Risk of Development of In Vitro Resistance to Amoxicillin, Clarithromycin, and Metronidazole in Helicobacter pylori

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1998, p Vol. 42, No /98/$ Copyright 1998, American Society for Microbiology Risk of Development of In Vitro Resistance to Amoxicillin, Clarithromycin, and Metronidazole in Helicobacter pylori MIKAEL SÖRBERG, 1 * HÅKAN HANBERGER, 2 MAUD NILSSON, 3 ANDERS BJÖRKMAN, 1 AND LENNART E. NILSSON 3 Department of Infectious Diseases, Danderyd Hospital, S Danderyd, 1 and Infectious Diseases 2 and Department of Clinical Microbiology, 3 University Hospital, S Linköping, Sweden Received 11 August 1997/Returned for modification 3 December 1997/Accepted 12 February 1998 We have studied initial killing, morphological alterations, the frequency of occurrence, and the selective growth of resistant subpopulations of Helicobacter pylori during exposure to amoxicillin, clarithromycin, or metronidazole by bioluminescence assay of intracellular ATP levels, microscopy, and a viable count assay. We found an induction of spheroplasts and a decrease in intracellular ATP levels after 21 h of exposure to high concentrations of amoxicillin. During clarithromycin exposure the onset of a decrease in intracellular ATP levels started after prolonged incubation, and with the highest concentration of clarithromycin an induction of coccoid forms was seen after 68 h. Metronidazole exposure resulted in the strongest initial decrease in intracellular ATP levels, and coccoid forms were seen after 21 h of exposure to high concentrations of metronidazole. Amoxicillin caused a low-level increase in resistant subpopulations, which indicates a need for surveillance of the amoxicillin susceptibility of H. pylori in order to detect decreasing susceptibility. No increase in the numbers of resistant subpopulations was demonstrated during clarithromycin exposure. Metronidazole selected resistant subpopulations, which caused high-level resistance in H. pylori. Helicobacter pylori infection is a principal cause of chronic gastritis type B (13) and is associated with gastric cancer (19, 20, 33). Eradication of H. pylori prevents relapse of duodenal ulcer, and treatment of this infection has now become standard for patients with peptic ulcer disease (22, 37). The regimen most widely used today to eradicate H. pylori is combination therapy with two antibiotics and bismuth (17, 27) or an acid pump inhibitor (3, 22). A major reason for H. pylori eradication failure is resistance to metronidazole (4, 5, 9, 17, 27, 29, 34, 36) or clarithromycin (11, 44). In contrast, H. pylori does not appear to develop resistance to amoxicillin (16). The prevalence of primary metronidazole resistance varies between 7 and 90% (2, 9, 14, 36), with the highest prevalences occurring in people from developing countries (2, 9, 14), after previous metronidazole ingestion (2, 14), and in women (2, 14, 36). The prevalence of primary clarithromycin resistance is between 4 and 7% (12, 16, 45). The development of resistance during therapy has been observed for both metronidazole (35, 36, 46) and clarithromycin (11, 26, 43). The aim of this study was to evaluate the initial killing, morphological alterations, and the frequency of occurrence and the selective growth of resistant subpopulations of H. pylori during exposure to amoxicillin, clarithromycin, or metronidazole. * Corresponding author. Mailing address: Department of Infectious Diseases, Danderyd Hospital, S Danderyd, Sweden. Phone: Fax: Mikael.Sorberg@kids.ki.se. MATERIALS AND METHODS Bacterial strain. H. pylori NCTC was used in the present study. Antibiotics. Amoxicillin and metronidazole were purchased from Sigma Chemical Co., St. Louis, Mo. Clarithromycin was kindly provided by Abbott Laboratories, Chicago, Ill. Growth medium. Mueller-Hinton broth (MHB; Gibco Limited, Renfrewshire, Scotland) supplemented with 50 mg of Ca 2 per liter, 25 mg of Mg 2 per liter, and 1% fetal calf serum was used as the growth medium. Bioluminescence assay of intracellular ATP levels. A 100- l sample from the bacterial culture was incubated at 37 C for 10 min with 100 l of the ATPhydrolyzing enzyme apyrase, purified grade I (Sigma Chemical Co.) in supplemented MHB, to eliminate extracellular ATP. A 50- l sample of the apyrasetreated sample was pipetted into 500 l of boiling 0.1 M Tris buffer (ph 7.75) containing 2 mm EDTA to release the intracellular ATP and inactivate the apyrase. After being heated for 90 s, the extracts were cooled before the intracellular ATP levels were assayed. This extraction was performed in an LKB Biocal 2030 incubator (LKB Products, Bromma, Sweden). Luciferase reagent (100 l) was added to 550 l of each cooled extract, and the light intensity was measured in a 1250 Luminometer (LKB Wallac, Turku, Finland) and was recorded on a 1250 Display (LKB Wallac). An ATP-monitoring reagent (Bio Orbit, Turku, Finland) was used in the assay of ATP levels. The ATP levels in the samples were calculated by using the results of assays of standard amounts of ATP as a reference. A correction was made for background luminescence. Extracts to which known amounts of ATP were added were used as internal standards in order to correct for inhibition of the luciferase reaction by the reagents. The coefficient of variation for the bioluminescence assay has been shown to vary between 1.7 and 6.5% (41). Monitoring of bacterial growth during antibiotic exposure in broth. The bacterial numbers were determined by the bioluminescence assay of bacterial ATP levels. As indicated by Thore et al. (42) and Molin et al. (30), 10 6 M ATP corresponded to approximately 10 9 bacteria/ml. From a culture with bacteria in the log phase diluted to 10 7 CFU/ml, 5 ml was transferred to 50-ml Erlenmeyer flasks containing 50 l of antibiotics at different concentrations. The concentrations tested were as follows: amoxicillin, to 0.25 g/ml; clarithromycin, to 0.25 g/ml; and metronidazole, 0.06 to 32 g/ml. Samples were taken daily for bioluminescence assay of intracellular bacterial ATP levels. The flasks were incubated at 37 C under microaerobic conditions (5% oxygen, 10% carbon dioxide, 85% nitrogen) in an incubator box (ASSAB with CO 2 and O 2 regulator; Kebo Biomed, Spånga, Sweden). The experiments were repeated three times. Morphology. The bacteria were exposed to amoxicillin, clarithromycin, and metronidazole and were studied by light microscopy at a magnification of 1,250 after being stained with acridine orange (28). The sensitivity of the staining method is 10 4 CFU/ml (28). Population analysis. Population analyses were performed with control cultures and regrowing cultures exposed to metronidazole or amoxicillin. Population analyses were also performed with cultures that grew in the presence of the highest concentration during clarithromycin exposure. The contents of the flasks were thoroughly mixed, and 0.1-ml portions were removed and diluted serially in 0.9-ml aliquots of phosphate-buffered saline. A total of 50 l from each dilution was dropped onto paper disc method (PDM) agar plus 5% defibrinated horse blood (AB Biodisk, Solna, Sweden) containing different concentrations of amoxicillin ( to 0.25 g/ml), clarithromycin ( to 0.25 g/ml), and metronidazole (0.06 to 32 g/ml). The drops were allowed to dry at room temperature. The plates were incubated for 5 days at 37 C under microaerobic conditions, and the colonies were then counted. The frequencies of occurrence of variants resistant to different concentrations of amoxicillin, clarithromycin, and metronidazole were calculated by dividing the number of colonies on plates with antibiotic 1222

2 VOL. 42, 1998 IN VITRO DRUG RESISTANCE IN H. PYLORI 1223 FIG. 1. Monitoring of growth of H. pylori assayed by bioluminescence assay of ATP levels in bacteria in an unexposed culture ( ) and cultures exposed to amoxicillin at (F), ( ), 0.03 (E), and 0.25 ({) g/ml. by the number of colonies on plates without antibiotic. The experiments were repeated three times. Ten passages of the metronidazole-resistant cultures were done in drug-free MHB. Susceptibility testing on agar plates was done with passaged cultures in antibiotic-free broth by the E-test (AB Biodisk). Determination of antibiotic concentrations in H. pylori cultures. Samples from H. pylori cultures exposed to 0.25 g of amoxicillin per ml, 0.25 g of clarithromycin per ml, or 32 g of metronidazole per ml were taken after 0, 21, 46, 68, 96, 118, 142, 166, 191, 214, and 267 h incubation at 37 C under microaerobic conditions. The samples were put in wells of a PDM agar (AB Biodisk) tray with Micrococcus luteus ATCC 9341 for determination of amoxicillin and clarithromycin concentrations and in wells of a PDM agar plus 5% defibrinated horse blood (AB Biodisk, Solna, Sweden) tray with Clostridium perfingens ATCC for determination of metronidazole concentration. The trays with M. luteus were incubated overnight under aerobic conditions, and the trays with C. perfringens were incubated overnight under anaerobic conditions. The resulting inhibition zones surrounding the wells were measured and were compared with those obtained by linear regression analysis with standard concentrations of drugs. RESULTS Growth and morphology of H. pylori during exposure to amoxicillin. Growth of the cultures with an inoculum of M ATP was monitored, and three growth patterns were seen. Intracellular ATP levels increased in the cultures exposed to low concentrations of amoxicillin ( g/ml) (Fig. 1). There was an initial growth inhibition in the cultures exposed to g of amoxicillin per ml (Fig. 1). The cultures exposed to g of amoxicillin per ml showed an initial decrease in intracellular ATP levels after 46 h (Fig. 1). Microscopy showed spheroplasts in cultures in which a decrease in intracellular ATP levels occurred. After 118 h a few bacillary forms were seen, and the numbers of these forms increased when the intracellular ATP level in these cultures increased. There was no decrease in the amoxicillin concentration in the broth cultures containing and g of amoxicillin per ml when regrowth occurred. In the culture exposed to 0.03 g of amoxicillin per ml, intracellular ATP levels increased after 191 h, and microscopy showed a mixed population of bacillary forms, spheroplasts, and coccoid cells. In this culture there was a reduction in the amoxicillin concentration, and at 191 h, when the culture regrew, only 50% of the initial amoxicillin concentration was left in the broth. At concentrations above 0.03 g of amoxicillin per ml there was no regrowth. Population analyses of cultures exposed to amoxicillin. Population analyses were performed with the unexposed cultures and cultures exposed to amoxicillin at concentrations of 0.008, 0.015, and 0.03 g/ml. The bacteria in the unexposed cultures were resistant to up to g/ml (Fig. 2). At higher concentrations on agar plates there was a reduction in the frequency of resistant variants for the unexposed broth cultures (Fig. 2). The cultures exposed to and g of amoxicillin per ml in broth were resistant to up to g/ml, and the frequency of resistant variants exposed to amoxicillin at 0.03 g/ml was 10 1 (Fig. 2). The frequency of resistant variants in the cultures exposed to 0.03 g of amoxicillin per ml was lower than that for the unexposed cultures (Fig. 2). Population analyses were performed with the control cultures on two different occasions with an interval of 3 days, and no reduction in the amoxicillin concentrations on the agar plates was found. Growth and morphology of H. pylori during exposure to clarithromycin. Growth of the cultures with an inoculum of M ATP was monitored. Intracellular ATP levels increased in the cultures exposed to low concentrations of clarithromycin ( 0.06 g/ml) (Fig. 3). There was a concentration-dependent inhibition of the increase in intracellular ATP levels, and after prolonged incubation there was a concentration-dependent decrease in intracellular ATP levels in the cultures (Fig. 3). Microscopy showed bacillary forms in the growing cultures, and a conversion from bacillary to coccoid forms was seen when the intracellular ATP levels decreased. No cultures showed regrowth. The clarithromycin concentrations in broth were stable throughout the experiment. Population analyses of cultures exposed to clarithromycin. Population analyses were performed with the unexposed cultures and cultures exposed to clarithromycin at concentrations of and g/ml. The unexposed cultures and the cultures exposed to g of clarithromycin per ml were resistant to up to g/ml (Fig. 4). At higher concentrations there was a reduction in the frequency of resistant variants to 10 1 after exposure to clarithromycin at 0.03 g/ml (Fig. 4). For cultures exposed to g of clarithromycin per ml the frequency of resistant variants was lower after exposure

3 1224 SÖRBERG ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 2. Population analysis of H. pylori in unexposed cultures ( and ) and cultures exposed to amoxicillin at (F), ( ) and 0.03 (E) g/ml. to clarithromycin at and 0.03 g/ml than that for the unexposed cultures and the cultures exposed to g of clarithromycin per ml (Fig. 4). Population analyses were performed with the control cultures on two different occasions with an interval of 3 days, and no reduction in the clarithromycin concentration in the agar plates was found. Growth and morphology of H. pylori during exposure to metronidazole. Growth of the cultures with an inoculum of M ATP was monitored, and two growth patterns were seen. Intracellular ATP levels increased in the cultures exposed to low concentrations of metronidazole ( 0.5 g/ml) (Fig. 5). The cultures exposed to 1 to 32 g of metronidazole per ml showed an initial decrease in intracellular ATP levels (Fig. 5), and microscopy showed a conversion from bacillary to coccoid forms after 21 h. When the intracellular ATP levels increased in the cultures exposed to 1 to 4 g of metronidazole per ml, a change in morphology was seen by microscopy, from coccoid forms to bacillary forms. In cultures with concentrations above 4 g of metronidazole per ml there was no regrowth (Fig. 5). The metronidazole concentrations in broth were stable throughout the experiment. Population analyses of cultures exposed to metronidazole. Population analyses were performed with the unexposed cultures and cultures exposed to metronidazole at concentrations of1to4 g/ml. The bacteria in the unexposed cultures were resistant to up to 0.25 g/ml (Fig. 6). With higher concentrations there was a reduction in the frequency of resistant variants for the unexposed cultures (Fig. 6). For the cultures ex- FIG. 3. Monitoring of growth of H. pylori assayed by bioluminescence assay of ATP levels in bacteria in an unexposed culture ( ) and cultures exposed to clarithromycin at to 0.25 g/ml ({, g/ml; }, g/ml; Œ, g/ml; E, g/ml;, g/ml; F, g/ml).

4 VOL. 42, 1998 IN VITRO DRUG RESISTANCE IN H. PYLORI 1225 FIG. 4. Population analysis of H. pylori in unexposed cultures ( and ) and cultures exposed to clarithromycin at (F) and ( ) g/ml. 5E-04, posed to 1 to 4 g of metronidazole per ml, resistant variants were resistant to up to 32 g/ml (Fig. 6). The selection of resistant variants was concentration dependent, and after exposure to the highest concentration (4 g of metronidazole per ml) all bacteria in the population were resistant to metronidazole at 32 g/ml (Fig. 6). Population analyses were performed with the control cultures on three different occasions with a total interval of 13 days, and no reduction in the metronidazole concentrations on the agar plates was found (Fig. 6). The resistance remained stable through 10 passages in MHB for all cultures in which metronidazole resistance developed. DISCUSSION This study showed an initial decrease in intracellular ATP levels during exposure of H. pylori to high concentrations of amoxicillin (Fig. 1), and this bactericidal effect of amoxicillin is in agreement with the effect found in a study by Berry et al. (6). During exposure of H. pylori to amoxicillin, microscopy showed spheroplasts after 21 h, which is in accordance with the findings of a study by Nilius et al. (32) and a previous study by us (40), in which we found a concentration-dependent induction of spheroplasts after only 2 h. Other investigators have reported a morphologic conversion of H. pylori during exposure to FIG. 5. Monitoring of growth of H. pylori assayed by bioluminescence assay of ATP levels in bacteria in an unexposed culture ( ) and cultures exposed to metronidazole at 0.5 to 4 and 32 g/ml (}, 32 g/ml; F, 4 g/ml;, 2 g/ml; E, 1 g/ml; {, 0.5 g/ml).

5 1226 SÖRBERG ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 6. Population analysis of H. pylori in an unexposed cultures (,, and Œ) and cultures exposed to metronidazole at 1 to 4 g/ml (E, 1 g/ml;, 2 g/ml; F, 4 g/ml). amoxicillin but have not distinguished between coccoid forms and spheroplasts (6, 7). Armstrong et al. (1) reported central clearing and vesiculation of H. pylori after a 24-h exposure to amoxicillin but did not discuss these findings in terms of coccoid forms or spheroplasts. No reports on the clinical significance of amoxicillin resistance in H. pylori have been published, and Glupczynski et al. (16) found an unchanged susceptibility of H. pylori to amoxicillin over a 5-year period. We found a small increase in the numbers of resistant subpopulations in all except one of the regrowing cultures exposed to amoxicillin. In one culture (containing 0.03 g of amoxicillin per ml), regrowth occurred after 191 h due to a decrease in the concentration of amoxicillin in the broth (Fig. 2). Our results are in agreement with those of Haas et al. (18), who found increased amoxicillin MICs during exposure to amoxicillin in several passages. Our results and those of Haas et al. (18) indicate that there is a need for surveillance of the amoxicillin susceptibility of H. pylori in order to detect decreasing levels of susceptibility. After a prolonged incubation, clarithromycin exposure resulted in a concentration-dependent decrease in intracellular ATP levels in H. pylori cultures (Fig. 3). Similar results were found by Flamm et al. (15), who demonstrated a bactericidal effect of clarithromycin on H. pylori after 8 h of exposure to clarithromycin. In an earlier study (40) we could not find any bactericidal effect after 5 h of exposure of H. pylori to clarithromycin, but this was probably due to a shorter exposure time. A conversion from bacillary to coccoid forms was seen in this study after 68 h of exposure of H. pylori to the highest concentration of clarithromycin. After the same exposure time, Nilius et al. (32) found an induction of coccoid forms during exposure of H. pylori to erythromycin. We found no regrowth or increase in the numbers of resistant subpopulations of H. pylori during clarithromycin exposure (Fig. 4). Haas et al. (18) found variation among different strains, with increasing MICs for H. pylori during exposure to erythromycin. This variation among different strains might explain why we did not find resistant subpopulations of the strain used in our study. The strongest initial decrease in intracellular ATP levels was seen during exposure of H. pylori to metronidazole (Fig. 5), and this bactericidal effect of metronidazole on H. pylori has also been demonstrated by Armstrong et al. (1). We found a conversion from bacillary to coccoid forms after 21 h in the cultures in which a decrease in intracellular ATP levels occurred. This is similar to the results of a study by Armstrong et al. (1), who reported a conversion to coccoid forms after 48 h of exposure of H. pylori to metronidazole. The mechanisms for metronidazole resistance are probably a decreased ability of metronidazole-resistant strains to achieve a sufficiently low redox potential under microaerobic conditions for the necessary reduction of metronidazole and that during short periods of anaerobic conditions these strains manage to reduce and store sufficient amounts of metronidazole so as to appear fully susceptible after subsequent incubation under microaerobic conditions (8, 38, 48). This leads to a slower uptake of metronidazole by resistant strains of H. pylori than by sensitive strains, which has been reported by several investigators (25, 31, 38). We found selection and regrowth of resistant subpopulations of H. pylori in all regrowing cultures during exposure to metronidazole (Fig. 6). During this regrowth a change in morphology from coccoid to bacillary forms was seen by microscopy. This change is probably due to a low frequency of occurrence of resistant subpopulations rather than a conversion from coccoid to bacillary forms. This caused a decreased susceptibility to metronidazole, which is in agreement with the findings of Haas et al. (18), who reported an increase in the MIC of metronidazole for H. pylori after several passages during exposure to metronidazole. When evaluating the development of resistance to metronidazole during treatment, it is important to study whether there is a selection of spontaneous resistant variants of the infecting strain or whether there is reinfection with an exogenous strain (23, 35). The metronidazole resistance in our study was stable during 10 passages, which is in agreement with the findings of a study by Haas et al. (18), but there have also been reports of unstable metronidazole resistance. Zwet et al. (48) found that metronidazole resistance induced in vitro was reversed in 30% of the isolates by further culture on antibiotic-free plates. The different results concerning the stability of metronidazole resistance might be explained by methodological differences such as the use of

6 VOL. 42, 1998 IN VITRO DRUG RESISTANCE IN H. PYLORI 1227 different inocula (21) and by the use of different incubation conditions (8, 38, 47). When studying the morphology of H. pylori it is important to differentiate between coccoid cells (1, 6) and spheroplasts (32). In our earlier study we found a rapid induction of spheroplasts during exposure of H. pylori to amoxicillin (40), while the rate of conversion to coccoid forms during exposure to clarithromycin and metronidazole in this study was slower. The spheroplasts are larger than the coccoid forms seen by microscopy (unpublished results). The coccoid forms changed color, from orange to green, during prolonged exposure to clarithromycin and metronidazole when acridine orange staining was used (unpublished results). A cell containing more RNA than DNA stains orange, and a cell in which much of the RNA has been degraded but still retains its DNA stains green (24). The degradation of RNA has been correlated with a loss of viability (10). The spheroplasts stained orange (unpublished results), and we found in an earlier study (40) that they reverted to bacillary forms. In another previous study (39) we found a low ATP level in the coccoid cell during prolonged incubation of H. pylori, but we were not able to demonstrate a conversion to bacillary forms. In conclusion, we found an induction of spheroplasts and a decrease in intracellular ATP levels after 21 h of exposure of H. pylori to high concentrations of amoxicillin. During clarithromycin exposure the onset of the decrease in intracellular ATP levels started after prolonged incubation, and with the highest concentration of clarithromycin an induction of coccoid forms was seen after 68 h. Metronidazole exposure resulted in the strongest initial decrease in intracellular ATP levels, and coccoid forms were seen after 21 h of exposure to high concentrations of metronidazole. Amoxicillin caused a low-level increase in the numbers of resistant subpopulations, which indicates that there is a need for surveillance of the amoxicillin susceptibility of H. pylori in order to detect decreasing susceptibility. No increase in resistant subpopulations was demonstrated during clarithromycin exposure. Metronidazole selected resistant subpopulations, which caused high-level resistance in H. pylori. REFERENCES 1. Armstrong, J. A., S. H. Wee, C. S. Goodwin, and D. H. Wilson Response of Campylobacter pyloris to antibiotics, bismuth and an acidreducing agent an ultrastructural study. J. Med. Microbiol. 24: Banatvala, N., G. R. Davies, Y. Abdi, A. Clements, D. S. Rampton, J. M. Hardie, and R. A. Feldman High prevalence of Helicobacter pylori metronidazole resistance in migrants to east London: relation with previous nitroimidazole exposure and gastroduodenal disease. Gut 35: Bazzoli, F., R. M. Zagari, S. Fossi, P. Pozzato, G. Alampi, P. Simoni, S. Sottili, A. Roda, and E. Roda Short-term low-dose triple therapy for the eradication of Helicobacter pylori. Eur. J. Gastroenterol. Hepatol. 6: Bell, G. D., K. Powell, S. M. Burridge, A. F. Bowden, W. Atoyebi, G. H. Bolton, P. H. Jones, and C. Browns Rapid eradication of Helicobacter pylori infection. Aliment. Pharmacol. Ther. 9: Bell, G. D., K. Powell, S. M. Burridge, A. Pallecaros, P. H. Jones, P. W. Gant, G. Harrison, and J. E. Trowell Experience with triple anti-helicobacter pylori eradication therapy: side effects and the importance of testing the pre-treatment bacterial isolate for metronidazole resistance. Aliment. Pharmacol. Ther. 6: Berry, V., K. Jennings, and G. Woodnutt Bacteriocidal and morphological effect of amoxicillin on Helicobacter pylori. Antimicrob. Agents Chemother. 39: Bode, G., F. Mauch, P. Malfertheiner The coccoid form of Helicobacter pylori. Criteria for their viability. Epidemiol. Infect. 111: Cederbrant, G., G. Kahlmeter, and Å. Ljungh Proposed mechanism for metronidazole resistance in Helicobacter pylori. J. Antimicrob. Chemother. 29: Ching, C. K., K. P. Leung, R. W. Yung, S. K. Lam, B. C. Wong, K. C. Lai, and C. L. Lai Prevalence of metronidazole resistance Helicobacter pylori strains among Chinese peptic ulcer disease patients and normal controls in Hong Kong. Gut 38: Davis, B. D., S. J. Lager, and P. C. Tai Role of ribosome degradation in the death of starved Escherichia coli cells. J. Bacteriol. 166: Debets-Ossenkopp, Y. J., M. Sparrius, J. G. Kusters, J. J. Kolkman, and C. M. Vandenbroucccke-Grauls Mechanism of clarithromycin resistance in clinical isolates of Helicobacter pylori. FEMS Microbiol. Lett. 142: De Koster, E., A. Cozzoli, C. Jonas, R. Ntounda, J. P. Butzler, and M. Detenre Six years resistance of Helicobacter pylori to macrolides and imidazoles. Gut 39(Suppl. 2):A5. (Abstract 1A:04.) 13. Dixon, M. F., and G. M. Sobala Gastritis and duodenitis: histopathological spectrum. Eur. J. Gastroenterol. Hepatol. 4(Suppl. 2): European Study Group on Antibiotic Susceptibility of Helicobacter pylori Results of a multicentre European survey in 1991 of metronidazole resistance in Helicobacter pylori. Eur. J. Microbiol. Infect. Dis. 11: Flamm, R. K., J. Beyer, S. K. Tanaka, and J. Clement Kill kinetics of antimicrobial agents against Helicobacter pylori. J. Antimicrob. Chemother. 38: Glupczynski, Y., S. Goutier, C. van den Borre, J. P. Butzler, and A. Burette Surveillance of Helicobacter pylori resistance to antimicrobial agents in Belgium from 1989 to Gut 37(Suppl. 1):A56. (Abstract 223.) 17. Graham, D. Y., G. M. Lew, H. M. Malaty, D. Evans, P. Klein, and L. Alpert Factors influencing the eradication of Helicobacter pylori with triple therapy. Gastroenterology 102: Haas, C. E., D. E. Nix, and J. J. Schentag In vitro selection of resistant Helicobacter pylori. Antimicrob. Agents Chemother. 34: Hansson, L. E., L. Engstrand, O. Nyren, D. J. Evans, A. Lindgren, and R. Bergström Helicobacter pylori infection: independent risk indicator of gastric adenocarcinoma. Gastroenterology 105: Hansson, L. E., L. Engstrand, O. Nyren, and A. Lindgren Prevalence of Helicobacter pylori infection in subtypes of gastric cancer. Gastroenterology 109: Henriksen, T. H., Ö. Brorson, R. Schöyen, and T. Thoresen Risks related to lack of standardization of tests to detect in vitro metronidazole resistance in Helicobacter pylori. Eur. J. Microbiol. Infect. Dis. 15: Hentchel, E., G. Brandsätter, B. Dragosics, A. M. Hirschl, H. Nemec, and K. Schutze Effect of ranitidine and amoxicillin plus metronidazole on the eradication of Helicobacter pylori and recurrence of duodenal ulcer. N. Engl. J. Med. 328: Jorgensen, M., G. Daskalopoulos, V. Warburton, H. M. Mitchell, and S. L. Hazell Multiple strain colonization and metronidazole resistance in Helicobacter pylori-infected patients: identification from sequential and multiple biopsy specimens. J. Infect. Dis. 174: Kasten, F. H Cytochemical studies with acridine orange and the influence of dye contaminants in staining of nucleic acid. Int. Rev. Cytol. 21: Lacey, S. L., S. F. Moss, and G. W. Taylor Metronidazole uptake by sensitive and resistant isolates of Helicobacter pylori. J. Antimicrob. Chemother. 32: Logan, R. P., P. A. Gummet, H. D. Schaufelberger, R. R. Greaves, G. M. Mendelson, and M. M. Walker Eradication of Helicobacter pylori with clarithromycin and omeprazole. Gut 35: Logan, R. P., P. A. Gummet, J. J. Misiewicz, Q. N. Karim, M. M. Walker, and J. H. Baron One week eradication regimen for Helicobacter pylori. Lancet 338: McCarthy, L. R., and J. E. Senne Evaluation of acridine orange stain for detection of microorganisms in blood cultures. J. Clin. Microbiol. 11: Midola, P. H., J. R. Lambert, and J. Turnidge Metronidazole resistance: a predictor of failure of Helicobacter pylori eradication by triple therapy. J. Gastroenterol. Hepatol. 11: Molin, Ö., L. Nilsson, and S. Ånsehn Rapid detection of bacterial growth in blood cultures by bioluminescent assay of bacterial ATP. J. Clin. Microbiol. 18: Moore, R. A., B. Beckthold, and L. E. Bryan Metronidazole uptake in Helicobacter pylori. Can. J. Microbiol. 41: Nilius, M., A. Ströhle, G. Bode, and P. Malfertheiner Coccoid like form(clf) of Helicobacter pylori. Enzyme activity and antigenicity. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 280: Passonet, J., G. D. Freidman, and D. P. Vandersteed Helicobacter pylori infection and the risk of gastric carcinoma. N. Engl. J. Med. 325: Powell, K. U., G. D. Bell, A. F. Bowden, J. E. Trowell, and P. H. Jones An effective one-week Helicobacter pylori eradication therapy using omeprazole, clarithromycin and metronidazole. Br. J. Clin. Res. 6: Rautelin, H., W. Tee, K. Seppälä, and T. U. Kosunen Ribotyping patterns and emergence of metronidazole resistance in paired clinical samples of Helicobacter pylori. J. Clin. Microbiol. 32: Rautelin, H., K. Seppälä, O.-V. Renkonen, U. Vainio, and T. U. Kosunen Role of metronidazole resistance in therapy of Helicobacter pylori infection. J. Antimicrob. Chemother. 36: Rauws, E. A., and G. N. Tytgat Cure of duodenal ulcer associated with eradication of Helicobacter pylori. Lancet 335:

7 1228 SÖRBERG ET AL. ANTIMICROB. AGENTS CHEMOTHER. 38. Smith, M. R., and D. I. Edwards The influence of microaerophilia and anaerobiosis on metronidazole uptake in Helicobacter pylori. J. Antimicrob. Chemother. 36: Sörberg, M., M. Nilsson, and L. E. Nilsson Morphologic conversion of Helicobacter pylori from bacillary form to coccoid form evaluated by bioluminescence, microscopy and viable count. Eur. J. Microbiol. Infect. Dis. 15: Sörberg, M., M. Nilsson, H. Hanberger, and L. E. Nilsson Pharmacodynamic effects of antibiotics and acid pump inhibitors on Helicobacter pylori. Antimicrob. Agents Chemother. 41: Sören, L., M. Nilsson, and L. E. Nilsson Quantitation of antibiotic effects on bacteria by bioluminescence, viable count and quantal analysis. J. Antimicrob. Chemother. 35: Thore, A., S. Ånsehn, A. Lundin, and S. Berman Detection of bacteriuria by luciferase assay of adenosine triphosphate. J. Clin. Microbiol. 1: Versalovic, J., D. Shortridge, K. Kimbler, M. V. Griffy, J. Beyer, R. K. Flamm, S. K. Tanaka, D. Y. Graham, and M. F. Go Mutations in 23S rrna are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 40: Xia, H., M. Buckley, D. Hyde, C. T. Kaene, and C. A. O Morain Effects of antibiotic-resistance on clarithromycin-combined triple therapy for Helicobacter pylori. Gut 37(Suppl. 1):A55. (Abstract 218.) 45. Xia, H., M. Buckley, C. T. Keane, and C. A. O Morain Clarithromycin resistance in Helicobacter pylori: prevalence in untreated dyspeptic patients and stability in vitro. J. Antimicrob. Chemother. 37: Xia, H., M. A. Daw, S. Sant, S. Beattie, C. T. Keane, and C. A. O Morain Clinical efficacy of triple therapy in Helicobacter pylori-associated duodenal ulcer. Eur. J. Gastroenterol. Hepatol. 5: Zwet, A. A., J. C. Thijs, W. Schievink-de Vries, J. Schiphuis, and J. A. M. Snijder In vitro studies on stability and development of metronidazole resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 38: Zwet, A. A., J. C. Thijs, and B. de Graaf Explanations for high rates of eradication with triple therapy using metronidazole in patients harboring metronidazole-resistant Helicobacter pylori strains. Antimicrob. Agents Chemother. 39: