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Influence of omeprazole on urease activity of Helicobacter pylori in vitro
Zbl. Bakt. 280, 273-278 (1993) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Influence of Omeprazole on Urease Activity of Helicobacter pylori in vitro KONST ANZE VOGT and HELMUT HAHN Institut fur Medizinische Mikrobiologie und Infektionsimmunologie der Freien Universitat Berlin, 13353 Berlin, Germany
With 3 Figures· Accepted March 17, 1993
Summary The influence of omeprazole on urease activity of 13 Helicobacter pylori strains was assessed in vitro employing different inocula of the bacteria and various concentrations of omeprazole. Bacteria were grown in liquid culture supplemented with omeprazole for 48 h. Afterwards, bacterial numbers were assessed and urease activity was measured in a spectrophotometric assay. In 10 strains, omeprazole had no influence on urease activity at concentrations up to 8 mg/l; higher concentrations had a bacteriostatic effect. Three strains were more resistant to omeprazole: These showed a marked diminution of urease activity although bacterial numbers were only slightly reduced. Thus a possible inhibitory effect of omeprazole should be taken into account when urease of Helicobacter pylori is measured for diagnostic purposes. Zusammenfassung Der EinfluB von Omeprazol auf die Ureaseaktivitat von 13 Helicobacter pylori-Stammen wurde in vitro anhand verschiedener Inokula und Omeprazolkonzentrationen untersucht. Die Bakterien wurden 48 Std. in omeprazol-angereicherten Fliissigkulturen inkubiert. Danach wurden die Keimzahlen bestimmt, und die Ureaseaktivitat wurde in einem spektrophotometrischen Ansatz gemessen. Bei 10 Stammen hatte Omeprazol bis zu 8 mg/l keinen EinfluB auf die Ureaseaktivitat; hohere Konzentrationen wirkten bakteriostatisch. Drei Stamme waren resistenter gegeniiber Omeprazol: Diese zeigten eine deutliche Verringerung der Ureaseaktivitat, obwohl die Keimzahlen nur leicht vermindert waren. Daher soUte ein moglicher inhibitorischer Effekt von Omeprazol in Betracht gezogen werden, wenn die Urease von Helicobacter pylori zu diagnostischen Zwecken untersucht wird. Introduction The unique urease activity of Helicobacter pylori (H. pylori) is an important microbiological characteristic (15). Moreover, it is exploited as a diagnostic tool: H. pylori infection can be diagnosed with the rapid urease test in gastric biopsies (2) or indirectly by measuring previously marked carbon which is liberated during the urea bre;lth test 18 Zbl. Bakt. 280/1-2
274
K. Vogt and H. Hahn
(10). Both procedures have a high degree of sensitivity and specificity (3). Furthermore, the urease test is a quick and simple method to control the efficacy of treatment regimens against H. pylori (11). The bacterial enzyme consists of two subunits with relative molecular masses of 66000 and 31000, and a close relationship was observed between the ureases of H. pylori, Klebsiella aerogenes and Proteus mirabilis (4). However, the reaction time of H. pylori was found to be significantly shorter than that of urease producing Klebsiella and Proteus strains (19). The urease of H. pylori can be competitively inhibited by acetohydroxamic acid (6). Antibiotics, however, have no influence on its activity (19). Among the acid-reducing substances commonly used in gastritis and ulcer therapy, only proton-pump inhibitors reveal moderate antibacterial activity against H. pylori: The density of H. pylori decreased significantly after treatment with omeprazole (10 mg or 20 mg daily), but not with the Hrreceptor antagonist ranitidine (7). Although an eradication of H. pylori is not achieved with omeprazole monotherapy (16), the substance appears to be a suitable partner for a combined antibiotic therapy of H. pylori infection. As the bacterial urease is suspected to be a virulence factor of H. pylori (4), we assessed the influence of omeprazole on urease activity of H.pylori in vitro in order to find out whether an inhibition of bacterial urease may render benefit to a combined antibacterial treatment. Materials and Methods 1. Bacteria. 13 strains of H. pylori were included, 12 recent patients' isolates from Klinikum Steglitz, Berlin, and one reference strain (NCTC 11637). The isolates were identified as H. pylori by typical growth morphology, positive urease, oxidase and catalase reaction, and by Gram staining. Cultivation was done on Columbia agar supplemented with 10% heated sheep blood, 10 mg/l vancomycin, 2500 I.E.!1 polymyxin,S mg/l trimethoprim, and 2 mg/l amphotericin B. They were incubated in a microaerophilic atmosphere (6% O2 , 10% CO2 ) and subcultured every 2-4 days. 2. Incubation with omeprazole. Different bacterial inocula were prepared (104 _10 9 CFUI ml) from colonies grown on Columbia agar as described above. These were inoculated into 50 ml-flasks containing 10 ml brain heart broth supplemented with 5 Ilg/l hemin, 2 mg/l amphotericin B, and various concentrations of omeprazole (0, 8, 16, 32 mg/l). Before incubation, bacterial counting was done by double lO-fold dilutions in sterile saline, plating 100 Ill-portions on Columbia agar plates and assessing the number of CFU/ml after 3 days of incubation as described above. Incubation took place in a microaerophilic atmosphere under horizontal rotation of 70 rpm (Certomat M, B. Braun, Melsungen) for 48 h. Afterwards, bacterial numbers were counted again as described above. All experiments were carried out in triplicate. 3. Urease assay. A pH indicator assay was used to assess urease activity (19). 100 !J.l of bacterial suspension were added to 900 !J.l of Christensen urea medium containing 2 % urea and 4 Ilg/l phenol red (1). Extinction at 520 nm was measured with a photometer (Ultrolab Calculating Absorptiometer, LKB, Bromma, Sweden) at 5 min, 10 min, 20 min, 30 min, 60 min, 120 min, and 180 min. All photometric measurements were done in duplicate.
Results The incubation of H. pylori with omeprazole for 48 h lead to an increase in bacterial numbers between 10 1 and 104 CFU/ml. Thus we decided to concentrate on the bacterial counts assessed immediately before the spectrophotometric assay.
Omeprazole and Urease of H.pylori
275
In the control group, there was no significant photometric reaction at low inocula (104 CFU/ml). An inoculum of 10 5 CFU/mllead to a rapid hydrolysis of urea with a median enzymatic half-time of 10-20 min. Higher inocula of 10 6-109 CFU/ml obviously accelerated the urease reaction. The incubation of H. pylori with omeprazole mostly lead to a dose-dependent reduction of bacterial numbers and thus to a delayed urease reaction. A typical example is i.llustrated in Fig. 1. Three patients' isolates, however, showed a higher resistance against omeprazole, i.e. bacterial numbers were not significantly reduced at omeprazole concentrations of 16 and 32 mg/l (a significant reduction would exceed 2 logarithmical steps). H. pylori strain No. 25 is illustrated in Fig.2: Here, a distinct delay in urease reaction is obvious at 16 mg/l and 32 mgll omeprazole. H. pylori strain No. 13, as illustrated in Fig. 3, shows an even more drastic decrease of urease activity which is significant at 32 mg/l (p < 0.05). Within the measuring period of 180 min, not even the half-time extinction was reached under the influence of omeprazole although the bacterial numbers are still 10 6 CFU/ml. Discussion The pathogenetic role of H. pylori urease is still controversely discussed. Whereas Mobley et al. (12) postulate a central role of the enzyme in the pathogenesis of gastritis and ulcer disease, Newell (14) mentions it as one of the factors enabling the organism to colonize the gastric epithelium, but not as a main property of H. pylori directly causing disease. Undoubtedly, hydrolysis of urea with generation of ammonia maintains the necessary microenvironment for the acid-sensitive organism in the gastric
E
_ _ =:=====:::======:::::::control lOB CFU/ml Omeprazol Bmg/l 10B CFU/mi Omeprazol 16 mgll 107 CFU/ml
Omeprazol 32 mg /I ~-----·104 CFU/ml
10 30
60
120
180
t/min
Fig. 1. Influence of omeprazole (8 mg/l, 16 mg/l, 32 mg/l) on urease activity of H. pylori, strain No. 7 Bacterial inocula are shown on the right side.
276
K. Vogt and H. Hahn
E 40
_ _-------------controI108CFU/ml Omeprazol 8mg/l 108 CFUlmi Omeprazol 16mg/ l 10 8 CFUlmi Omeprazol 32mg/l
30
106 CFU/ml
20
10
10 30
60
120
180 t/min
Fig. 2. Influence of omeprazole on urease activity of H. pylori, strain No. 25. Bacterial inocula (shown on the right) are only slighly reduced, whereas urease reaction is delayed at 32 mg/I omeprazole.
E
-::::::::=====:::====:::::cont rol 108 CFU/I11I Omeprazol8mg ll 1Q8 CFU/ml
40
Omeprazol 16 mg 107CFU/mi
30
Omeprazol 32 mg/l -----~106CFU/ml
30
60
120
180
tlmin
Fig. 3. Influence of omeprazole on urease activity of H. pylori, strain No. 13. Bacterial inocula (shown on the right) are only slightly reduced. At 16 mg/I omeprazole, urease reaction is evidently delayed. At 32 mg/I, the half-time extinction is not reached at all.
Omeprazole and Urease of H. pylori
277
. mucosa. Furthermore, the ammonia generated by urea hydrolysis is present at site in high concentrations and may have a cytotoxic effect on gastric epithelial cells (12). The effect of omeprazole on H. pylori is still unclear. Suerbaum et al. (18) reported a direct antibacterial effect of the substance at concentration between 8 and 32 mg/l. It is questionable, however, whether these concentrations are reached within the gastric lumen and the mucosal layer where H. pylori is located. Stolte and Bethke (17) postulate that the reduced density of H. pylori is due to bacterial overgrowth following omeprazole-induced rising of gastric pH values. This would deny any direct antibacterial activity of omeprazole on the bacteria. However, the accumulation of omeprazole within the parietal cell during a four week-therapy may possibly lead to a saturation of all Na +K+ -ATPase sites followed by an active excretion into the gastric lumen or - as supposed by Nagaya et al. (13) - a transformation to other active compounds which have been analyzed by Iwahi et al. (8) and found to have antibacterial properties against H. pylori. Regardless of direct antibacterial action, our results demonstrate that omeprazole may have an influence on H. pylori urease. The fact that the diminution of the enzyme becomes obvious in strains which are relatively resistant against omeprazole may be due to a higher concentration of omeprazole necessary to inhibit bacterial urease. Concentrations of 16 mg or lower inhibit growth of susceptible strains, so the urease inhibition does not become apparent. The mechanism by which omeprazol might lead to inhibition of the urease enzyme is unclear. It might be assumed that omeprazole as an inhibitor of the proton pump activity may also have an effect on the uptake of hydrogen ions into the cell. These, however, are necessary for the urea hydrolysis so that a reduction might lengthen the reaction time. For clinical settings, the possible influence of omeprazole on H. pylori urease must be taken into account for the rapid urease test and the urea breath test. Both tests should not be performed during omeprazole therapy or immediately after cessation as they might lead to a falsely negative result. As shown by Wagner et al. (20), a monotherapy with omeprazole is unable to eradicate H. pylori. The combination with one antibiotic is also ineffective (9), so that at least triple therapeutic regimens seem to be necessary (5). In such a combination, the use of omeprazole could be beneficial in three aspects: Firstly, the rise of gastric pH enables several antibiotics to reach full antibacterial activity. Secondly, the antibacterial activity of the substance itself may contribute to eradication. Thirdly, a possible influence on bacterial urease in some strains may at least make recolonization more difficult.
Acknowledgement. We thank Petra Wiedersatz for skilful technical assistance.
References 1. Christensen, W. B.: Urea decomposition as a means of differentiating Proteus and Paracolon cultures from each other and from Salmonella and Shigella types. J. Bact. 52 (1946) 461-466 2. Das, S. S., L. A. Bain, Q. N. Karim, L. G. Coelho, and]. H. Baron: Rapid diagnosis of Campylobacter pyloridis infection. J. Clin. Path. 40 (1987) 701-702
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3. Deltenre, M., Y. Glupczynski, C. de Pres, j. F. Nyst, A. Burette, M. Labbe, C. jonas, and E. Dekoster: The reliability of urease tests, histology and culture in the diagnosis of Campylobacter pylori infection. Scand. J. Gasttoenterol. 24, Suppl. 160 (1989) 19-24 4. Evans, D. j., D. G. Evans, S. S. Kirkpatrick, and D. Y. Graham: Characterization of the Helicobacter pylori urease and purification of its subunits. Microb. Pathogen. 10 (1991) 15-26 5. Glupczynski, Y. and A. Burette: Eradicating Helicobacter pylori. Lancet I (1992) 54-55 6. Glupczynski, Y., M. Labbe, and F. Thibaumont: Interaction between acetohydroxamic acid - a potent urease inhibitor - and various antimicrobial agents against Campylobacter pylori. Proc. First Meeting of the Eur. Camp. pylori Study Group, Bordeaux, France, 7-8 Oct. 1988, pp. 157-160 7. Hui, W. M., S. K. Lang, j. Ho, C. L. Lai, A. S. F. Lok, M. M. T. Ng, W. Y. Lau, and F. j. Branicki: Effect of omeprazole on duodenal ulcer-associated antral gastritis and Helicobacter pylori. Dig. Dis. Sci. 36 (1991) 577-582 8. Iwahi, T., H. Satoh, M. Nakao, T. Iwasaki, T. Yamasaki, K. Kubo, T. Tamura, and A. Imada: Lansoprazole, a novel benzimidazole proton pump inhibitor, and its related compounds have selective activity against Helicobacter pylori. Antimicrob. Agents Chemother. 35 (1990) 490-496 9. Labenz, j., E. Gyenes, U. Peitz und G. Borsch: Ciprofloxacin-Omeprazol-Kombinationstherapie zur Eradikation von Helicobacter pylori. Z. Gastroenterol. 29 (1991) 143-145 10. Marshall, B. j. and 1. Surveyer: Carbon-14 urea breath test for the diagnosis of Campylobacter pylori associated gastritis. J. Nucl. Med. 29 (1988) 11-16 11. Marshall, B. j., j. R. Warren, G. j. Francis, S. R. Langton, C. S. Goodwin, and E. D. Blincow: Rapid urease test in the management of Campylobacter pyloridis-associated gastritis. Am. J. Gastroenterol. 82 (1987) 200-210 12. Mobley, H. L. T., L.- T. Hu, and P. A. Foxall: Helicobacter pylori urease: Properties and role in pathogenesis. Scand. J. Gastroenterol. 26, Suppl. 187 (1991) 39-46 13. Nagaya, H., H. Satoh, K. Kubo, and Y. Maki: Possible mechanism for the inhibition of gastric (H+ + K+)-adenosine triphosphatase by the proton pump inhibitor AG-1749. J. Pharmacol. Exp. Ther. 248 (1989) 799-803 14. Newell, D. G.: Virulence factors of Helicobacter pylori. Scand. J. Gastroenterol. 26, Suppl. 187 (1991) 31-38 15. Owen, R. j., S. R. Martin, and P. Borman: Rapid urea hydrolysis by gastric campylobacters. Lancet 1(1985) 111 16. Rauws, E. A. j., W. Langenberg, A. Bosma, j. Dankert, and G. N. j. Tytgat: Lack of eradication of Helicobacter pylori after omeprazole. Lancet I (1991) 1093 17. Stolte, M. and B. Bethke: Elimination of Helicobacter pylori under treatment with omeprazole. Z. Gastroenterol. 28 (1990) 271-274 18. Suerbaum, S., H. Leying, K. Klemm, and W. Opferkuch: Antibacterial activity of pantoprazole and omeprazole against Helicobacter pylori. Eur. J. Clin. Microbiol. Infect. Dis. 10 (1991) 92-93 19. Vogt, K. and H. Hahn: Urease production by Helicobacter (Campylobacter) pylori. Zbl. Bakt. 275 (1991) 63-72 20. Wagner, S., M. Varrentrapp, K. Haruma, P. Lange, M. j. Milller, T. Schorn, B. Soudah, W. Bar, and M. Gebel: The role of omeprazole (40 mg) in the treatment of gastric Helicobacter pylori infection. Z. Gastroenterol. 29 (1991) 595-598 Dr. Kostanze Vogt, Institut fur Medizinische Mikrobiologie und Infektionsimmunologie der Freien Universitiit Berlin, Universitiitsklinikum Rudolf Virchow, Augustenburger Platz 1. D-13353 Berlin
Influence of Omeprazole on Urease Activity of Helicobacter pylori in vitro KONST ANZE VOGT and HELMUT HAHN Institut fur Medizinische Mikrobiologie und Infektionsimmunologie der Freien Universitat Berlin, 13353 Berlin, Germany
With 3 Figures· Accepted March 17, 1993
Summary The influence of omeprazole on urease activity of 13 Helicobacter pylori strains was assessed in vitro employing different inocula of the bacteria and various concentrations of omeprazole. Bacteria were grown in liquid culture supplemented with omeprazole for 48 h. Afterwards, bacterial numbers were assessed and urease activity was measured in a spectrophotometric assay. In 10 strains, omeprazole had no influence on urease activity at concentrations up to 8 mg/l; higher concentrations had a bacteriostatic effect. Three strains were more resistant to omeprazole: These showed a marked diminution of urease activity although bacterial numbers were only slightly reduced. Thus a possible inhibitory effect of omeprazole should be taken into account when urease of Helicobacter pylori is measured for diagnostic purposes. Zusammenfassung Der EinfluB von Omeprazol auf die Ureaseaktivitat von 13 Helicobacter pylori-Stammen wurde in vitro anhand verschiedener Inokula und Omeprazolkonzentrationen untersucht. Die Bakterien wurden 48 Std. in omeprazol-angereicherten Fliissigkulturen inkubiert. Danach wurden die Keimzahlen bestimmt, und die Ureaseaktivitat wurde in einem spektrophotometrischen Ansatz gemessen. Bei 10 Stammen hatte Omeprazol bis zu 8 mg/l keinen EinfluB auf die Ureaseaktivitat; hohere Konzentrationen wirkten bakteriostatisch. Drei Stamme waren resistenter gegeniiber Omeprazol: Diese zeigten eine deutliche Verringerung der Ureaseaktivitat, obwohl die Keimzahlen nur leicht vermindert waren. Daher soUte ein moglicher inhibitorischer Effekt von Omeprazol in Betracht gezogen werden, wenn die Urease von Helicobacter pylori zu diagnostischen Zwecken untersucht wird. Introduction The unique urease activity of Helicobacter pylori (H. pylori) is an important microbiological characteristic (15). Moreover, it is exploited as a diagnostic tool: H. pylori infection can be diagnosed with the rapid urease test in gastric biopsies (2) or indirectly by measuring previously marked carbon which is liberated during the urea bre;lth test 18 Zbl. Bakt. 280/1-2
274
K. Vogt and H. Hahn
(10). Both procedures have a high degree of sensitivity and specificity (3). Furthermore, the urease test is a quick and simple method to control the efficacy of treatment regimens against H. pylori (11). The bacterial enzyme consists of two subunits with relative molecular masses of 66000 and 31000, and a close relationship was observed between the ureases of H. pylori, Klebsiella aerogenes and Proteus mirabilis (4). However, the reaction time of H. pylori was found to be significantly shorter than that of urease producing Klebsiella and Proteus strains (19). The urease of H. pylori can be competitively inhibited by acetohydroxamic acid (6). Antibiotics, however, have no influence on its activity (19). Among the acid-reducing substances commonly used in gastritis and ulcer therapy, only proton-pump inhibitors reveal moderate antibacterial activity against H. pylori: The density of H. pylori decreased significantly after treatment with omeprazole (10 mg or 20 mg daily), but not with the Hrreceptor antagonist ranitidine (7). Although an eradication of H. pylori is not achieved with omeprazole monotherapy (16), the substance appears to be a suitable partner for a combined antibiotic therapy of H. pylori infection. As the bacterial urease is suspected to be a virulence factor of H. pylori (4), we assessed the influence of omeprazole on urease activity of H.pylori in vitro in order to find out whether an inhibition of bacterial urease may render benefit to a combined antibacterial treatment. Materials and Methods 1. Bacteria. 13 strains of H. pylori were included, 12 recent patients' isolates from Klinikum Steglitz, Berlin, and one reference strain (NCTC 11637). The isolates were identified as H. pylori by typical growth morphology, positive urease, oxidase and catalase reaction, and by Gram staining. Cultivation was done on Columbia agar supplemented with 10% heated sheep blood, 10 mg/l vancomycin, 2500 I.E.!1 polymyxin,S mg/l trimethoprim, and 2 mg/l amphotericin B. They were incubated in a microaerophilic atmosphere (6% O2 , 10% CO2 ) and subcultured every 2-4 days. 2. Incubation with omeprazole. Different bacterial inocula were prepared (104 _10 9 CFUI ml) from colonies grown on Columbia agar as described above. These were inoculated into 50 ml-flasks containing 10 ml brain heart broth supplemented with 5 Ilg/l hemin, 2 mg/l amphotericin B, and various concentrations of omeprazole (0, 8, 16, 32 mg/l). Before incubation, bacterial counting was done by double lO-fold dilutions in sterile saline, plating 100 Ill-portions on Columbia agar plates and assessing the number of CFU/ml after 3 days of incubation as described above. Incubation took place in a microaerophilic atmosphere under horizontal rotation of 70 rpm (Certomat M, B. Braun, Melsungen) for 48 h. Afterwards, bacterial numbers were counted again as described above. All experiments were carried out in triplicate. 3. Urease assay. A pH indicator assay was used to assess urease activity (19). 100 !J.l of bacterial suspension were added to 900 !J.l of Christensen urea medium containing 2 % urea and 4 Ilg/l phenol red (1). Extinction at 520 nm was measured with a photometer (Ultrolab Calculating Absorptiometer, LKB, Bromma, Sweden) at 5 min, 10 min, 20 min, 30 min, 60 min, 120 min, and 180 min. All photometric measurements were done in duplicate.
Results The incubation of H. pylori with omeprazole for 48 h lead to an increase in bacterial numbers between 10 1 and 104 CFU/ml. Thus we decided to concentrate on the bacterial counts assessed immediately before the spectrophotometric assay.
Omeprazole and Urease of H.pylori
275
In the control group, there was no significant photometric reaction at low inocula (104 CFU/ml). An inoculum of 10 5 CFU/mllead to a rapid hydrolysis of urea with a median enzymatic half-time of 10-20 min. Higher inocula of 10 6-109 CFU/ml obviously accelerated the urease reaction. The incubation of H. pylori with omeprazole mostly lead to a dose-dependent reduction of bacterial numbers and thus to a delayed urease reaction. A typical example is i.llustrated in Fig. 1. Three patients' isolates, however, showed a higher resistance against omeprazole, i.e. bacterial numbers were not significantly reduced at omeprazole concentrations of 16 and 32 mg/l (a significant reduction would exceed 2 logarithmical steps). H. pylori strain No. 25 is illustrated in Fig.2: Here, a distinct delay in urease reaction is obvious at 16 mg/l and 32 mgll omeprazole. H. pylori strain No. 13, as illustrated in Fig. 3, shows an even more drastic decrease of urease activity which is significant at 32 mg/l (p < 0.05). Within the measuring period of 180 min, not even the half-time extinction was reached under the influence of omeprazole although the bacterial numbers are still 10 6 CFU/ml. Discussion The pathogenetic role of H. pylori urease is still controversely discussed. Whereas Mobley et al. (12) postulate a central role of the enzyme in the pathogenesis of gastritis and ulcer disease, Newell (14) mentions it as one of the factors enabling the organism to colonize the gastric epithelium, but not as a main property of H. pylori directly causing disease. Undoubtedly, hydrolysis of urea with generation of ammonia maintains the necessary microenvironment for the acid-sensitive organism in the gastric
E
_ _ =:=====:::======:::::::control lOB CFU/ml Omeprazol Bmg/l 10B CFU/mi Omeprazol 16 mgll 107 CFU/ml
Omeprazol 32 mg /I ~-----·104 CFU/ml
10 30
60
120
180
t/min
Fig. 1. Influence of omeprazole (8 mg/l, 16 mg/l, 32 mg/l) on urease activity of H. pylori, strain No. 7 Bacterial inocula are shown on the right side.
276
K. Vogt and H. Hahn
E 40
_ _-------------controI108CFU/ml Omeprazol 8mg/l 108 CFUlmi Omeprazol 16mg/ l 10 8 CFUlmi Omeprazol 32mg/l
30
106 CFU/ml
20
10
10 30
60
120
180 t/min
Fig. 2. Influence of omeprazole on urease activity of H. pylori, strain No. 25. Bacterial inocula (shown on the right) are only slighly reduced, whereas urease reaction is delayed at 32 mg/I omeprazole.
E
-::::::::=====:::====:::::cont rol 108 CFU/I11I Omeprazol8mg ll 1Q8 CFU/ml
40
Omeprazol 16 mg 107CFU/mi
30
Omeprazol 32 mg/l -----~106CFU/ml
30
60
120
180
tlmin
Fig. 3. Influence of omeprazole on urease activity of H. pylori, strain No. 13. Bacterial inocula (shown on the right) are only slightly reduced. At 16 mg/I omeprazole, urease reaction is evidently delayed. At 32 mg/I, the half-time extinction is not reached at all.
Omeprazole and Urease of H. pylori
277
. mucosa. Furthermore, the ammonia generated by urea hydrolysis is present at site in high concentrations and may have a cytotoxic effect on gastric epithelial cells (12). The effect of omeprazole on H. pylori is still unclear. Suerbaum et al. (18) reported a direct antibacterial effect of the substance at concentration between 8 and 32 mg/l. It is questionable, however, whether these concentrations are reached within the gastric lumen and the mucosal layer where H. pylori is located. Stolte and Bethke (17) postulate that the reduced density of H. pylori is due to bacterial overgrowth following omeprazole-induced rising of gastric pH values. This would deny any direct antibacterial activity of omeprazole on the bacteria. However, the accumulation of omeprazole within the parietal cell during a four week-therapy may possibly lead to a saturation of all Na +K+ -ATPase sites followed by an active excretion into the gastric lumen or - as supposed by Nagaya et al. (13) - a transformation to other active compounds which have been analyzed by Iwahi et al. (8) and found to have antibacterial properties against H. pylori. Regardless of direct antibacterial action, our results demonstrate that omeprazole may have an influence on H. pylori urease. The fact that the diminution of the enzyme becomes obvious in strains which are relatively resistant against omeprazole may be due to a higher concentration of omeprazole necessary to inhibit bacterial urease. Concentrations of 16 mg or lower inhibit growth of susceptible strains, so the urease inhibition does not become apparent. The mechanism by which omeprazol might lead to inhibition of the urease enzyme is unclear. It might be assumed that omeprazole as an inhibitor of the proton pump activity may also have an effect on the uptake of hydrogen ions into the cell. These, however, are necessary for the urea hydrolysis so that a reduction might lengthen the reaction time. For clinical settings, the possible influence of omeprazole on H. pylori urease must be taken into account for the rapid urease test and the urea breath test. Both tests should not be performed during omeprazole therapy or immediately after cessation as they might lead to a falsely negative result. As shown by Wagner et al. (20), a monotherapy with omeprazole is unable to eradicate H. pylori. The combination with one antibiotic is also ineffective (9), so that at least triple therapeutic regimens seem to be necessary (5). In such a combination, the use of omeprazole could be beneficial in three aspects: Firstly, the rise of gastric pH enables several antibiotics to reach full antibacterial activity. Secondly, the antibacterial activity of the substance itself may contribute to eradication. Thirdly, a possible influence on bacterial urease in some strains may at least make recolonization more difficult.
Acknowledgement. We thank Petra Wiedersatz for skilful technical assistance.
References 1. Christensen, W. B.: Urea decomposition as a means of differentiating Proteus and Paracolon cultures from each other and from Salmonella and Shigella types. J. Bact. 52 (1946) 461-466 2. Das, S. S., L. A. Bain, Q. N. Karim, L. G. Coelho, and]. H. Baron: Rapid diagnosis of Campylobacter pyloridis infection. J. Clin. Path. 40 (1987) 701-702
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