, 1990
Summary A combination of clindamycin and colloidal bismuth subcitrate was evaluated for synergistic inhibition of Helicobaeter pylori employing the agar dilution method. A total of 47 clinical isolates of Helieobaeter pylori were examined. Synergistic interaction was observed in 36%, additive behaviour in 64% of the strains. No antagonism could be detected. Zusammenfassung Die Kombination von Clindamycin und kolloidalem Wismutsubcitrat wurde mit der Agardilutionstechnik auf synergistische Wechselwirkungen bei 47 Isolaten von Helicobacter pylori untersucht. Bei 36% der Stamme wurde ein Synergismus beobachtet, bei 64% eine additive Interaktion. Ein Antagonismus zwischen den beiden Substanzen trat nicht auf. Introduction
Campylobaeter pylori - recently renamed Helieobaeter pylori (H. pylori) (1) - is nowadays accepted as a possible etiologic agent in chronic gastritis and peptic ulcer disease (2). Elimination of the bacteria with antibiotic substances leads to both clinical and histological improvement (7). The therapeutic regimen of choice is still unknown; antibiotic monotherapy, e.g. with ampicillin, results in short-term elimination rates of 68% immediately after treatment (7). Long-term studies, however, show a relapse rate for H. pylori infection of 67% after 1 year (7). A similar experience has been made with bismuth preparations (6). Combinations of antibiotics have resulted in lower long-term relapse rates. A combined therapy with ampicillin and colloidal bismuth subcitrate (CBS), for example, has resulted in a relapse rate of only 39% after one year (7). Similar results were reported for a CBS/metronidazole combination (12). In the present study, we have investigated the interaction of colloidal bismuth subcitrate and clindamycin against H. pylori in vitro. Clindamycin which is mostly
Clindamycin and CBS against H. pylori
247
employed for the treatment of coccal or anaerobic infections inhibits bacterial protein synthesis and acts specifically on the 50 S subunit of the bacterial ribosome (5). It is well absorbed after oral administration. Furthermore, the presence of food in the stomach does not impair its absorption (5). Its in vitro activity against H. pylori has been studied by Goodwin and coworkers; they found an MIC so of 0.5 mg/I (3). Materials and Methods
47 recent clinical isolates of H. pylori were tested. The bacteria were grown, identified and subcultured according to conventional methods described before (3). Clindamycin was supplied by Upjohn GmbH, Heppenheim, colloidal bismuth subcitrate by Byk Gulden, Konstanz. Synergy testing was performed with an agar dilution technique with Mueller-Hinton agar supplemented with 10% sheep blood and 1 mg/I amphotericin B. 1 ml clindamycin solution (0.0625 mg/l up to 4 mg/l) and 1 ml CBS solution (0.5 mg/l to 32 mg/l) were added to 18 ml agar according to the checkerboard scheme. For inoculation, the bacteria were resuspended in 3 ml brain heart broth supplemented with 5 ~g/l hemin to give a concentration of 107/ml. Plates were inoculated with a Steer multipoint inoculator (transfer volume 4 ~l), incubated at 3rC in a microaerophilic atmosphere (6% 02> 10% CO 2 ), and read after 72 h. The results obtained were recorded and the fractional inhibitory index (FIC index) was calculated employing the scheme of Krogstad and Moellering (4): FIC values of < 0.5 were considered synergistic; such between 0.51 and 1.0 were considered additive while FIC values of > 2 were thought to be antagonistic. Results Among the 47 H. pylori strains tested, synergistic action of clindamycin and CBS was found in 36% (17 strains) of the isolates under study. All other strains (64% = 30 strains) showed an additive behaviour. A typical result of synergistic action has been illustrated in Fig. 1: The MIC of clindamycin for H. pylori (4 mg/I) was reduced to 0.5 mg/I in the presence of 4 mg/I CBS.
4
2
1
0.5 0.25 ~.125 0,063
Clindamycin mg/l
32 16
0.56
+
+
8
0.31
+
+
+
+
+
4
0.38 0.25
2
0.56
+
+
+
+
+
1
0.53
+
+
+
+
+
+ i+
+
+
+
+
0.5
CBS mgll
Fig. 1. Checkerboard scheme with clindamycin and colloidal bismut subcitrate (CBS), H. pylori strain 169. For the zones of bacterial growth inhibition, the FIC index is calculated. The combination is highly synergistic at the concentrations for clindamycin of 0.5 mg/l and CBS of 4 mg/1. + = bacterial growth 17 ZbL Bakt. 274/2
248
K. Vogt and H. Hahn
Discussion Bismuth preparations are a well-proven therapeutic principle for chronic gastritis and peptic ulcer disease (9). Although they are not completeley soluble, significant levels are reached in gastric juice which exceed the MIC for H. pylori (10). Nevertheless, the bacteria often regrow in their sheltered niche underneath the gastric mucosal barrier which may lead to a relapse (7). A combined antibiotic therapy is apparently more effective, provided that both components reach the bacteria in their habitat. In establishing such combinations, clindamycin seems to be a suitable partner since it can be administered orally, is well distributed in body fluids and is acid-stable (5). The results of our study show a high rate of synergistic interaction of clindamycin and CBS in vitro. The FIC index calculation of Krogstad and Moellering employed (4), however, does not separate indifferent from additive interaction. Ullmann (11) has modified the respective definition by calculating an FIC index of 0.51-1.0 for addition and 1.1-1.9 for indifference. To find out whether the remaining 30 out of 47 strains were additive or indifferent, we used this more precise interpretation of results. It could be shown that the 30 strains were still attached in an additive mode by the two substances. Presumably, there is no antagonism between CBS and clindamycin. The molecular basis of the reported interaction is unknown. It is possible that the aggregation of CBS underneath the bacterial cell wall as described by Rauws and Tytgat (8) enhances the penetration of clindamycin into the bacteria. This might increase its bactericidal action on H. pylori. Clinical follow-up studies should be done to determine whether these promising invitro results can be applied to in-vivo conditions and whether H. pylori is just temporarily reduced by this combined treatment or in fact eliminated from the gastric mucosa.
Acknowledgement. We thank Petra Wiedersatz for technical assistance. References 1. Goodwin, C. S., J. A. Armstrong, T. Chi/vers, M. Peters, M. D. Collins, L. Sly, W. McConnell, and W. E. S. Harper: Transfer of Campylobaeter pylori and Campylobaeter mustelae to Helieobaeter gen. nov. as. Helieobaeter pylori comb. nov. and Helieobaeter mustelae comb. nov., respectively. Int. J. System. Bact. 39 (1989) 397-405 2. Goodwin, C. S., J. A. Armstrong, and B. J. Marshall: Campylobaeter pyloridis, gastritis, and peptic ulceration. J. Clin. Path. 39 (1986) 353-365 3. Goodwin, C. S., P. Blake, and E. Blineow: The minimum inhibitory and bactericidal concentrations of antibiotics and anti-ulcer agents against Campylobaeter pyloridis. J. Antimicrob. Chemother. 17 (1986) 309-314 4. Krogstad, D. and R. C. Moellering: Combination of antibiotics, mechanisms of interaction against bacteria. In: Antibiotics in Laboratory Medicine, 1st ed. (V. Lorian ed.), pp. 298-341. Williams and Wilkins, Baltimore-London (1980) 5. Kueers, A. and N. MeK. Bennett: Lincomycin and clindamycin. In: The Use of Antibiotics, 4th edition (A. Kueers and N. MeK. Bennett, eds.), pp. 819-850. William Heinemann Medical Books, London (1987) 6. Lee, F. 1., 1. M. Samloff, and M. Hardman: Comparison of tri-potassium di-citrate bismuthate tablets with ranitidine in healing and relapse of duodenal ulcers. Lancet I (1985) 1299-1301
Clindamycin and CBS against H. pylori
249
7. Rauws, E. A. j., W. Langenberg, H. J. Houthoff, H. C. Zanen, and C. N. j. Tytgat: Campylobaeter pyloridis-associated chronic active antral gastritis. A prospective study 8. 9. 10. 11. 12.
of its prevalence and the effects of antibacterial and antiulcer treatment. Gastroenterol. 94 (1988) 33-40 Rauws, E. A. j. und C. N. Tytgat: Elektronenmikroskopische Befunde wiihrend einer Behandlung Campylobaeter-pylori-positiver Gastritiden mit Wismutsalzen. Z. Gastroenterol. 25 (S4 (1987) 41-43 Rosch, W.: Therapie des peptischen Ulcus und der chronischen Gastritis mit Wismutsalzen. Z. Gastroenterol. 25 S4 (1987) 34-40 Skoglund, M.L. and K. Watters: Bismuth concentration at site of Campylobaeter pylori colonization. Gastroenterol. 94 (1988) A430 Ullmann, U.: Synergism between ciprofloxacin and fosfomycin in vitro. Infection 15 (1987) 264 Weil, j., C. D. Bell, P. Cant, J. Trowell, and C. Harrison: High success rate for eradication of Campylobaeter pylori infection using a colloidal bismuth sub citrate (CBS) metronidazole combination. Gut 30 (1989) A733-734
Dr. Konstanze Vogt, Institut fiir Medizinische Mikrobiologie der Freien Universitiit Berlin, Hindenburgdamm 27, D-lOOO Berlin 45