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or bismuth against clarithromycin-susceptible and clarithromycin-resistant strains of Helicobacter pylori
ANTIMICROBIAL SUSCEPTIBILITY STUDIES
The Bactericidal Activity of Clarithromycin Versus Ampicillin Alone and in Combination with Omeprazole and/or Bismuth Against Clarithromycin-susceptible and Clarithromycin-resistant Strains of Helicobacter pylori Philip E. Coudron and Charles W. Stratton
We evaluated the in vitro bactericidal effect of clarithromycin versus ampicillin alone and in combination against clarithromycin-sensitive and clarithromycin-resistant strains of Helicobacter pylori. No combination containing clarithromycin achieved complete bactericidal effect against clarithromycin-resistant strains. Complete bactericidal effect was achieved for all strains by triple-agent combinations that
contained bismuth, omeprazole, and relatively high concentrations of ampicillin. These in vitro results demonstrate the additive bactericidal activity provided by a combination of agents for the eradication of H. pylori. Bismuth may play a particularly important role in the bactericidal effect. © 1998 Elsevier Science Inc.
INTRODUCTION
1993; Labenz and Borsch 1994). Eradication of this organism from the stomach of patients with peptic ulcer disease substantially reduces the incidence of ulcer recurrence within one year from more than 90% to less than 10% (Forbes et al. 1994; Graham et al. 1992; Sung et al. 1995). Accordingly, an N.I.H. Consensus Development Panel (Feldman 1994; NIH Consensus Development Panel 1994) has recommended that ulcer patients with H. pylori infection be treated with antimicrobial agents in addition to antisecretory agents on first presentation or recurrence of the illness. Despite over a decade of in vitro susceptibility testing and clinical therapeutic trials directed at determining the optimal regimen for the complete eradication of H. pylori from the human gastrointestinal tract, this optimal therapy has yet to be defined (De Boer and Tytgat 1995; Graham 1995; Marshall 1993; Unge 1996; Van der Hulst et al. 1996). Triple
Although the exact details of the pathogenesis of Helicobacter pylori in peptic ulcer disease remains unclear (Blazer 1992; DeCross and Marshall 1993; Labenz and Borsch 1994), a causal relationship has now been well established (Blazer 1992; Chiba et al. 1992; DeCross and Marshall 1993; Fennerty 1994; Graham From the Pathology and Laboratory Medicine Service/113, McGuire Veterans Affairs Medical Center, Richmond, Virginia, and the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee. Address reprint requests to Philip E. Coudron, Ph.D., Pathology and Laboratory Medicine Service/113, McGuire Veterans Affairs Medical Center, 1201 Broad Rock Blvd., Richmond, Virginia 23249-0001, USA. These data were presented in part at The American Gastroenterological Association and Digestive Disease Week, Washington, D.C., May 11–14, 1997. Received 17 December 1997; revised and accepted 5 April 1998
DIAGN MICROBIOL INFECT DIS 1998;31:39 –44 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
0732-8893/98/$19.00 PII S0732-8893(98)00054-6
P.E. Coudron and C.W. Stratton
40 therapy (bismuth, metronidazole, tetracycline) has been considered the gold standard for many years (NIH Consensus Development Panel 1994; Van der Hulst et al. 1996). Clarithromycin is a relatively new semisynthetic analog of erythromycin with a single C-6 methoxy substitution. It is the most effective single agent for eradication of H. pylori, and achieved a 54% eradication rate after 14 days of therapy (Peterson et al. 1993). Omeprazole is an antisecretory drug that inhibits the H1/K1-ATPase enzyme in human parietal cells (Shamburek and Schubert 1992) and also exerts antimicrobial activity against H. pylori (Iwahi et al. 1991). It is often included in H. pylori eradication regiments and with two or three other agents has achieved eradication rates up to 95% (Bazzoli et al. 1994; Borody et al. 1995; De Boer et al. 1995; Labenz et al. 1995; Van der Hulst et al. 1996). We have previously demonstrated the usefulness of bactericidal methods for in vitro susceptibility testing of H. pylori using antimicrobial agents alone and in combination (Coudron and Stratton 1995a, 1995c). In this study, we selected two new antimicrobial agents, clarithromycin and omeprazole, as well as two older agents, ampicillin and bismuth, all of which are used in therapeutic regimens for H. pylori infections (Axon 1994; Chiba et al. 1992; Van der Hulst et al. 1996). The bactericidal effects of these agents were assessed alone and in various combinations against clarithromycin-susceptible and clarithromycin-resistant strains of H. pylori.
MATERIALS AND METHODS
gram of antrum tissue) (Lambert 1991). We tested for complete bactericidal effect (i.e., complete eradication in vitro) of combination agents by withdrawing a relatively large sample volume (1 mL) and inoculating it directly onto agar medium. Agar may neutralize toxic byproducts that are present in the liquid milieu of spent medium (personal observation).
Micro-organisms For comprehensive testing, we studied three strains of H. pylori, the ATCC 43504 strain, which was tested previously (Coudron and Stratton 1995a, 1995b, 1995c) and two clinical isolates that were clarithromycin resistant, Hp 113-4 and Hp 110-2. The latter isolate was recovered from a patient who failed clarithromycin monotherapy. In addition, five clinical isolates that were clarithromycin sensitive were tested against triple combination agents only, using relatively high, although tissue achievable, concentrations of ampicillin or clarithromycin.
Antimicrobial Agents The antimicrobial agents and concentrations (mg/ mL) tested included ampicillin (0.008, 0.016, 0.031, 0.125, and 0.25; Sigma Chemical Company, St. Louis, MO, USA), clarithromycin (0.016, 0.031, 0.25, 0.50, 1.0, and 2.0; Abbott Laboratories, Abbott Park, IL, USA), omeprazole (8, 16, 32, and 64; (Astra Hassle AB, Molndal, Sweden), and colloidal bismuth subcitrate (8, and 16; Brocades Pharma bv, Delft, The Netherlands).
Study Design
Inoculum Preparation
This study was designed to address several issues. Because of stability properties, it is recommended that in vitro susceptibility testing of omeprazole be performed in liquid media (P. Lorentzon (Astre Hassle, Sweden), personal communication). The concentrations of antimicrobial agents for in vitro susceptibility testing of combinations typically range from several dilutions below the MIC to twice the MIC of each drug. We initially determined the bactericidal activity of single agents against three strains. We then assessed the activity of combination agents by testing drugs at concentrations that generally yielded minimal changes in CFU/mL at 24 h as single agents. We also tested drugs alone and in combination at levels equal to or less than known or presumably achievable tissue concentrations. The concentration of antimicrobial agents in the antrum has been documented for ampicillin (0.04 to 1.71 mg per milliliter of antral homogenate) (Cooreman at al. 1993), clarithromycin (5 to 20 mg per gram of antrum tissue) (Gustavson et al. 1995), and bismuth (8 to 375 mg per
Inocula for MIC testing and bactericidal studies were prepared where organisms were initially grown overnight at 37°C in 10% CO2 in cation-adjusted Mueller-Hinton broth containing 10% fetal calf serum. Samples from these overnight broth cultures were inoculated onto agar medium (Coudron and Kirby 1989) and plates were incubated overnight at 37°C under microaerobic conditions. Final cell suspensions were prepared from agar plates and contained motile cells with typical curved morphology when viewed by phase microscopy.
MIC Determinations MICs were performed by a modification of standard agar procedures (NCCLS 1997) as previously described (Coudron and Stratton 1995a). Agar plates contained Mueller-Hinton agar and 0.5% starch and either clarithromycin or ampicillin. The inocula per spot were 105 CFU. MICs were read after 3 to 5 days of incubation at 37°C under microaerobic conditions.
Clarithromycin versus Ampicillin Activity
41
As reported previously (Coudron and Stratton 1995c), bismuth MICs are difficult to read using this method. Moreover, omeprazole is unstable in agar medium. Therefore, MICs were not performed by agar dilution methods for these two agents.
RESULTS MIC Results The agar dilution susceptibility results showed that the ATCC strain was susceptible to clarithromycin (MIC, 0.016 mg/mL) and ampicillin (MIC, 0.06 mg/ mL). In contrast, Hp 110-2 and Hp 113-4 isolates were resistant to clarithromycin (MICs, 8.0 mg/mL) and susceptible to ampicillin (MICs, 0.13 mg/mL). The five other clinical isolates tested were susceptible to both clarithromycin (MICs #0.25 mg/mL) and ampicillin (MICs #0.13 mg/mL).
Bactericidal Studies Bactericidal studies were modified from methods previously described (Coudron and Stratton 1995c). These studies were performed in cation-adjusted Mueller-Hinton broth containing 0.5% starch, which has been shown to support the growth of H. pylori without suppressing the activity of antimicrobial agents (Coudron and Stratton 1995b). The inoculation of media-containing sealed bottles was done with a syringe and yielded approximately 5 3 106 CFU/mL. Following inoculation, the bottles were flushed with a microaerophilic mixture of gases (oxygen, carbon dioxide, and nitrogen) and shaken continuously at 150 rpm at 37°C. Samples of H. pylori were removed at 24 h, serially diluted, and plated (20 mL onto each of two plates) for colony counts. Undiluted samples of 200 and 1000 mL were inoculated directly onto agar medium. Antimicrobial carryover was addressed by adding penicillinase (for ampicillin) or EDTA (for bismuth salts) to the samples before dilution (Coudron and Stratton 1995c). After 4 to 6 days of incubation under microaerobic conditions at 37°C, plates were read. The change in the log10 CFUs per milliliter after 24-h incubation relative to the log10 CFUs per milliliter at time 0 was determined. Maximal killing was defined as less than 30 CFU recovered from a 1.0 mL-aliquot.
Results of Bactericidal Studies Results of the bactericidal studies are summarized in Tables 1 to 3. All bottles containing the lowest concentration of a single agent showed a minimal effect (slight increased or decreased growth) at 24 h except for one bottle containing Hp 113-4 with bismuth, which showed greater than one log decrease of CFU/mL. The data also indicate that clarithromycin appears to be more active than ampicillin for the ATCC strain (Table 1), and that both agents were bactericidal at a concentration of 0.25 mg/mL. At 2 mg/mL, however, clarithromycin had little effect on the clarithromycin-resistant isolates (Tables 2 and 3). At 0.25 mg/mL, amoxicillin exerted about a 3 log10 decrease in CFUs/mL at 24 h for the ATCC and Hp 110-2 strains, whereas bismuth salts alone required at least 16 mg/mL and omeprazole alone required 32 mg/mL or greater for a similar magnitude of killing. The Hp 113-4 isolate was generally more susceptible to bismuth and ampicillin than were the other two strains.
TABLE 1 Bactericidal Effects of Antimicrobial Agents Alone and In Combination Against a Clarithromycin-susceptible Strain of H. pylori (ATCC 43504)a Single Agents (mg/mL) 0b
Bi (8)
Bi (16)
Om (8)
Om (16)
Om (32)
Om (64)
Am (0.016)
Am (0.031)
Am (0.125)
Am (0.25)
Cl (0.016)
Cl (0.031)
Cl (0.25)
11.6
11.4
22.6
11.4
10.7
20.3
22.7
11.0
21.7
22.7
23.2
10.3
21.9
24.9
Dual Agents (mg/mL) Am (0.016) Bi (8)
Am (0.016) Om (8)
Cl (0.016) Bi (8)
Cl (0.016) Om (8)
Bi (8) Om (8)
11.4
10.1
22.2
10.1
23.1
Triple Agents (mg/mL) Am (0.016) Bi (8) Om (8)
Am (0.25) Bi (8) Om (8)
Cl (0.016) Bi (8) Om (8)
Cl (0.25) Bi (8) Om (8)
23.0
$25.0
$ 25.0
$ 25.0
a b
The values listed represent the change in log10 CFU/mL after 24-h incubation with agents relative to log10 CFU/mL at time zero. 0, no antibiotics; Bi, bismuth; Cl, clarithromycin; Om, omeprazole; Am, ampicillin.
P.E. Coudron and C.W. Stratton
42
TABLE 2 Bactericidal Effects of Antimicrobial Agents Alone and In Combination Against a Clarithromycin-resistant Clinical Strain of H. pylori (Hp 110-2)a Single Agents (mg/mL) 0b
Bi (8)
Bi (16)
Om (8)
Om (16)
Om (32)
Am (0.008)
Am (0.016)
Am (0.031)
Am (0.25)
Cl (0.5)
Cl (1.0)
Cl (2.0)
11.3
20.6
22.6
10.3
20.3
23.6
10.8
21.1
22.8
22.9
10.6
10.1
20.2
Dual Agents (mg/mL) Am (0.008) Bi (8)
Am (0.008) Om (8)
Cl (0.5) Bi (8)
Cl (0.5) Om (8)
Bi (8) Om (8)
10.4
20.6
23.9
20.2
22.5
Triple Agents (mg/mL) Am (0.008) Bi (8) Om (8)
Am (0.25) Bi (8) Om (8)
Cl (0.5) Bi (8) Om (8)
24.1
$ 25.4
24.9
a b
The values listed represent the change in log10 CFU/mL after 24-h incubation with agents relative to log10 CFU/mL at time zero. 0, no antibiotics; Bi, bismuth; Cl, clarithromycin; Om, omeprazole.
TABLE 3 Bactericidal Effects of Antimicrobial Agents Alone and In Combination Against a Clarithromycin-resistant Clinical Strain of H. pylori (Hp 113-4)a Single Agents (mg/mL) b
0
Bi (8)
Bi (16)
Om (8)
Am (0.008)
Am (0.016)
Cl (0.5)
Cl (1.0)
Cl (2.0)
10.3
21.5
$25.0
20.2
20.5
22.2
10.3
10.2
10.1
Dual Agents (mg/mL) Am (0.008) Bi (8)
Am (0.008) Om (8)
Cl (0.5) Bi (8)
Cl (0.5) Om (8)
Bi (8) Om (8)
22.8
20.2
21.8
0.0
22.6
Triple Agents (mg/mL) Am (0.008) Bi (8) Om (8)
Am (0.25) Bi (8) Om (8)
Cl (0.5) Bi (8) Om (8)
$25.0
$25.0
24.5
a b
The values listed represent the change in log10 CFU/mL after 24-h incubation with agents relative to log10 CFU/mL at time zero. 0, no antibiotics; Bi, bismuth; Cl, clarithromycin; Om, omeprazole.
All bottles with two agents contained viable organisms at 24 h. Bismuth and omeprazole achieved substantial bactericidal effect against all three strains, but surprisingly, bismuth and clarithromycin also achieved substantial killing of the clarithromycinresistant strain, Hp 110-2 (Table 2). Omeprazole showed some additive activity when combined with ampicillin for the ATCC and Hp 110-2 strains. Complete bactericidal effect against all three strains occurred with the combination of bismuth and omeprazole and at a relatively high concentration of ampicillin (Tables 1 to 3). At a relatively low concentration, clarithromycin in combination with bismuth and omeprazole also achieved complete bactericidal effect against the clarithromycinsusceptible strain (Table 1). However, clarithromycin
at a higher concentration in combination with bismuth and omeprazole failed to achieve complete bactericidal effect against both clarithromycinresistant strains (Tables 2 and 3). Complete bactericidal effect was achieved against the five other clinical isolates that were susceptible to clarithromycin, when tested against the combination agents, bismuth (8 mg/mL), omeprazole (8 mg/mL), and a relatively high concentration (0.25 mg/mL) of either ampicillin or clarithromycin.
DISCUSSION An advantage of in vitro susceptibility testing of H. pylori is the ability to focus solely on the effects of
Clarithromycin versus Ampicillin Activity antimicrobial agents in the absence of the influence of physiological factors, such as the acid milieu of the stomach, etc. Several results of this study are noteworthy. The minimal bactericidal concentrations of omeprazole, amoxicillin, and clarithromycin were reported previously for the H. pylori strain 43504 as follows: 64 mg/mL (Sjostrom et al. 1996), .0.1 mg/mL and .0.1 mg/mL (Sjostrom and Larsson 1996), respectively. These results are consistent with our time-kill results for the same strain, which indicate that at or near these concentrations, the corresponding single agents were bactericidal (Table 1). The results of testing triple-agent combinations against the clarithromycin-resistant strains of H. pylori (Tables 2 and 3) show that when clarithromycin was added to bismuth and omeprazole, an additional 2.4 and 1.9 log10 decrease in CFU/mL was achieved for Hp 110-2 and Hp 113-4, respectively, relative to that observed with only the latter two agents in combination. Although complete bactericidal eradication for these strains was not achieved, the data suggest that resistance to clarithromycin alone does not totally negate the bactericidal effect of combination agents. Clinical studies have also shown successful eradication (63%) of metronidazoleresistant strains by triple-agent regimens that contain metronidazole (Lerang et al. 1997; Rautelin et al. 1992). A paucity of data exists for in vitro susceptibility testing of omeprazole against H. pylori in liquid media (Sjostrom and Larsson 1996; Sjostrom et al. 1996). In an earlier report, omeprazole and one other agent, amoxicillin, were tested in combination against the 43504 strain by the broth microdilution method (Sjostrom and Larsson 1996). These agents were additive and not synergistic. Results from this report agree with data from this study shown in Table 1. We believe that bismuth is an important component of any therapeutic regimen seeking complete in
43 vivo eradication of H. pylori. Results of this study demonstrate that the complete bactericidal effect was achievable with triple-agent combinations containing bismuth. Recent work in our laboratory (Stratton and Coudron 1997) suggests that the mechanism of action for bismuth salts involves the disruption of the glycocalyx/cell wall of the H. pylori organism and is the result of a physiochemical interaction. The development of microbial resistance to polycationic agents has been described in vitro for mutant microorganisms that have diminished production of glycocalyx (Nicas and Hancock 1980). However, this resistance is rare and is not likely to become an important clinical problem because microorganisms that have diminished glycocalyx quality are generally poor pathogens with low virulence. This may explain why no H. pylori isolates with resistance to bismuth have been reported to date. In summary, a bactericidal effect against both clarithromycin-sensitive and clarithromycin-resistant strains of H. pylori was achieved with appropriate double or triple combinations of antimicrobial agents. The combination of bismuth, omeprazole, and relatively high concentrations of ampicillin achieved complete bactericidal effect against all strains tested. No combination containing clarithromycin achieved complete bactericidal effect against the clarithromycin-resistant strains. These in vitro results demonstrate the bactericidal effect provided by a combination of agents including omeprazole for the eradication of H. pylori. Bismuth appears to play a particularly important role in the bactericidal effect of multiple agents.
This work was supported in part by a grant from Abbott Laboratories, Abbott Park, IL.
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analysis of the efficacy of antibiotic therapy in eradicating Helicobacter pylori. Am J Gastroenterol 87:1716–1727. Cooreman MP, Krausgrill P, Hengels KJ (1993) Local gastric and serum amoxicillin concentrations after different oral application forms. Antimicrob Agents Chemother 37: 1506–1509. Coudron PE, Kirby DF (1989) Comparison of rapid urease tests, staining techniques, and growth on different solid media for detection of Campylobacter pylori. J Clin Microbiol 27:1527–1530. Coudron PE, Stratton CW (1995a) Utilization of time-kill kinetic methodologies for assessing the bactericidal activities of ampicillin and bismuth, alone and in combination, against Helicobacter pylori in stationary and log-
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arithmic growth phases. Antimicrob Agents Chemother 39:66–69. Coudron PE, Stratton CW (1995b) Factors affecting growth and susceptibility testing of Helicobacter pylori in liquid media. J Clin Microbiol 33:1028–1030. Coudron PE, Stratton CW (1995c) Use of time-kill methodology to assess antimicrobial combinations against metronidazole- susceptible and metronidazole-resistant strains of Helicobacter pylori. Antimicrob Agents Chemother 39:2641–2644. De Boer WA, Tytgat GNJ (1995) The best therapy for Helicobacter pylori infection: should efficacy or sideeffect profile determine our choice? Scand J Gastroenterol 30:401–407. De Boer W, Driessen W, Jansz A, Tytgat G (1995) Effect of acid suppression on efficacy of treatment for Helicobacter pylori infection. Lancet 345:817–820. DeCross AJ, Marshall BJ (1993) The role of Helicobacter pylori in acid-peptic disease. Am J Med Sci 306:381–392. Feldman M (1994) The acid test. Making clinical sense of the Consensus Conference on Helicobacter pylori. JAMA 272:70–71. Fennerty MB (1994) Helicobacter pylori Arch Intern Med 154: 721–727. Forbes GM, Glaser ME, Cullen DJE, Warren JR, Christiansen KJ, Marshall BJ, Collins BJ (1994) Duodenal ulcer treated with Helicobacter pylori eradication: seven year follow-up. Lancet 343:258–260. Graham DY (1993) Treatment of peptic ulcers caused by Helicobacter pylori. N Engl J Med 328:349–350. Graham DY (1995) A reliable cure for Helicobacter pylori infection? Gut 37:154–156. Graham DY, Lew GM, Klein PD, Evans DG, Evans DJ, Saeed ZA, Malaty HM (1992) Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. A randomized, controlled study. Ann Intern Med 116:705–708. Gustavson LE, Kaiser JF, Edmonds AL, Locke CS, DeBartolo ML, Schneck DW (1995) Effect of omeprazole on concentrations of clarithromycin in plasma and gastric tissue at steady state. Antimicrob Agents Chemother 39: 2078–2083. Iwahi T, Satoh H, Nakao M, Iwasaki T, Yamazaki T, Kubo K, Tamura T, Imada A (1991) Lansoprazole, a novel benzimidazole proton pump inhibitor, and its related compounds have selective activity against Helicobacter pylori. Antimicrob Agents Chemother 35:490–496. Labenz J, Borsch G (1994) Evidence for the essential role of Helicobacter pylori in gastric ulcer disease. Gut 35:19–22. Labenz J, Stolte M, Ruhl GH, Becker T, Tillenburg B, Sollbohmer M, Borsch G (1995) One-week, low-dose triple for the eradication of Helicobacter pylori infection. Eur J Gastroenterol Hepatol 7:9–11.
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Lambert JR (1991) Pharmacology of bismuth-containing compounds. Rev Infect Dis 13(suppl 8):S691–S695. Lerang F, Moum B, Haug JB, Tolas P, Breder O, Aubert E, Hoie O, Soberg T, Flaaten B, Farup P, Berge T (1997) Highly effective twice-daily triple therapies for Helicobacter pylori infection and peptic ulcer disease: does in vitro metronidazole resistance have any clinical relevance? Am J Gastroenterol 92:248–253. Marshall BJ (1993) Treatment strategies for Helicobacter pylori infection. Gastroenterol Clin North Am 22:183–198. National Committee for Clinical Laboratory Standards (NCCLS) (1997) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A4. 4th ed. Villanova, PA: NCCLS. Nicas TI, Hancock REW (1980) Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin. J Bacteriol 143:872– 878. NIH Consensus Development Panel (1994) Helicobacter pylori in peptic ulcer disease: JAMA 272:65–69. Peterson WL, Graham DY, Marshall B, Blaser MJ, Genta RM, Klein PD, Stratton CW, Drnec J, Prokocimer P, Siepman N (1993) Clarithromycin as monotherapy for eradication of Helicobacter pylori: a randomized, doubleblind trial. Am J Gastroenterol 88:1860–1864. Rautelin H, Seppala K, Renkonen OV, Vainio U, Kosunen TU (1992) Role of metronidazole resistance in therapy of Helicobacter pylori infections. Antimicrob Agents Chemother 36:163–166. Shamburek RD, Schubert ML (1992) Control of gastric acid secretion: histamine H2-receptor antagonist and H1K1ATPase inhibitors. Gastrointest Pharmacol 21:527–550. Sjostrom JE, Larsson H (1996) Factors affecting growth and antibiotic susceptibility of Helicobacter pylori: effect of pH and urea on the survival of a wild-type strain and a urease-deficient mutant. J Med Microbiol 44:425–433. Sjostrom JE, Fryklund J, Kuhler T, Larsson H (1996) In vitro antibacterial activity of omeprazole and its selectivity for Helicobacter spp. are dependent on incubation conditions. Antimicrob Agents Chemother 40:621–626. Stratton CW, Coudron PE (1997) Bismuth-mediated disruption of the glycocalyx/cell wall of Helicobacter pylori: evidence for a mechanism of action for bismuth salts. Gastroenterology 112(AGA abstr):A1098. Sung JJY, Chung SCS, Ling TKW, Yung MY, Leung VKS, Ng EKW, Li MKK, Cheng AFB, Li AKC (1995) Antibacterial treatment of gastric ulcers associated with Helicobacter pylori. N Engl J Med 332:139–142. Unge P (1996) Review of Helicobacter pylori eradication regimens. Scand J Gastroenterol 31(suppl 215):74–81. Van der Hulst RWM, Keller JJ, Rauws EAJ, Tytgat GNJ (1996) Treatment of Helicobacter pylori infection: a review of the world literature. Helicobacter 1:6–19.-
The Bactericidal Activity of Clarithromycin Versus Ampicillin Alone and in Combination with Omeprazole and/or Bismuth Against Clarithromycin-susceptible and Clarithromycin-resistant Strains of Helicobacter pylori Philip E. Coudron and Charles W. Stratton
We evaluated the in vitro bactericidal effect of clarithromycin versus ampicillin alone and in combination against clarithromycin-sensitive and clarithromycin-resistant strains of Helicobacter pylori. No combination containing clarithromycin achieved complete bactericidal effect against clarithromycin-resistant strains. Complete bactericidal effect was achieved for all strains by triple-agent combinations that
contained bismuth, omeprazole, and relatively high concentrations of ampicillin. These in vitro results demonstrate the additive bactericidal activity provided by a combination of agents for the eradication of H. pylori. Bismuth may play a particularly important role in the bactericidal effect. © 1998 Elsevier Science Inc.
INTRODUCTION
1993; Labenz and Borsch 1994). Eradication of this organism from the stomach of patients with peptic ulcer disease substantially reduces the incidence of ulcer recurrence within one year from more than 90% to less than 10% (Forbes et al. 1994; Graham et al. 1992; Sung et al. 1995). Accordingly, an N.I.H. Consensus Development Panel (Feldman 1994; NIH Consensus Development Panel 1994) has recommended that ulcer patients with H. pylori infection be treated with antimicrobial agents in addition to antisecretory agents on first presentation or recurrence of the illness. Despite over a decade of in vitro susceptibility testing and clinical therapeutic trials directed at determining the optimal regimen for the complete eradication of H. pylori from the human gastrointestinal tract, this optimal therapy has yet to be defined (De Boer and Tytgat 1995; Graham 1995; Marshall 1993; Unge 1996; Van der Hulst et al. 1996). Triple
Although the exact details of the pathogenesis of Helicobacter pylori in peptic ulcer disease remains unclear (Blazer 1992; DeCross and Marshall 1993; Labenz and Borsch 1994), a causal relationship has now been well established (Blazer 1992; Chiba et al. 1992; DeCross and Marshall 1993; Fennerty 1994; Graham From the Pathology and Laboratory Medicine Service/113, McGuire Veterans Affairs Medical Center, Richmond, Virginia, and the Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee. Address reprint requests to Philip E. Coudron, Ph.D., Pathology and Laboratory Medicine Service/113, McGuire Veterans Affairs Medical Center, 1201 Broad Rock Blvd., Richmond, Virginia 23249-0001, USA. These data were presented in part at The American Gastroenterological Association and Digestive Disease Week, Washington, D.C., May 11–14, 1997. Received 17 December 1997; revised and accepted 5 April 1998
DIAGN MICROBIOL INFECT DIS 1998;31:39 –44 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
0732-8893/98/$19.00 PII S0732-8893(98)00054-6
P.E. Coudron and C.W. Stratton
40 therapy (bismuth, metronidazole, tetracycline) has been considered the gold standard for many years (NIH Consensus Development Panel 1994; Van der Hulst et al. 1996). Clarithromycin is a relatively new semisynthetic analog of erythromycin with a single C-6 methoxy substitution. It is the most effective single agent for eradication of H. pylori, and achieved a 54% eradication rate after 14 days of therapy (Peterson et al. 1993). Omeprazole is an antisecretory drug that inhibits the H1/K1-ATPase enzyme in human parietal cells (Shamburek and Schubert 1992) and also exerts antimicrobial activity against H. pylori (Iwahi et al. 1991). It is often included in H. pylori eradication regiments and with two or three other agents has achieved eradication rates up to 95% (Bazzoli et al. 1994; Borody et al. 1995; De Boer et al. 1995; Labenz et al. 1995; Van der Hulst et al. 1996). We have previously demonstrated the usefulness of bactericidal methods for in vitro susceptibility testing of H. pylori using antimicrobial agents alone and in combination (Coudron and Stratton 1995a, 1995c). In this study, we selected two new antimicrobial agents, clarithromycin and omeprazole, as well as two older agents, ampicillin and bismuth, all of which are used in therapeutic regimens for H. pylori infections (Axon 1994; Chiba et al. 1992; Van der Hulst et al. 1996). The bactericidal effects of these agents were assessed alone and in various combinations against clarithromycin-susceptible and clarithromycin-resistant strains of H. pylori.
MATERIALS AND METHODS
gram of antrum tissue) (Lambert 1991). We tested for complete bactericidal effect (i.e., complete eradication in vitro) of combination agents by withdrawing a relatively large sample volume (1 mL) and inoculating it directly onto agar medium. Agar may neutralize toxic byproducts that are present in the liquid milieu of spent medium (personal observation).
Micro-organisms For comprehensive testing, we studied three strains of H. pylori, the ATCC 43504 strain, which was tested previously (Coudron and Stratton 1995a, 1995b, 1995c) and two clinical isolates that were clarithromycin resistant, Hp 113-4 and Hp 110-2. The latter isolate was recovered from a patient who failed clarithromycin monotherapy. In addition, five clinical isolates that were clarithromycin sensitive were tested against triple combination agents only, using relatively high, although tissue achievable, concentrations of ampicillin or clarithromycin.
Antimicrobial Agents The antimicrobial agents and concentrations (mg/ mL) tested included ampicillin (0.008, 0.016, 0.031, 0.125, and 0.25; Sigma Chemical Company, St. Louis, MO, USA), clarithromycin (0.016, 0.031, 0.25, 0.50, 1.0, and 2.0; Abbott Laboratories, Abbott Park, IL, USA), omeprazole (8, 16, 32, and 64; (Astra Hassle AB, Molndal, Sweden), and colloidal bismuth subcitrate (8, and 16; Brocades Pharma bv, Delft, The Netherlands).
Study Design
Inoculum Preparation
This study was designed to address several issues. Because of stability properties, it is recommended that in vitro susceptibility testing of omeprazole be performed in liquid media (P. Lorentzon (Astre Hassle, Sweden), personal communication). The concentrations of antimicrobial agents for in vitro susceptibility testing of combinations typically range from several dilutions below the MIC to twice the MIC of each drug. We initially determined the bactericidal activity of single agents against three strains. We then assessed the activity of combination agents by testing drugs at concentrations that generally yielded minimal changes in CFU/mL at 24 h as single agents. We also tested drugs alone and in combination at levels equal to or less than known or presumably achievable tissue concentrations. The concentration of antimicrobial agents in the antrum has been documented for ampicillin (0.04 to 1.71 mg per milliliter of antral homogenate) (Cooreman at al. 1993), clarithromycin (5 to 20 mg per gram of antrum tissue) (Gustavson et al. 1995), and bismuth (8 to 375 mg per
Inocula for MIC testing and bactericidal studies were prepared where organisms were initially grown overnight at 37°C in 10% CO2 in cation-adjusted Mueller-Hinton broth containing 10% fetal calf serum. Samples from these overnight broth cultures were inoculated onto agar medium (Coudron and Kirby 1989) and plates were incubated overnight at 37°C under microaerobic conditions. Final cell suspensions were prepared from agar plates and contained motile cells with typical curved morphology when viewed by phase microscopy.
MIC Determinations MICs were performed by a modification of standard agar procedures (NCCLS 1997) as previously described (Coudron and Stratton 1995a). Agar plates contained Mueller-Hinton agar and 0.5% starch and either clarithromycin or ampicillin. The inocula per spot were 105 CFU. MICs were read after 3 to 5 days of incubation at 37°C under microaerobic conditions.
Clarithromycin versus Ampicillin Activity
41
As reported previously (Coudron and Stratton 1995c), bismuth MICs are difficult to read using this method. Moreover, omeprazole is unstable in agar medium. Therefore, MICs were not performed by agar dilution methods for these two agents.
RESULTS MIC Results The agar dilution susceptibility results showed that the ATCC strain was susceptible to clarithromycin (MIC, 0.016 mg/mL) and ampicillin (MIC, 0.06 mg/ mL). In contrast, Hp 110-2 and Hp 113-4 isolates were resistant to clarithromycin (MICs, 8.0 mg/mL) and susceptible to ampicillin (MICs, 0.13 mg/mL). The five other clinical isolates tested were susceptible to both clarithromycin (MICs #0.25 mg/mL) and ampicillin (MICs #0.13 mg/mL).
Bactericidal Studies Bactericidal studies were modified from methods previously described (Coudron and Stratton 1995c). These studies were performed in cation-adjusted Mueller-Hinton broth containing 0.5% starch, which has been shown to support the growth of H. pylori without suppressing the activity of antimicrobial agents (Coudron and Stratton 1995b). The inoculation of media-containing sealed bottles was done with a syringe and yielded approximately 5 3 106 CFU/mL. Following inoculation, the bottles were flushed with a microaerophilic mixture of gases (oxygen, carbon dioxide, and nitrogen) and shaken continuously at 150 rpm at 37°C. Samples of H. pylori were removed at 24 h, serially diluted, and plated (20 mL onto each of two plates) for colony counts. Undiluted samples of 200 and 1000 mL were inoculated directly onto agar medium. Antimicrobial carryover was addressed by adding penicillinase (for ampicillin) or EDTA (for bismuth salts) to the samples before dilution (Coudron and Stratton 1995c). After 4 to 6 days of incubation under microaerobic conditions at 37°C, plates were read. The change in the log10 CFUs per milliliter after 24-h incubation relative to the log10 CFUs per milliliter at time 0 was determined. Maximal killing was defined as less than 30 CFU recovered from a 1.0 mL-aliquot.
Results of Bactericidal Studies Results of the bactericidal studies are summarized in Tables 1 to 3. All bottles containing the lowest concentration of a single agent showed a minimal effect (slight increased or decreased growth) at 24 h except for one bottle containing Hp 113-4 with bismuth, which showed greater than one log decrease of CFU/mL. The data also indicate that clarithromycin appears to be more active than ampicillin for the ATCC strain (Table 1), and that both agents were bactericidal at a concentration of 0.25 mg/mL. At 2 mg/mL, however, clarithromycin had little effect on the clarithromycin-resistant isolates (Tables 2 and 3). At 0.25 mg/mL, amoxicillin exerted about a 3 log10 decrease in CFUs/mL at 24 h for the ATCC and Hp 110-2 strains, whereas bismuth salts alone required at least 16 mg/mL and omeprazole alone required 32 mg/mL or greater for a similar magnitude of killing. The Hp 113-4 isolate was generally more susceptible to bismuth and ampicillin than were the other two strains.
TABLE 1 Bactericidal Effects of Antimicrobial Agents Alone and In Combination Against a Clarithromycin-susceptible Strain of H. pylori (ATCC 43504)a Single Agents (mg/mL) 0b
Bi (8)
Bi (16)
Om (8)
Om (16)
Om (32)
Om (64)
Am (0.016)
Am (0.031)
Am (0.125)
Am (0.25)
Cl (0.016)
Cl (0.031)
Cl (0.25)
11.6
11.4
22.6
11.4
10.7
20.3
22.7
11.0
21.7
22.7
23.2
10.3
21.9
24.9
Dual Agents (mg/mL) Am (0.016) Bi (8)
Am (0.016) Om (8)
Cl (0.016) Bi (8)
Cl (0.016) Om (8)
Bi (8) Om (8)
11.4
10.1
22.2
10.1
23.1
Triple Agents (mg/mL) Am (0.016) Bi (8) Om (8)
Am (0.25) Bi (8) Om (8)
Cl (0.016) Bi (8) Om (8)
Cl (0.25) Bi (8) Om (8)
23.0
$25.0
$ 25.0
$ 25.0
a b
The values listed represent the change in log10 CFU/mL after 24-h incubation with agents relative to log10 CFU/mL at time zero. 0, no antibiotics; Bi, bismuth; Cl, clarithromycin; Om, omeprazole; Am, ampicillin.
P.E. Coudron and C.W. Stratton
42
TABLE 2 Bactericidal Effects of Antimicrobial Agents Alone and In Combination Against a Clarithromycin-resistant Clinical Strain of H. pylori (Hp 110-2)a Single Agents (mg/mL) 0b
Bi (8)
Bi (16)
Om (8)
Om (16)
Om (32)
Am (0.008)
Am (0.016)
Am (0.031)
Am (0.25)
Cl (0.5)
Cl (1.0)
Cl (2.0)
11.3
20.6
22.6
10.3
20.3
23.6
10.8
21.1
22.8
22.9
10.6
10.1
20.2
Dual Agents (mg/mL) Am (0.008) Bi (8)
Am (0.008) Om (8)
Cl (0.5) Bi (8)
Cl (0.5) Om (8)
Bi (8) Om (8)
10.4
20.6
23.9
20.2
22.5
Triple Agents (mg/mL) Am (0.008) Bi (8) Om (8)
Am (0.25) Bi (8) Om (8)
Cl (0.5) Bi (8) Om (8)
24.1
$ 25.4
24.9
a b
The values listed represent the change in log10 CFU/mL after 24-h incubation with agents relative to log10 CFU/mL at time zero. 0, no antibiotics; Bi, bismuth; Cl, clarithromycin; Om, omeprazole.
TABLE 3 Bactericidal Effects of Antimicrobial Agents Alone and In Combination Against a Clarithromycin-resistant Clinical Strain of H. pylori (Hp 113-4)a Single Agents (mg/mL) b
0
Bi (8)
Bi (16)
Om (8)
Am (0.008)
Am (0.016)
Cl (0.5)
Cl (1.0)
Cl (2.0)
10.3
21.5
$25.0
20.2
20.5
22.2
10.3
10.2
10.1
Dual Agents (mg/mL) Am (0.008) Bi (8)
Am (0.008) Om (8)
Cl (0.5) Bi (8)
Cl (0.5) Om (8)
Bi (8) Om (8)
22.8
20.2
21.8
0.0
22.6
Triple Agents (mg/mL) Am (0.008) Bi (8) Om (8)
Am (0.25) Bi (8) Om (8)
Cl (0.5) Bi (8) Om (8)
$25.0
$25.0
24.5
a b
The values listed represent the change in log10 CFU/mL after 24-h incubation with agents relative to log10 CFU/mL at time zero. 0, no antibiotics; Bi, bismuth; Cl, clarithromycin; Om, omeprazole.
All bottles with two agents contained viable organisms at 24 h. Bismuth and omeprazole achieved substantial bactericidal effect against all three strains, but surprisingly, bismuth and clarithromycin also achieved substantial killing of the clarithromycinresistant strain, Hp 110-2 (Table 2). Omeprazole showed some additive activity when combined with ampicillin for the ATCC and Hp 110-2 strains. Complete bactericidal effect against all three strains occurred with the combination of bismuth and omeprazole and at a relatively high concentration of ampicillin (Tables 1 to 3). At a relatively low concentration, clarithromycin in combination with bismuth and omeprazole also achieved complete bactericidal effect against the clarithromycinsusceptible strain (Table 1). However, clarithromycin
at a higher concentration in combination with bismuth and omeprazole failed to achieve complete bactericidal effect against both clarithromycinresistant strains (Tables 2 and 3). Complete bactericidal effect was achieved against the five other clinical isolates that were susceptible to clarithromycin, when tested against the combination agents, bismuth (8 mg/mL), omeprazole (8 mg/mL), and a relatively high concentration (0.25 mg/mL) of either ampicillin or clarithromycin.
DISCUSSION An advantage of in vitro susceptibility testing of H. pylori is the ability to focus solely on the effects of
Clarithromycin versus Ampicillin Activity antimicrobial agents in the absence of the influence of physiological factors, such as the acid milieu of the stomach, etc. Several results of this study are noteworthy. The minimal bactericidal concentrations of omeprazole, amoxicillin, and clarithromycin were reported previously for the H. pylori strain 43504 as follows: 64 mg/mL (Sjostrom et al. 1996), .0.1 mg/mL and .0.1 mg/mL (Sjostrom and Larsson 1996), respectively. These results are consistent with our time-kill results for the same strain, which indicate that at or near these concentrations, the corresponding single agents were bactericidal (Table 1). The results of testing triple-agent combinations against the clarithromycin-resistant strains of H. pylori (Tables 2 and 3) show that when clarithromycin was added to bismuth and omeprazole, an additional 2.4 and 1.9 log10 decrease in CFU/mL was achieved for Hp 110-2 and Hp 113-4, respectively, relative to that observed with only the latter two agents in combination. Although complete bactericidal eradication for these strains was not achieved, the data suggest that resistance to clarithromycin alone does not totally negate the bactericidal effect of combination agents. Clinical studies have also shown successful eradication (63%) of metronidazoleresistant strains by triple-agent regimens that contain metronidazole (Lerang et al. 1997; Rautelin et al. 1992). A paucity of data exists for in vitro susceptibility testing of omeprazole against H. pylori in liquid media (Sjostrom and Larsson 1996; Sjostrom et al. 1996). In an earlier report, omeprazole and one other agent, amoxicillin, were tested in combination against the 43504 strain by the broth microdilution method (Sjostrom and Larsson 1996). These agents were additive and not synergistic. Results from this report agree with data from this study shown in Table 1. We believe that bismuth is an important component of any therapeutic regimen seeking complete in
43 vivo eradication of H. pylori. Results of this study demonstrate that the complete bactericidal effect was achievable with triple-agent combinations containing bismuth. Recent work in our laboratory (Stratton and Coudron 1997) suggests that the mechanism of action for bismuth salts involves the disruption of the glycocalyx/cell wall of the H. pylori organism and is the result of a physiochemical interaction. The development of microbial resistance to polycationic agents has been described in vitro for mutant microorganisms that have diminished production of glycocalyx (Nicas and Hancock 1980). However, this resistance is rare and is not likely to become an important clinical problem because microorganisms that have diminished glycocalyx quality are generally poor pathogens with low virulence. This may explain why no H. pylori isolates with resistance to bismuth have been reported to date. In summary, a bactericidal effect against both clarithromycin-sensitive and clarithromycin-resistant strains of H. pylori was achieved with appropriate double or triple combinations of antimicrobial agents. The combination of bismuth, omeprazole, and relatively high concentrations of ampicillin achieved complete bactericidal effect against all strains tested. No combination containing clarithromycin achieved complete bactericidal effect against the clarithromycin-resistant strains. These in vitro results demonstrate the bactericidal effect provided by a combination of agents including omeprazole for the eradication of H. pylori. Bismuth appears to play a particularly important role in the bactericidal effect of multiple agents.
This work was supported in part by a grant from Abbott Laboratories, Abbott Park, IL.
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