Heterogeneity in Susceptibility to Metronidazole among Helicobacter pylori Isolates from Patients with Gastritis or Peptic Ulcer Disease

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1996, p Vol. 34, No /96/$ Copyright 1996, American Society for Microbiology Heterogeneity in Susceptibility to Metronidazole among Helicobacter pylori Isolates from Patients with Gastritis or Peptic Ulcer Disease J. F. L. WEEL, 1 *R.W.M.VAN DER HULST, 2 Y. GERRITS, 1 G. N. J. TYTGAT, 2 A. VAN DER ENDE, 1 AND J. DANKERT 1 Departments of Medical Microbiology 1 and Gastroenterology, 2 Academic Medical Center, Amsterdam, The Netherlands Received 5 February 1996/Returned for modification 25 March 1996/Accepted 10 June 1996 Combination therapies that include metronidazole (MTZ) are the most successful therapies used in eradicating Helicobacter pylori. In this study, the prevalence and the relevance of heterogeneity in susceptibility to MTZ among H. pylori populations of 156 patients were evaluated. The results of this study show that 37 patients (24%) were infected with MTZ-resistant H. pylori (MIC > 8 g/ml). Furthermore, 33% (52 of 156) of the patients were found to be infected with H. pylori populations heterogeneous for their susceptibility to MTZ. The reassessment of the MICs of MTZ for these 52 H. pylori populations revealed MTZ resistance in 28 of them, increasing the number of MTZ-resistant H. pylori populations among the 156 patients to 65 (42%). Out of 20 isolates, 2 (10%) heterogeneous in their susceptibility to MTZ also appeared to be heterogeneous at the genome level as determined by randomly amplified polymorphic DNA fingerprinting. In conclusion, the results show the limitations and risk of possible misinterpretations when only a single colony, picked from the primary H. pylori populations isolated from patients, is analyzed for its susceptibility to MTZ. Helicobacter pylori is a gram-negative, spiral-shaped bacterium which grows optimally under microaerophilic conditions. Although not all persons infected with the bacterium suffer from gastroduodenal disease, H. pylori is now recognized to cause active chronic gastritis (14), is generally accepted to play a causative role in the pathogenesis of gastric and duodenal ulceration (10), and is suspected to be involved in the pathogenesis of both mucosa-associated lymphoid tissue lymphoma (17) and gastric adenocarcinoma (6). For these reasons, therapy aimed at eradicating the bacterium is given to those patients. So far, different therapeutic regimens have been reported to be successful in eliminating the bacterium (19). Many of these therapies include metronidazole (MTZ), a 5 nitroimidazole which has its optimal activity in anaerobic environments (3). Resistance to MTZ affects the success rate of the MTZ-including therapies negatively. For triple therapies consisting of either a bismuth compound, amoxicillin or tetracycline, and MTZ (11) or a proton pump inhibitor, clarithromycin, and MTZ (8, 11), the success rate in patients infected with MTZ-susceptible (MTZ-S) H. pylori is 30% higher than that in patients infected with MTZ-resistant (MTZ-R) H. pylori. Until recently disk diffusion and an agar dilution technique have been used to determine the MICs for H. pylori strains. However, in comparison with these methods, the Epsilometer (E)-test has been found to be a more accurate and easy-to-use method in assessing the MICs of all kinds of antibiotics, including MTZ (5), for H. pylori. In general, strains are regarded as susceptible to MTZ if the MICs for the bacteria are 8 g/ml (4, 11). Recently, patients infected with strains having an intermediate resistance to MTZ (4 g/ml MIC 8 g/ml) have shown responses to an MTZ-including triple therapy as * Corresponding author. Mailing address: Department of Medical Microbiology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Phone: , beeper no Fax: poor as those of patients infected with strains for which the MICs were 8 g/ml (23), calling into question the 8- g/ml cutoff value between MTZ susceptibility and resistance. Pilot experiments showed that patients can be infected with a population of H. pylori strains heterogeneous for their susceptibility to MTZ. In this study the prevalence and the relevance of heterogeneity in susceptibility to MTZ among H. pylori populations in 156 patients were evaluated. The results of this study show that 37 patients (24%) were infected with MTZ-R H. pylori (MIC 8 g/ml). Furthermore, 33% (52 of 156) of the patients were infected with H. pylori strains with various MTZ susceptibilities. Reassessment of the MICs of MTZ for these 52 H. pylori populations revealed MTZ resistance in 28 of these H. pylori populations. Out of 20 isolates, 2 (10%) heterogeneous in their susceptibility to MTZ also appeared to be heterogeneous at the genome level as determined by randomly amplified polymorphic DNA (RAPD) fingerprinting. MATERIALS AND METHODS Patients and strains. From among adult patients with dyspeptic complaints who were undergoing upper gastrointestinal endoscopy and who had H. pyloripositive cultures, 156 consecutive H. pylori patients were enrolled in this study. In 77 of them either an active ulcer or scars from ulcers were seen. The other 79 patients suffered from functional dyspepsia (20, 22). Seventeen of them had been treated with anti-h. pylori therapies before (20). The biopsy specimens were transported in 2 ml of phosphate-buffered saline and inoculated onto Columbia agar plates containing 7% (vol/vol) horse blood and onto Columbia agar (CM 331; Oxoid, Unipath Ltd., Basingstoke, England) plates containing 7% (vol/vol) horse blood and Skirrow (Oxoid) supplement. Colonies that exhibited characteristic morphologies were identified as H. pylori if they were urease, catalase, and oxidase positive. The antrum and corpus specimens were collected separately with swabs for cultures. Each swab was shaken in 8% glycerol peptone, and the suspensions were stored at 70 C (Fig. 1). These bacterial suspensions are referred to in this work as the frozen primary cultures (22). Determination of antimicrobial susceptibility by disk diffusion test and E-test. MTZ susceptibilities were determined by the disk (Oxoid) diffusion method and by the E-test (AB Biodisk, Solna, Sweden). The frozen primary cultures (see above) from both antrum and corpus specimens were inoculated onto fresh Columbia agar plates containing 7% (vol/vol) horse blood and cultured under 2158

2 VOL. 34, 1996 HETEROGENEITY IN MTZ-S H. PYLORI ISOLATES 2159 Chromosomal DNA (20 ng) and one of the primer pairs, 1254 (CCGCAGC CAA) and 1281 (AACGCGCAAC) (5 pmol), were used (1). The PCR was carried out in 25 l containing 10 mm Tris-HCl (ph 8.8), 50 mm KCl, 3.0 mm MgCl 2, and bovine serum albumin (0.1 mg/ml). The reaction conditions were as follows: 3 cycles of 5 min at 94 C, 5 min at 36 C, and 5 min at 72 C, followed by 29 cycles of 1 min at 94 C, 1 min at 36 C, and 2 min at 72 C, followed by 10 min at 72 C. The PCR fragments were analyzed by horizontal agarose (1%) gel electrophoresis as described before (7, 12). RESULTS FIG. 1. Schematic representation of H. pylori assessment and MTZ susceptibility testing. BA, blood agar; DMEM, Dulbecco s modified Eagle medium. microaerophilic conditions at 37 C (Campypak; BBL, Cockeysville, Md.). After 3 days, the H. pylori strains grown were collected with cotton swabs and resuspended in 2 ml of Dulbecco s modified Eagle medium for cell culture. From this suspension, 100 l, containing 10 7 to 10 8 CFU of H. pylori per ml, was flooded on Columbia agar plates containing 7% (vol/vol) horse blood. According to the instructions of the manufacturer, the E-test strip was placed on the plate when the surface of the plate was dry. All plates were incubated for 3 and 5 days at 37 C under microaerophilic conditions. In the E-test, the MIC was defined as the concentration indicated on the E-test strip that was closest to the point of intersection with growth on the plate (per the manufacturer s instructions). Colonies growing within the zone of growth inhibition of the bacterial lawn were subjected to one of the following treatments: (i) either they were resuspended in 100 l of DMEM and their MICs were directly retested by the E-test, or (ii) they were subcultured for 3 days on blood agar before the assessment of their MTZ MICs was performed (Fig. 1). In the disk diffusion test using MTZ disks containing 5 g of MTZ, H. pylori strains with inhibition zone diameters of less than 20 mm, 20 to 26 mm, and more than 26 mm were reported as resistant, intermediate, and susceptible, respectively (23). For 12 H. pylori isolates, the effect of 24 h of anaerobic preincubation on their susceptibility to MTZ was studied. After the bacteria and E-test strips had been applied to the blood agar plates, the plates were incubated anaerobically (Gaspak anaerobic system; Becton Dickinson, Cockeysville, Md.) at 35 to 37 C for 24 h. Then, the plates were transferred to microaerophilic conditions and the incubation was prolonged for 3 and 5 days before the MIC of MTZ was read. As a control for the 24-h anaerobic incubation, a blood agar plate was inoculated with the obligate anaerobe Bacteroides fragilis and incubated together with the plates inoculated with H. pylori. H. pylori chromosomal DNA isolation. From 20 patients infected with H. pylori populations differing in MTZ susceptibility (ranging from susceptible to resistant), 10 colonies were recultured on horse blood agar plates. After 3 days of growth under microaerophilic conditions, these H. pylori colonies were harvested and DNA was isolated according to the method described by Oudbier et al. (12). In short, after lysing was performed the bacterial suspension RNase was added (final concentration, 20 g/ml) and the mixture was incubated for 2hat55 C. The solution was extracted with equal volumes of phenol, phenol-chloroformisoamyl alcohol (25:24:1), and chloroform-isoamyl alcohol (24:1). Thereafter, 1/10 of a volume of 3 M sodium acetate was added. The DNA was precipitated with 2 volumes of ethanol, washed with 70% ethanol, and dissolved in 100 l of distilled water. RAPD fingerprinting. RAPD or arbitrary-primer PCR is a PCR-based DNA fingerprinting method described by Akyopantz et al. (1) for detecting DNA sequence diversity among H. pylori isolates. By this method, for 20 patients the DNA fingerprints of 10 single colonies, differing in MTZ susceptibility, were studied. MIC of MTZ for H. pylori assessed by disk diffusion and E-test. The susceptibility to MTZ of the primary H. pylori populations from 156 patients was assessed by disk diffusion as well as by E-test. In the disk diffusion test, 110 isolates (70%) were found to be susceptible to MTZ, 9 (6%) were intermediately resistant to MTZ, and 37 (24%), including isolates from 13 of the 17 patients treated in the past with anti-h. pylori, MTZ-including therapies, were resistant to MTZ. Initially, for the E-test the concentration on the E-test strip closest to the edge of the bacterial lawn on the plate was regarded as the MIC. Determined in this way, the MICs for H. pylori populations from 106 patients (68%) were less than 4 g/ml. The MICs of 81 of these H. pylori populations were less than 1 g/ml. The MICs for 13 out of 156 isolates (8%) were between 4 and 8 g/ml. Out of 156 H. pylori populations, 37 (24%) were resistant to MTZ (MIC 8 g/ml). The MICs for 35 of these 37 H. pylori populations were more than 32 g/ml (Table 1). However, close examination of the E-test plates revealed that in 52 of the 156 H. pylori populations, either small or large Helicobacter colonies were growing within the zones of growth inhibition of the bacterial lawn. After the incubation was prolonged from 3 to 5 days, the colonies became even more clearly visible (Fig. 2). In the disk diffusion test plates, in 40 of these 52 cases colonies were within the zone of growth inhibition of the bacterial lawn. Reassessment of the MIC of MTZ for colonies growing at the edge of the inhibition zone in the 52 H. pylori populations with colonies within the zone of inhibition revealed MIC patterns identical to those in the initial test. Again, MTZ-R and MTZ-S H. pylori populations were found. To see if induction of resistance could occur under these in vitro conditions, for 25 H. pylori populations susceptible to MTZ (MIC, 0.38 to 1.0 g/ml) and without colonies growing within the zone of growth inhibition of the bacterial lawn, the MIC of MTZ was reassessed with bacteria growing at the edge of the inhibition zone. No significant change in MIC was observed and the zone of growth inhibition remained clear, indicating that MTZ resistance was not induced in these H. pylori populations. Determination of MIC of MTZ for H. pylori growing within the zone of growth inhibition. Fifty-two patients were found to be infected with H. pylori populations heterogeneous in their susceptibility to MTZ. For all these patients, either the H. MIC of MTZ for H. pylori ( g/ml) TABLE 1. Summary of MICs of MTZ assessed by E-test in this study No. of tested H. pylori populations found to have: Clear zone of inhibition Colonies within zone of inhibition Total no. of H. pylori populations Total

3 2160 WEEL ET AL. J. CLIN. MICROBIOL. FIG. 2. Examples of H. pylori populations showing no heterogeneity (A) and different levels of heterogeneity (B and C) in MTZ susceptibility. pylori colonies growing within the zone of growth inhibition were resuspended in 100 l of Dulbecco s modified Eagle medium and the MICs of MTZ were directly tested or the colonies were first subcultured for another 3 days on blood agar plates before susceptibility testing (E-test) was performed. Both test methods gave the same results. Furthermore, the estimation of the MIC for the H. pylori colonies growing within the zone of growth inhibition perfectly agreed with that found in this reassessment analysis. No increase in the MIC was found. When the MICs for the reassessed colonies were taken into consideration, the number of MTZ-S Helicobacter isolates decreased from 106 to 77 and the number of resistant isolates increased from 37 to 65. Comparison of DNA fingerprints of MTZ-S and MTZ-R H. pylori from populations heterogeneous in their susceptibility to MTZ. To determine whether MTZ-R and MTZ-S H. pylori strains isolated from the same patient also differed genetically, chromosomal DNA of the H. pylori strains was analyzed by RAPD fingerprinting. From 20 out of 28 H. pylori populations heterogeneous in their susceptibility to MTZ, at least 10 colonies with various MTZ susceptibilities were analyzed. In only 2 of 20 patients (10%) were the Helicobacter populations found to contain bacteria with distinct RAPD profiles. In those two cases, only one colony had an RAPD profile different from those of the other nine colonies tested (Fig. 3). Furthermore, in one of these two cases the MIC of MTZ for the bacterium was more than 32 g/ml. Effect of 24 h of anaerobic preincubation on MTZ susceptibility of H. pylori. The effect of 24 h of anaerobic preincubation on the MTZ susceptibility in MTZ-S and MTZ-R H. pylori populations isolated from one patient was studied and compared with the effect of anaerobic preincubation on H. pylori populations that were all MTZ-S or MTZ-R. Out of the 156 H. pylori populations, 12 (3 susceptible, 6 heterogeneous in their susceptibility, and 3 resistant to MTZ) were tested. All 12 populations were found to be very susceptible to MTZ (MIC, 0.38 to 1.0 g/ml). Furthermore, growth of colonies within the zone of growth inhibition of the bacterial lawn was not observed. This finding is consistent with that reported by Cederbrant et al. (2). shown that resistance to MTZ (MIC 8 g/ml) has a strong negative effect on the efficacy of MTZ-including therapeutic regimens (8, 11). Eradication rates were similarly poor when patients were infected with intermediately resistant H. pylori (MIC between 4 and 8 g/ml) (23). Accurate and precise determination of the MIC therefore is of major importance in deciding whether MTZ can be used as an effective antibiotic in Helicobacter eradication therapy. The aim of this study was to determine, by the E-test and disk diffusion, the exact MICs of MTZ for H. pylori strains isolated from peptic ulcer and functional dyspepsia patients. With the E-test 68, 8, and 24% of the isolates were susceptible, intermediately resistant, and resistant to MTZ, respectively. Initially, a discrepancy between the E-test and disk diffusion results was found for four H. pylori populations. However, use of the E-test revealed that 52 patients carried H. pylori populations heterogeneous in their susceptibility to MTZ. Of these H. pylori populations, 28 contained MTZ-S as well as MTZ-R bacteria. When these findings were taken into account, no longer 4 but 13 MICs determined by disk diffusion appeared to be underestimated. The finding that 33% of the H. pylori populations from infected patients are heterogeneous in their susceptibility to MTZ shows that the determinations of the MIC of MTZ should be based on the total primary H. pylori populations DISCUSSION Combination therapies that include MTZ are the most successful therapies used in eradicating H. pylori. It was previously FIG. 3. RAPD patterns of 10 H. pylori colonies growing within the zone of growth inhibition. The pattern in lane a is clearly distinct from those in lanes b to j. Lane k, 100-bp ladder.

4 VOL. 34, 1996 HETEROGENEITY IN MTZ-S H. PYLORI ISOLATES 2161 present in biopsy specimen cultures. Using suspensions prepared from single colonies from such H. pylori populations will reveal false-negative cases of MTZ susceptibility at high rates. In our study, 28 cases of MTZ resistance would otherwise have been missed. The impact of this finding is a topic of further study; however, it is likely that this small number of MTZ-R bacteria among so many MTZ-S bacteria will affect the efficacy of the MTZ-including therapy given to these patients. The outcome of such a study would reveal whether a precise determination of the MIC of MTZ for the H. pylori population from each individual patient is indeed demanded. Our results show that for the H. pylori populations heterogeneous in their susceptibility to MTZ, the E-test allows a more precise determination of the MIC of MTZ for clinical H. pylori isolates than does the disk diffusion test. Of the H. pylori populations heterogeneous in their susceptibility to MTZ, 10% were also heterogeneous at the genomic level as determined by PCR-based RAPD fingerprinting. This indicates that in general, H. pylori isolates with identical DNA fingerprints can be susceptible as well as resistant to MTZ. These findings are in agreement with the findings by Owen et al. (13) and Taylor et al. (18), which showed identical DNA fingerprints of MTZ-S and MTZ-R H. pylori strains isolated before and after treatment with MTZ. Furthermore, the results show the limitations and risk of possible misinterpretations of analysis of single colonies picked from the primary culture of H. pylori populations isolated from patients. The findings show that for patients treated with MTZ-including therapies, MTZ-R H. pylori isolates having RAPD profiles identical to those of the MTZ-S H. pylori isolates determined before the patients had undergone such therapy were not necessarily the results of induction of resistance during the course of MTZ-including therapy. Such resistance could have already been present in the pretreatment populations. On the other hand, when an MTZ-R H. pylori strain with an RAPD profile different from that found in the pretreatment isolate is isolated post-mtz-including therapy one can conclude that the patient is reinfected with a new MTZ-R H. pylori strain. Again, our results show that before MTZ treatment the H. pylori population can already consist of MTZ-R and MTZ-S H. pylori isolates having different DNA fingerprints. In anaerobic environments, MTZ, a nitroimidazole, is activated by reduction of its nitro group to a radical anion, which causes damage to DNA by a yet unknown mechanism (3). H. pylori isolated and analyzed in this study and resistant to MTZ under microaerophilic conditions becomes susceptible to this drug when preincubated for 24 h under anaerobic conditions. This finding not only confirms the results of Cederbrant et al. (2) but also shows the disappearance of H. pylori isolates that are heterogeneous in their susceptibility to MTZ after anaerobic preincubation. The mechanism causing the heterogeneity in MTZ susceptibility of H. pylori is unclear but may be compared to the heterogeneity in methicillin susceptibility for Staphylococcus aureus. However, this heterogeneous resistance of S. aureus to methicillin is unstable and depends on environmental conditions like temperature and osmolarity (9). On the other hand, mutations causing high-level resistance to methicillin in a subpopulation of an S. aureus culture have been described previously (15). Studying the effect of anaerobicity on susceptibility to MTZ clearly reveals that H. pylori can survive at least 24 h under anaerobic conditions. In our hands, however, culturing H. pylori under anaerobic conditions was never successful (unpublished observations). Recently, a minimum oxygen concentration of 0.5% has been found to allow poor growth (16). Optimal growth of H. pylori, however, occurs under microaerophilic conditions, and higher oxygen concentrations have not been found to affect H. pylori outgrowth negatively (24). Under microaerophilic conditions, the reduced MTZ is converted back to its parent compound by a process called futile cycling, thereby generating the formation of superoxide. In the end, hydroxyl free radicals are formed, causing cellular damage and death of the bacterium. It has been suggested that the relative levels of enzymes (superoxide dismutase and catalase) mediating protection from these toxic radicals by removing them differ in sensitive and resistant strains (2). Growing susceptible H. pylori in the presence of sub-mics of MTZ has been found to result in resistant bacteria, and the induction of superoxide dismutase and catalase has been postulated to occur, rendering susceptible bacteria resistant (21). However, the futile cycling theory was recently seriously questioned (16) when high and low oxygen tensions were found to have no effect on the activity of MTZ. In addition, in that study superoxide dismutase and catalase levels showed no correlation with resistance patterns. In our study, no increase in the MIC was found, even when bacteria growing at the edge of the inhibition zone (sub- MIC) were reassessed, indicating that under the conditions tested resistance to MTZ is not induced. 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