Omeprazole Therapy for Helicobacter p y Zori Infection d

P. SHERMAN, B. SHAMES, V. LOO, A. MATLOW, B. DRUMM & J . PENNER mivision1 of Gastroenterolog3nd Microbiology, Research Institute, Theaospital for Sick Children2 and Depts. of Pediatrics and Microbiology, University of Toronto,@onto, C a n a a and Dept. of Pediatrics, Our Lady’s Hospital for Sick Children (Crumlin), Dublin, Ireland Sherman P, Shames B, Loo V, Matlow A, Drumm B, Penner J . Omeprazole therapy for Helicohacter pvlori infection. Scand J Gastroenterol 1992;27:1018-1022. determine whether omeprazole eradicates Helicobacter pylori infection of the gastric antrum, six adolescents and one adult with H . pylori colonization of the antrum were entered into a clinical, open trial of medical therapy. Histologic evidence of antral gastritis and three complementary methods to document H. pylori colonization of the stomach (silver stain, urease testing, and culture of antrum) were obtained before and after an 8-week course of omeprazole. In vitro susceptibility to omeprazole and restriction endonuclease analysis were performed on H. pylori isolates obtained from patients before and after omeprazole therapy. Each of the seven patients treated with omeprazole had continued active inflammation in the antrum and one or more features indicative of persisting H. pylori colonization. Minimum inhibitory concentrations and DNA fingerprints of H. pylori isolated after therapy were identical to those of the pre-treatment bacterial isolates in each of the four subjects examined. We conclude that omeprazole therapy alone did not eradicate H . pylori infection of the human antrum. Continued bacterial colonization was not related to either acquired bacterial resistance to the drug or reinfection of the stomach with a different H . pylori strain

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_J Key words: Gastritis; Helicobucter pylori; minimum inhibitory concentrations; omeprazole; restriction endonucleases; ulcers

Philip Sherman, M.D., FRCPC, Division of Gasrroenrerology, The Hospital for Sick Children, 555 University Avenue, Toronto, Onrario, Canada M5G 1.H

Gram negative spiral-shaped bacteria, currently referred to as Helicobacter pylori ( l ) , are associated with chronic-active gastritis, duodenal ulcer disease, and (to a lesser extent) gastric ulcers ( 2 , 3). Supporting evidence for an etiologic role of the organism in the pathogenesis of chronic-active gastritis is provided by studies indicating specificity of the infection for primary gastritis (4-6), improvement in the severity of gastritis with clearing of the infection ( 7 ) , case reports of infection of human volunteers (8,9), and animal models of human disease (10). The etiopathogenetic role of the organism in peptic ulcer disease is less well established (2, 3). The major evidence supporting H . pylori infection as a factor in duodenal ulceration is a series of studies demonstrating a reduction in the relapse rate for duodenal ulcers when therapy is directed towards eradicating H . pylori colonization of the proximal gastrointestinal tract (11 , 12). However, these treatment protocols involve multiple therapeutic agents with accompanying concerns about side effects, patient compliance, and costs (13). A report by Mainguet et al. (14) indicated that the substituted benzimidazole omeprazole is effective therapy for both ulcer healing and clearing of H . pylori colonization of the antrum. Subsequently, reports have appeared which support (15, 16) and refute (17, 18) this initial observation. We therefore placed seven patients with H . pylori infection in an open trial of omeprazole for 8 weeks. Repeat endoscopy and mucosal biopsies, performed at least 4 weeks after

completing omeprazole therapy, were then undertaken to evaluate both healing of gastritis and eradication of H . pylori. When infection persisted after treatment, the in vitro susceptibility of H . pylori to omeprazole was tested to determine whether acquired microbial resistance t o the therapeutic agent might account for treatment failures. Restriction endonuclease analysis of D N A extracted from H . pylori strains isolated before and after a course of omeprazole therapy was undertaken t o determine whether reinfection with another H . pylori strain, rather than a failure of treatment, accounted for persisting bacterial infection in the antrum.


Pa tients Seven patients (six children and one adult) with symptoms of dyspepsia underwent upper endoscopy plus mucosal biopsies at this hospital. Each of the patients was treated with 20 mg ( n = 3) to 40 mg ( n = 4) of omeprazole once daily for 8 weeks. No other medications were administered during this period. Four weeks ( n = 6) to 12 weeks ( n = 1) after completing the medical therapy, follow-up endoscopy and mucosal biopsies were undertaken for clinical indications. Evaluation of gastritis and silver stain, culture, and urease activity of antral biopsy specimens for evidence of H .

Omeprazole Therapy of H. pylori Infection

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pylori colonization was performed, as described previously (4-7). Restriction enzyme analysis DNA fingerprinting was performed on H . pylori isolates using methods described previously (19, 20). In brief, H . pylori strains were grown on blood agar plates supplemented with 5% horse serum for 3 days at 37°C under microaerobic conditions. Bacteria were pelleted at 8700 g for 10 min, washed twice in sterile phosphate-buffered saline (PBS) and twice in Tris-ethylenediaminetetraacetic acid (EDTA) (pH8.5), lysed in lysozyme (3 mg/ml) and detergent (1% sodium dodecyl sulfate) at 37°C for 10 min, and treated with RNAase A (0.05 mg/ml, Boehringer Mannheim Biochemicals, Indianapolis, Ind., USA) for 1h, followed by proteinase K (0.5 mg/ml; Boehringer Mannheim) and pronase (0.8 mg/ml) overnight at 37°C. The DNA was extracted with phenol/chloroform (1: 1vol/vol), precipitated overnight in sodium acetate (0.3 M) and 100% ethanol (2:l vol), and pelleted by centrifugation at 12,000g for 60min at 4°C. DNA was resuspended in sterile distilled water and quantitated spectrophotometrically using ultraviolet light at a wavelength of 260 nm (21). To exclude significant protein contamination, the DNA samples were also examined by ultraviolet spectroscopy at 280 nm (21). Approximately 20 pg of H . pylori DNA was digested to completion with each of the restriction enzymes Hae 111, Hind 111, and Pvu I1 (Boehringer Mannheim) in excess for 4 h at 37°C. DNA digests were separated by horizontal agarose (0.8%) gel electrophoresis for 16h at 30V and then stained with ethidium bromide (1 pg/ml). Reference 1 kilobase molecular weight standards (Bethesda Research Laboratories, Gaithersburg, Md., USA) were run in parallel with each of the DNA digests.

In vitro susceptibilities Minimum inhibitory concentrations (MIC) for omeprazole against H . pylori isolates were determined by the agar dilution method (22). Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), and Escherichia coli (ATCC 25922, American Type Culture Collection, Rockville, Md., USA) were used for comparisons. Bacteria were grown overnight at 37°C under microaerobic conditions on horse blood agar plates (Columbia agar base, Quelab, Montreal, Quebec) with 5% defibrinated horse blood (Woodlyn Lab., Guelph, Ontario). Bacteria were swabbed and suspended in Mueller-Hinton broth (Oxoid Ltd., Hampshire, England) to a 1.0 McFarland barium sulfate standard. After a 1:lO dilution in sterile PBS, 104CFU in 0.02ml were spotted onto Mueller-Hinton agar plates (Oxoid) supplemented with 5% horse blood and omeprazole (Astra Pharmaceuticals, Mississauga, Qntario) in concentrations between 8mg/l and 1024mg/l. Plates were then incubated at 37°C under microaerophilic conditions for both 48 h and 72 h. Assays were performed in duplicate on two


Table I. In vitro susceptibility of bacteria to omeprazole, tested by the agar dilution method (22), both before and after an 8-week course of omeprazole therapy Bacteria


MIC (mg/l)

Staphylococcus aureus no. 29213 Enterococcus faecalis no. 29212 Escherichia coli no. 25922 Helicobacter pylori


>lo24 >lo24 >lo24

Patient 4 Patient 4 t Patient 5 Patient 5 t Patient 6 Patient 6 Patient 6t Patient 7 Patient 7 t

32,64 32 32 32 32 32 32 32 32

LC 17 LC 19 LC 24 LC 29 LC 16 LC 25 LC 28 LC 9 LC 26

* Bacterial strains obtained from the American Type Culture Collection, Rockville, Md., USA. t H . pylori isolates cultured from the antrum after the patient had received an 8-week course of therapy with omeprazole.

separate occasions, and all inocula verified by viability counts. RESULTS

Patients Seven patients colonized with H. pylori, ranging in age from 11 to 32 years, were placed on an 8-week course of omeprazole. The patients included four male and two female adolescents who were otherwise in good health and one adult male with an underlying diagnosis of cystic fibrosis. A positive history of duodenal ulcer disease in a first-degree relative was elicited in three of the seven patients. Six of the seven subjects were evaluated for symptoms of episodic and nocturnal epigastric pain consistent with dyspepsia. Patient 1 was evaluated because of two episodes of massive upper gastrointestinal hemorrhage which had occurred over a period of 2 years. Four of seven patients had previously received at least one course of medical therapy with the H2 receptor antagonist ranitidine. Fasting serum gastrin levels were measured in three of seven subjects. In each case levels were below 100ng/ml. None of the subjects had received either oral corticosteroids or nonsteroidal anti-inflammatory agents. Five of the seven subjects had endoscopic evidence of a duodenal ulcer. Antral biopsy specimens demonstrated a chronic-active gastritis in each subject. Six of seven patients had positive silver stain, positive urease test, and positive culture of antral mucosa as confirmatory evidence of H . pylori colonization of the antrum. One patient had histologic evidence of chronic-active gastritis, and staining with silver demonstrated the presence of Helicobacter-like organisms. However, culture of the antrum was negative. In each of the seven patients the severity of antral gastritis was not affected by omeprazole therapy. Silver stain, urease testing, and

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P. Sherman et al.

Fig. 1. Agarose gel electrophoresis of genomic D N A extracted from Helicohacter pylori isolates and cut with the restriction enzyme Hind-111. H. pylori isolates from Patient 4 before (lane 1) and after (lane 2) treatment with omeprazole, Patient 5 before (lane 3) and after (lane 4) omeprazole therapy, Patient 6 6 months (lane 5) and I month (lane 6) before therapy and 1 month after completing 8 weeks of omeprazole treatment (lane 7), and Patient 7 before (lane 8) and after (lane 9) treatment with omeprazole. Isolates from each of the four patients show distinct restriction enzyme profiles. However, the D N A fingerprints for H. pylori isolated from each patient were indistinguishable despite an 8-week course of treatment with omeprazole. Lane 10 contains a 1-kb D N A ladder (Bethesda Research Lab., Bethesda, Md., USA) with sizes between 0.516 and 12.216 kb pairs as a reference standard.

culture confirmed continued infection of the antrum by H. pylori.

In vitro susceptibilities None of the three reference bacteria were inhibited by omeprazole at concentrations u p to 1024 mg/l (Table I). In contrast, each of the H. pylori strains tested demonstrated inhibition of growth in the presence of omeprazole. As shown in Table I, the MIC was between 32mg/l and 64 mg/l. However, the MIC was not affected by prior treatment of patients with omeprazole. Therefore, resistance of the organisms to omeprazole did not develop after chronic in vivo exposure to the drug. Results identical to those shown

Fig. 2. Restriction endonuclease analysis of Helicohacfer pylori genomic D N A using Hae-111. The contents of each lane are identical to those described in Fig. 1. Profiles for H . pylori D N A from each of the patients differ, but the D N A fingerprints for D N A isolated from H . pylori before and after treatment were virtually identical. Genomic D N A from the three H . pylori isolates from Patient 6 were not cut by this restriction enzymc (lanes 5 , 6, and 7).

in Table I were obtained when the in vitro incubation period was extended from 48 h to 72 h. Restriction endonuclease analysis H. pylori was successfully cultured from the antrum in five of the seven patients both before and after omeprazole therapy. When undigested D N A was subjected to electrophoresis in agarose gels, there was no evidence of plasmid D N A in any of the eight isolates (data not shown). As shown in Fig. 1, D N A extracted from H . pylori isolates and cut with the restriction endonuclease Hind-I11 demonstrated unique fingerprints for each of the four subjects examined in this study. However, sequential H . pylori isolates from each individual showed identical digest patterns. These findings were confirmed when two additional restriction endonucleases, Hae-I11 (Fig. 2 ) and Pvu I1 (Fig. 3). were utilized for D N A fingerprinting. These results confirm that the H . pylori strains isolated before and after omeprazole therapy were identical.

Omeprazole Therapy of H . pylori Infection

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DISCUSSION The results Of this study demonstrate that omeprazole has antibacterial activity in vitro. A standard antimicrobial sensitivity assay for microorganisms was used (22). In addition, reference bacterial strains were used in the assays for comparisons. Antimicrobial activity has been reported recently (23,24), even though a previous report indicated that omeprazole does not inhibit growth of H. pylori in vitro (25). However, in vitro antimicrobial activity does not necessarily indicate that the agent in question will demonstrate an effect in vivo. Previous studies have shown susceptibility of H . pylori strains t o multiple antimicrobials in vitro but a lack of benefit after treatment of infected human subjects (26). The drug level achieved in the local gastric microenvironment may be an important factor accounting for the discrepancy between in vitro and in vivo findings (27,28). The possible beneficial effect of omeprazole in eradicating H . pylori infection has been controversial, with studies reporting both beneficial effects (14-16) and, as in this study, no positive effect (17, 18). The reason for the discordant findings is uncertain. In this study we have shown that acquired resistance of H . pylori to omeprazole does not


2 3 4 5 6 7 8 9 10

Fig, 3 . pvu-11 digests of He[jcobacter pylori isolates, Each lane contains samples as described in Fig. 1. Restriction endonucleasc profiles are virtually the same for genomic DNA extracted from H . pylori cultured from the antrum before and after an 8-week course of omeprazole.


account for persisting bacterial infection in vivo. This possibility was considered because previous reports showed acquired resistance to antimicrobials such as metronidazole ,qn,


A further possible explanation for persisting H. pylori infection is that omeprazole did eradicate the organism but the patients became reinfected from an, as yet unidentified, environmental source. The time interval to follow-up endoscopy was at least 4 weeks after completing a full 8-week course of omeprazole therapy. Some commentators feel that this time frame confirms eradication, rather than suppression or temporary clearing, of H. pylori colonization of the antrum (30). The problem with this time interval before reevaluation is that it is possible that continued infection could represent reinfection of a susceptible host by exposure to H . pylori either from infected family members or an environmental reservoir (4). Restriction endonuclease analysis of H . pylori genomic DNA was used to resolve these possibilities, as we have previously shown that D N A fingerprints differ between patients, whereas those obtained from a single individual over time without therapy remain constant (19). The restriction digests shown in this study clearly indicate that the same H . p y f o r i strains were isolated from the antrum of patients before and after omeprazole theranv. It is possible that the antral infection was temporarily clearcd by omeprazole and that, in the time interval before reassessment, the stomach became reinfected with the identical ff. pylori strain. Dental plaque has been shown as u reservoir for H . pylori with identical DNA profile t o that identified in the antrum of the same patient (20). Therefore, it might be postulated that omeprazole cleared H . pvlori infection in the stomach but that it had no effect on bacteria residing in the oral cavitv. Once omeprazole was withdrawn, swallowed organisms might have recolonized susceptible gahtric mucosa. Omeprazole may yet prove t o have a role in the treatment of H . pylori infections because it is effective in healing associated duodenal ulceration (31). In addition, omeprazole could affect gastric microflora ( 3 2 ) atid gastric acidity (31) sufficiently to promote the bactericidal action of antimicrobial agents. Raising the p H of the lunienal contents of the stomach might promote an increase in the delivery of acid-sensitive antibiotics to the gastric niucus and surface epithzlial cells that serve as the habitat for H. pylori colonization of the stomach (2,3). In summary, the results of this study indicate that onicprazole, as single therapy for H . pylori infection in the antrum, is not an effective therapeutic strategy. H . pylori infection is most likely the result of persisting infection and not related to either reinfection by other bactcrial strains or acquired resistance to the therapeutic agent. Future studies will examine the merits of omeprazole therapy provided in conjunction with antibiotics such as ampicillin and metronidazole. 1 ,


P. Sherman et a1

ACKNOWLEDGEMENTS P. Sherman is the recipient of a Career Scientist Award from the Ontario Ministry of Health. T h e authors thank Ms. Margaret Roscoe for excellent advice and technical assistance.


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1. Goodwin CS, Armstrong JA, Chilvers T, et al. Transfer of

Campylobacter pylori and Campylobacter mustelae to Helicobacter gen. nov. as Helicobacter pylori comb. nov. and Helicobacter mustelae comb. nov.. respectively. Int J Syst Bacteriol 1989;39:397-405. 2. Axon AR. Duodenal ulcer: the villain unmasked? Br Med J 1991;3O2:9 19-2 1. 3. Drumm B. Helicobacter pylori. Arch Dis Childh 1990;65:127882. 4. Drumm B, Perez-Perez GI, Blaser MJ, Sherman P. Intrafamilial clustering of Helicobacter pylori infection. N Engl J Med 1990; 322~359-63. 5. Drumm B, Sherman P, Cutz E, Karmali M. Association of Campylobacter pylori on the gastric mucosa with antral gastritis in children. N Engl J Med 1987;316:1557-61. 6 . Drumm B , O'Brien A, Cutz E, Sherman P. Campylobacter pyloridis-associated primary gastritis in children. Pediatrics 1987;XO:192-5. 7. Drumm B, Sherman P, Chiasson D, Karmali M, Cutz E. Treatment of Cumpylobacter pylori-associated antral gastritis in children with bismuth subsalicylate and ampicillin. J Pediatr 1988;113:Y8(b 12. 8. Marshall BJ, Armstrong JA, McGechie DB, Glancy RJ. Attempt to fulfill Koch's postulates for pyloric campylobacter. Med J Austr 1985;142:436-9. 9. Morris A, Nicholson G. Ingestion of Campylobacter pyloridis causes gastritis and raised fasting gastric pH.Arn J Gastroenterol I987 $2: 192-9. 10. Fox JG, Lee A. Gastric campylobacter-like organisms: their role in gastric disease in laboratory animals. Lab Anim Sci 1989;39:543-53. 11. Rauws EAJ, Tytgat GNJ. Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet 1990;335:12335. 12. George LL, Borody TJ, Andrews P, et al. Cure of duodenal ulcer after eradication of Helicobacter pylori. Med J Austr 1990;153 : 145-9. 13. Elta GH. Duodenal ulcer disease-to heal or to cure? [selected summary]. Gastroenterology 1991;100:573-5. 14. Mainguet P, Delmee M, Debongnie JC. Omeprazole, Campylobacter pylori, and duodenal ulcer [letter]. Lancet 1989; 2 :389-90. 15. Bisco G , Miglioli M, Barbara L, Corinaldesi R, DiFebo G . Received 15 April 1992 Accepted 9 July 1992

Omeprazolc, Helicobacter pylori, gastritis and duodenal ulcer (letter]. Lancet 1989;2:1403. 16. Hui WM, Lam SK, Ho J, et al. Effect of omeprazole on duodenal ulcer-associated antral gastritis and Helicobacter pylori. Dig Dis Sci 1991;36:577-82. 17. Unge P, Gad A , Gnarpe H, Olsson J. Does omeprazole improve antimicrobial therapy directed towards gastric Campylobacter pylori in patients with antral gastritis? Scand J Gastroenterol 1989;24 SUPPI 167:49-54. 18. Rauws EAJ, Langenberg W, Bosma A, Dankert J, Tytgat GNJ. Lack of eradication of Helicobacter pylori after omeprazole [letter]. Lancet 1991;337:1093. 19. Simor AE, Shames B, Drumm 8, Sherman P, Low D, Penner J . Typing of Campylobacter pylori by bacterial DNA restriction endonuclease analysis and determination of plasmid profile. J Clin Microbiol 1990;28:8.3-6. 20. Shames B, Krajden S , Fuksa M, Babida C, Penner JL. Evidence for occurrence of the same strain of Campylobacter pylori in the stomach and dental plaque. J Clin Microbiol 1989;27:2849-50. 21. Sambrook J , Fritsch EF, Maniatis T. Molecular cloning. A laboratory manual. 2nd ed. Cold Spring Harbor: Laboratory Press, 1989. 22. Sahm DF, Washington JA. Antibacterial susceptibility tests: dilution methods. In: Balows A, Hausler WJ, Herrmann KL, lsenberg HD, Shadomy HJ, editors. Manual of clinical microbiology. 5th ed. Washington: ASM Press, 1991:1105-16. 23. Iwahi T, Satoh H, Nakao M, et al. Lansoprazole, a novel benzimidazole proton pump inhibitor, and its related compounds have selective activity against Helicobucter pylori. Antirnicrob Agents Chemother 1991;35:49&6. 24. Megraud F, Boyanova L, Lamouliatte H. Activity of lansoprazole against Helicobacter pylori (letter]. Lancet 1991; 337: 1486. 25. Ghelani AM, Hale S , Coleman H, Radziwonik H, Robertson C, Atkinson M. Lack of in vitro activity of omeprazole against Cumpylobacter pylori [letter]. J Clin Pathol 1990;43:171-2. 26. McNulty CAM, Gearty JC, Crump B, et al. Campylobacter pyloridis and associated gastritis: investigator blind, placebo controlled trial of bismuth salicylate and erythromycin ethylsuccinate. Br Med J 1986;293:645-9. 27. Westblom T U , Duriex DE. Pharmacokinetics of cefuroxime and ciprofloxacin in gastric mucosa: comparison to in vitro inhibitory concentrations against Helicobacter pylori. Microbiologica 1991 ;14:37-43. 28. McNulty CAM, Dent JC, Ford GA, Wilkinson SP. Inhibitory antimicrobial concentrations against Carnpylobacfer pylori in gastric mucosa. J Antimicrob Chemother 1988;22:729-38. 29. Glupczynski Y, Burette A, DeKoster E, et al. Metronidazole resistance in Helicobacter pylori [letter]. Lancet 1990;1:9767. 30. Bell GD. Anti-Helicobacfer pylori therapy: clearance, elimination, or eradication? [letter]. Lancet 1991;337:310-1. 31. Maton PN. Omeprazole. N Engl J Med 1991;324:%575. 32. Sharma BK, Santana IA, Wood EC, et al. Intragastric bacterial activity and nitrosation before, during, and after treatment with omeprazole. Br Med J 1984;289:717-9.

Omeprazole therapy for Helicobacter pylori infection.

To determine whether omeprazole eradicates Helicobacter pylori infection of the gastric antrum, six adolescents and one adult with H. pylori colonizat...
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