Comparison of primary and secondary antimicrobial minimum inhibitory concentrations for Helicobacter pylori isolated from Korean patients

International Journal of Antimicrobial Agents 28 (2006) 6–13

Comparison of primary and secondary antimicrobial minimum inhibitory concentrations for Helicobacter pylori isolated from Korean patients Jung Mogg Kim a,∗ , Joo Sung Kim b , Nayoung Kim b , Sang Gyun Kim b , Hyun Chae Jung b , In Sung Song b a

b

Department of Microbiology and Institute of Biomedical Science, Hanyang University College of Medicine, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, South Korea Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul 110-744, South Korea Received 28 December 2005; accepted 13 February 2006

Abstract In this study we assessed minimum inhibitory concentration (MIC) values and resistance rates of several antibiotics in 65 primary and 324 secondary Helicobacter pylori isolates from Korean patients. Primary resistance to amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, ciprofloxacin, levofloxacin and moxifloxacin was 18.5%, 13.8%, 66.2%, 12.3%, 32.3%, 33.8%, 21.5% and 21.5%, respectively. Secondary resistance was 31.3%, 85.1%, 70.1%, 0%, 89.6%, 35.8%, 32.8% and 32.8%, respectively. Sequence analysis of pbp1A in H. pylori strains with an amoxicillin MIC ≥2 ␮g/mL revealed C206T (Asp69 → Val), C1667G (Thr556 → Ser), A1684T (Asn562 → Tyr), A1777G (Thr593 → Ala) and C1798A (Pro600 → Thr) substitutions. Eleven (16.4%) of 67 treatment failures showed mixed infections with antibioticsusceptible and -resistant H. pylori. The most common multidrug resistance profile was to clarithromycin, metronidazole and azithromycin. These results indicate that MIC values of secondary isolates were higher than those of primary isolates and that resistance to amoxicillin is probably mediated through mutations in pbp1A. © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Antimicrobial resistance; MIC; Multidrug resistance

1. Introduction Helicobacter pylori is thought to infect >50% of the world’s population and may well be considered the most common chronic bacterial infection in humans [1]. In addition, H. pylori infection is recognised as a causal factor of chronic gastritis, peptic ulcer and gastric cancer [2]. As a result, H. pylori should be eradicated in patients with peptic ulceration to accelerate ulcer healing and to prevent longterm ulcer relapse [3,4]. Treatment regimens incorporating a proton pump inhibitor (PPI) and a combination of two or more antibiotics, including amoxicillin, clarithromycin, metronidazole or tetracycline, are considered to be most ∗

Corresponding author. Tel.: +82 2 2220 0645; fax: +82 2 2282 0645. E-mail address: [email protected] (J.M. Kim).

efficacious [5]. However, antibiotic resistance is a growing problem [6–11]. The frequencies of resistance to antibiotics have varied widely according to geographical regions and subgroups within study populations [6,7,12]. For example, metronidazole resistance is found in 10–50% of H. pylori-positive adults in the developed world [6,7,12] whereas virtually all strains are resistant in developing countries [11]. The clarithromycin resistance rate, which is relatively low (2–15%) [6,7,9,13], has also been increasing. In addition, resistance to metronidazole and clarithromycin following treatment failure (secondary resistance) has increased to 40–70% for metronidazole, 50–70% for clarithromycin and 30–90% for both antibiotics [14–17]. We recently reported that primary resistant strains for several antibiotics, such as clarithromycin, amoxicillin, metronidazole, tetracycline

0924-8579/$ – see front matter © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2006.02.015

J.M. Kim et al. / International Journal of Antimicrobial Agents 28 (2006) 6–13

and fluoroquinolones (including ciprofloxacin, levofloxacin and moxifloxacin), had continuously increased from 1987 to 2003 in Korean patients [9,10]. Consistent with this, eradication rates for first-line therapy decreased to less than 75% in Korea [18]. Treatment for H. pylori infection is usually initiated on an empirical basis and a resistant infecting strain hampers successful eradication. Such outcomes clearly indicate a need for exploring the status of antibiotic resistance in H. pylori. Nevertheless, little information is available regarding the distribution of the minimum inhibitory concentrations (MICs) of primary and secondary H. pylori isolates in Korea. The aim of this study was to assess MICs and antibiotic resistance rates in primary and secondary H. pylori isolates from Korean patients.

2. Materials and methods 2.1. Patients and H. pylori strains For the determination of MIC values in secondary isolates, 324 strains of H. pylori were isolated from 67 patients (aged 59.3 ± 2.4 years; 46 male, 21 female) in Seoul National University Hospital, South Korea, from 2004 to 2005. All patients received PPI triple therapy consisting of amoxicillin (1000 mg twice a day (bid)) and clarithromycin (500 mg bid) for 1 week. A total of six specimens (three specimens from different sites of the antrum and three specimens from different sites of the gastric body) were obtained from each patient. In the first culture isolated from the biopsy specimen, one colony was picked up. Only 324 H. pylori strains were isolated from 67 patients because bacteria could not be isolated from 78 biopsy specimens. Thus, 324 strains from 67 patients were isolated from different sites of each patient. The diagnoses for the patients with secondary isolates included gastric ulcer (n = 42) and duodenal ulcer (n = 25). For the determination of MIC values in primary isolates, 65 strains of H. pylori were isolated from antral gastric mucosal biopsy specimens of 65 patients in Seoul National University Hospital (aged 58.1 ± 10.2 years; 37 male, 28 female) [9]. Only one H. pylori strain was isolated from each patient. The diagnoses for the patients with primary isolates included gastritis (n = 4), gastric ulcer (n = 13), duodenal ulcer (n = 47) and gastric cancer (n = 1). No patient had taken antibiotics, PPIs or non-steroidal anti-inflammatory drugs during the preceding 3 months. The H. pylori strains were cultured under microaerobic conditions (5% O2 , 10% CO2 , 85% N2 ) as previously described [19]. All stock cultures were stored at −70 ◦ C in Brucella broth supplemented with 15% glycerol. These preparations were thawed and subcultured for experiments. 2.2. Determination of MICs The MIC values of the H. pylori isolates to amoxicillin (Sigma Chemical Co., St Louis, MO), clarithromycin (Abbott

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Laboratories, Abbott Park, IL), metronidazole (Sigma), tetracycline (Sigma), azithromycin (Groton Laboratories, Pfizer Inc., Groton, CT), ciprofloxacin (Sigma), levofloxacin (Sigma) and moxifloxacin (Bayer AG Pharmaceuticals, Germany) were examined using the serial two-fold agar dilution method as described previously [9,20]. Briefly, bacteria were subcultured on Mueller–Hinton agar supplemented with 5% defibrinated sheep blood for 48 h. The bacterial suspension, adjusted to 1 × 107 colony-forming units, was inoculated directly onto each antibiotic-containing agar dilution plate. After 72 h of incubation, the MIC of each antibiotic was determined. Quality control was performed using H. pylori ATCC 43504. The resistance breakpoints for amoxicillin, metronidazole and tetracycline were defined as ≥0.5, >8 and >4 ␮g/mL, respectively [12]. The breakpoint for clarithromycin was set at >1.0 ␮g/mL [20] and those for azithromycin, ciprofloxacin, levofloxacin and moxifloxacin were provisionally defined as >1.0 ␮g/mL. If secondary resistance to the antibiotics occurred in at least one of the six isolates, the patient was designated as a patient with antibioticresistant H. pylori. 2.3. Polymerase chain reaction (PCR) amplification and nucleotide sequence for mutations in penicillin-binding protein (PBP) 1A Helicobacter pylori genomic DNA was extracted as reported previously [9]. To detect mutations in PBP1A gene (pbp1A), the following oligonucleotide primers were used: pbp1A-1, for identification of mutation at Asp69 (5 GCC ATT CTT ATC GCT CAA GTT-3 and 5 -TCT CGT GTG AGC ACC ATG TT-3 (341 bp)); and pbp1A-2 for identification of mutations at Thr556, Asn562, Thr593 and Pro600 (5 -CGC TAG CAT GAT AGT TAC AGA CAC GAG C-3 and 5 -CGT TCT TCG CTA TCG TCT GTT C-3 (975 bp)). PCR cycling conditions consisted of 35 cycles of 1 min denaturation at 94 ◦ C, 1 min annealing at 57 ◦ C and 1 min extension at 72 ◦ C. Sequencing was performed on both strands of the non-restricted amplicons using an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA). The nucleotide sequence for the pbp1A genes from amoxicillin-susceptible H. pylori 69A was deposited in GenBank under accession number AF315503 [21]. 2.4. Detection of β-lactamase The chromogenic cephalosporin method was used to test for production of ␤-lactamase [22]. Cefinase disks (Becton Dickinson Microbiology Systems, Cockeysville, MD) impregnated with nitrocefin, a chromogenic cephalosporin, were moistened with a drop of sterile distilled water and then several well isolated fresh colonies from brain–heart infusion agar plates containing 8 ␮g/mL amoxicillin were selected and smeared on the disk surface. ␤-Lactamase activity was read as positive by a change in colour of the chro-

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mogenic cephalosporin after 12 h of incubation at room temperature.

3. Results and discussion 3.1. Prevalence of secondary antibiotic resistance and MICs MICs were determined for four classes of antibiotics: ␤-lactams such as amoxicillin; macrolides such as clarithromycin and azithromycin; tetracycline; and fluoroquinolones such as ciprofloxacin, levofloxacin and moxifloxacin. The distribution of amoxicillin MIC values ranged from 0.0625 ␮g/mL to 2 ␮g/mL in the secondary isolates. Interestingly, amoxicillin MIC values in the secondary H. pylori isolates had two peaks (≤0.0625 ␮g/mL and 0.5 ␮g/mL), indicating a kind of dual distribution (Fig. 1A). Although two strains in the primary isolates showed MICs of 8 ␮g/mL (Fig. 1A), the MIC values of secondary isolates showed a shift to higher concentrations compared with primary isolates. Currently, the official breakpoint for amoxicillin resistance has not been designated for H. pylori isolates by the National Committee for Clinical Laboratory Standards (NCCLS). However, several studies have reported that the secondary resistance rate of amoxicillin was 0–1.0% when 0.5 ␮g/mL was designated as the provisional breakpoint of amoxicillin resistance [23,24]. According to this provisional breakpoint, we found a secondary resistance rate to amoxicillin of 31.3% (Table 1). This secondary resistance rate is higher than that found in other countries; the rates were reported to be 1.0% in Italy [24], 0% in Eastern Europe [23] and 0% in European countries [11]. In addition, primary resistance to amoxicillin in Korean patients (18.5%) is higher than that in other countries [23,24]. Table 1 Prevalence of primary and secondary antibiotic resistance among patients with Helicobacter pylori isolatesa Breakpoint for resistance (␮g/mL)

Amoxicillin Clarithromycin Metronidazole Tetracycline Azithromycin Ciprofloxacin Levofloxacin Moxifloxacin

≥0.5 >1.0 >8.0 >4.0 >1.0 >1.0 >1.0 >1.0

% Resistance (number of patients with resistant H. pylori) Secondary (n = 67)b

Primary (n = 65)

31.3 (21) 85.1 (57) 70.1 (47) 0 (0) 89.6 (60) 35.8 (24) 32.8 (22) 32.8 (22)

18.5 (12) 13.8 (9) 66.2 (43) 12.3 (8) 32.3 (21) 33.8 (22) 21.5 (14) 21.5 (14)

a Minimum inhibitory concentrations were determined by the agar dilution method. b If secondary resistance to the antibiotic occurred in at least one of the six isolates, the patient was designated as a patient with antibiotic-resistant H. pylori.

The pattern of secondary clarithromycin MICs demonstrated a typical bimodal distribution, with the MICs of clarithromycin-resistant strains ranging from 4 ␮g/mL to ≥256 ␮g/mL (Fig. 1B). In addition, the MIC values of secondary isolates showed a definite shift to higher concentrations compared with primary isolates. This is typical evidence of antibiotic resistance induced by continuous exposure to clarithromycin in vivo. In vitro resistance of H. pylori to clarithromycin has been found to equate with in vivo resistance [7]. Moreover, clarithromycin administration may select a resistant strain and this acquired resistance to clarithromycin is stable [11], which could explain the bimodal distribution of clarithromycin MICs. The NCCLS has designated the official breakpoint for clarithromycin resistance as 1 ␮g/mL [20]. According to this official breakpoint, the prevalence of secondary clarithromycin resistance was 85.1% compared with a primary resistance rate of 13.8% (Table 1). The distribution of metronidazole MICs followed a continuous spectrum ranging from 0.25 ␮g/mL to ≥256 ␮g/mL. The peak MIC of secondary isolates was 32 ␮g/mL compared with 64 ␮g/mL in primary isolates (Fig. 1C). In contrast, the secondary resistance rate was 70.1% compared with a primary resistance rate of 66.2% using a provisional breakpoint for metronidazole resistance of 8 ␮g/mL (Table 1). Based on this result, metronidazole may not be used therapeutically in Korean patients following treatment failure. Although primary resistance to tetracycline is known to be rare [25], the present study showed that the primary resistance rate to tetracycline in Korean patients was 12.3% when the provisional breakpoint for tetracycline resistance was 4 ␮g/mL (Table 1). Strangely, there were no H. pylori strains with a tetracycline MIC > 4 ␮g/mL in the patients with treatment failure (Fig. 1D). There is no obvious explanation for this reduction in resistance in the secondary H. pylori isolates. Further study is necessary to clarify tetracycline resistance in Korean patients. Azithromycin, ciprofloxacin, levofloxacin and moxifloxacin have been proposed as components of a triple PPIbased regimen [18,26,27]. In particular, fluoroquinolones have been widely used in second-line therapy for H. pylori eradication in Korea [18]. In the present study, the azithromycin MIC values in secondary isolates were higher than those in primary isolates (Fig. 1E). The peak MIC of secondary isolates was 8 ␮g/mL compared with 0.5 ␮g/mL in primary isolates. Consistent with this, the prevalence of secondary azithromycin resistance was 89.6% compared with primary resistance of 32.3%, provisionally assigning 1 ␮g/mL as the breakpoint for azithromycin resistance (Table 1). This finding may be due to cross-resistance with clarithromycin. Fluoroquinolones have been recommended for treatment of H. pylori infection in Korea [18]. In this study, the MIC patterns of fluoroquinolones, including ciprofloxacin, levofloxacin and moxifloxacin, showed typical bimodal distributions (Fig. 1F–H). Specifically, the MIC patterns

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Fig. 1. Minimum inhibitory concentration (MIC) distributions of antimicrobial agents for Helicobacter pylori. Sixty-five patients who had not taken antibiotics, proton pump inhibitors (PPIs) or non-steroidal anti-inflammatory drugs during the preceding 3 months (primary isolates) and 67 patients following failure of PPI triple therapy incorporating amoxicillin (1000 mg twice a day (bid)) and clarithromycin (500 mg bid) for 1 week (secondary isolates) were evaluated. MICs were determined by the agar dilution method. (A) Amoxicillin; (B) clarithromycin; (C) metronidazole; (D) tetracycline; (E) azithromycin; (F) ciprofloxacin; (G) levofloxacin; and (H) moxifloxacin.

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of levofloxacin and moxifloxacin were similar, as shown in Fig. 1G and H. However, the MICs in secondary isolates were higher than those in primary isolates. The peak MIC of secondary isolates was 0.5 ␮g/mL compared with 0.25 ␮g/mL in primary isolates. The proportions of secondary H. pylori strains with a MIC >1 ␮g/mL for ciprofloxacin, levofloxacin and moxifloxacin were 35.8%, 32.8% and 32.8%, respectively (Table 1). These secondary resistance rates are higher than those of the primary isolates (33.8%, 21.5% and 21.5%, respectively). Overall, MIC values of amoxicillin, clarithromycin, metronidazole, azithromycin and ciprofloxacin were higher in secondary isolates than in primary isolates in H. pylori strains isolated from Korean patients. 3.2. Mutations in H. pylori gene pbp1A and resistance to amoxicillin A recent study demonstrated that amoxicillin resistance was due in part to an increased diffusional barrier to ␤lactam antibiotics in the resistant strain [28]. In this study, ␤-lactamase activity was not detected in the amoxicillinresistant strains or the amoxicillin-susceptible strains, suggesting that amoxicillin resistance was not due to the presence of ␤-lactamase activity. Since a pbp1 gene mutation is known to be involved in amoxicillin resistance in H. pylori isolates [21,22,29,30], we asked whether pbp1A gene mutations may be associated with high amoxicillin MIC values. We examined pbp1A mutations using four secondary strains with an amoxicillin MIC of 2.0 ␮g/mL and two primary strains with an amoxicillin MIC of 8.0 ␮g/mL. As shown in Table 2, the mutations in primary H. pylori isolates were at C206T (Asp69 → Val), C1667G (Thr556→Ser), A1684T (Asn562→Tyr) and A1777G (Thr593 → Ala). One strain among the secondary H. pylori isolates showed an additional mutation of C1798A (Pro600 → Thr). No mutations were found in strains with an amoxicillin MIC < 0.125 ␮g/mL. These results suggest that mutations of pbp1A may contribute to amoxicillin resistance in H. pylori. However, transformation with the mutated pbp1 gene rendered bacteria only moderately amoxicillin resistant (MICs 0.5–1 ␮g/mL) [30], suggesting that the pbp1 mutation is not the sole cause of acquired resistance. Future studies are necessary to investigate genetic mutations that cause amoxicillin resistance in H. pylori.

3.3. Distribution of antimicrobial-resistant H. pylori according to the site of isolation Several studies reported the possibility that a given host may be infected with antibiotic heteroresistant H. pylori [31–33]. In addition, mixed infection by clarithromycinsusceptible and -resistant H. pylori strains could prevent eradication of H. pylori by the triple therapy and result in selection for resistant strains [33]. To determine the distribution of resistant H. pylori strains by the site of isolation, a total of six specimens (three from different sites of the antrum and three from different sites of the gastric body) were obtained from each patient following therapy failure. Patients with resistant H. pylori strains were then divided into three groups: the Ra/Sb group (patients with antibiotic-resistant strains isolated from the antrum and antibiotic-susceptible strains isolated from the body); the Sa/Rb group (patients with antibiotic-susceptible strains isolated from the antrum and antibiotic-resistant strains isolated from the body); and the Ra/Rb group (patients with antibiotic-resistant strains isolated both from the antrum and the body). Eleven (16.4%) of the 67 patients showed mixed infection with antibioticsusceptible and -resistant H. pylori. As shown in Table 3, 11 patients (16.4%) had amoxicillin-resistant strains both in antrum and body sites. However, seven patients (10.4%) had resistant strains in the antrum but not in the body, and three patients (4.5%) had resistant strains in the body but not in the antrum. In the case of clarithromycin resistance, 11.9% of patients had clarithromycin-resistant strains in the antrum but not in the body, and 4.5% had resistant strains in the body but not in the antrum. This phenomenon was also observed in metronidazole-, ciprofloxacin-, levofloxacinand moxifloxacin-resistant strain infected cases (Table 3). These results indicate that mixed infection with antibioticsusceptible and -resistant H. pylori strains exists within the stomach of individual patients. Considering that isogenic variation of an H. pylori strain leading to heteroresistant antibacterial phenotypes has been reported in a single host [31], it is possible that developing genomic diversity in a single strain could modulate an antibiotic heteroresistant H. pylori in vivo. The study of location-specific genotypes will be necessary to understand further the evolution of resistance within the host. In the present study, the secondary resistance rates may be falsely increased as the isolation of one resistant strain from

Table 2 Amino acid changes in penicillin-binding protein (PBP) 1A in amoxicillin-resistant Helicobacter pylori strainsa Strain

Amoxicillin MIC (␮g/mL)a

Substitution in PBP1A

Primary isolate #1 Primary isolate #2 Secondary isolate #3 Secondary isolate #4 Secondary isolate #5 Secondary isolate #6

8 8 2 2 2 2

Asp69 → Val; Thr556 → Ser; Asn562 → Tyr; Thr593 → Ala Asp69 → Val; Thr556 → Ser; Asn562 → Tyr; Thr593 → Ala Asp69 → Val; Thr556 → Ser; Asn562 → Tyr; Thr593 → Ala; Pro600 → Thr Asp69 → Val; Thr556 → Ser; Asn562 → Tyr; Thr593 → Ala Asp69 → Val; Thr556 → Ser; Asn562 → Tyr; Thr593 → Ala Asp69 → Val; Thr556 → Ser; Asn562 → Tyr; Thr593 → Ala

a

Minimum inhibitory concentrations (MICs) were determined by the agar dilution method.

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Table 3 Distribution of secondary antimicrobial-resistant Helicobacter pylori strains according to the site of isolationa Antimicrobial agent (breakpoint)

Amoxicillin (≥0.5 ␮g/mL) Clarithromycin (>1.0 ␮g/mL) Metronidazole (>8.0 ␮g/mL) Azithromycin (>1.0 ␮g/mL) Ciprofloxacin (>1.0 ␮g/mL) Levofloxacin (>1.0 ␮g/mL) Moxifloxacin (>1.0 ␮g/mL)

No. of patients with resistant isolates (% of 67 patients) Ra/Sb

Sa/Rb

Ra/Rb

Total

7 (10.4) 8 (11.9) 5 (7.5) 0 2 (3.0) 1 (1.5) 1 (1.5)

3 (4.5) 3 (4.5) 2 (3.0) 0 3 (4.5) 2 (3.0) 2 (3.0)

11 (16.4) 46 (68.7) 40 (59.7) 60 (89.6) 19 (28.4) 19 (28.4) 19 (28.4)

21 (31.3) 57 (85.1) 47 (70.1) 60 (89.6) 24 (35.8) 22 (32.8) 22 (32.8)

Ra/Sb, antimicrobial-resistant H. pylori isolates from the antrum and susceptible isolates from the body; Sa/Rb, antimicrobial-susceptible H. pylori isolates from the antrum and resistant isolates from the body; Ra/Rb, antimicrobial-resistant H. pylori isolates both from the antrum and body sites. a Minimum inhibitory concentrations were determined by the agar dilution method.

any gastric site defined the patient as resistant. However, considering that mixed infection with clarithromycin-susceptible and -resistant H. pylori strains could prevent the eradication of H. pylori by triple therapy [33], there is a possibility that

existence of a resistant strain in any site of the stomach may be one of the reasons for treatment failure in a patient receiving antibiotic treatment. Our study may support this hypothesis. Therefore, regarding antibiotic treatment, the existence of a

Table 4 Multidrug resistance of Helicobacter pylori isolatesa Type of multidrug resistance

AMX + MT AMX + CIP CLR + AZM MT + AZM MT + CIP MT + TET AMX + CLR + AZM AMX + MT + AZM AMX + AZM + LVX CLR + MT + AZM CLR + AZM + CIP MT + LVX + MXF CIP + LVX + MXF AMX + CLR + MT + AZM AMX + AZM + LVX + MXF CLR + MT + AZM + CIP MT + TET + AZM + CIP MT + AZM + LVX + MXF MT + CIP + LVX + MXF AMX + CLR + MT + AZM + LVX AMX + CLR + MT + AZM + MXF AMX + MT + TET + AZM + CIP AMX + MT + CIP + LVX + MXF AMX + AZM + CIP + LVX + MXF CLR + AZM + CIP + LVX + MXF CLR + MT + AZM + LVX + MXF MT + AZM + CIP + LVX + MXF AMX + CLR + MT + CIP + LVX + MXF AMX + CLR + AZM + CIP + LVX + MXF AMX + MT + AZM + CIP + LVX + MXF CLR + MT + AZM + CIP + LVX + MXF AMX + CLR + MT + AZM + CIP + LVX + MXF

No. of patients (% of total) Secondary (n = 67)b

Primary (n = 65)

– – 9 (13.4) 3 (4.5) – – 1 (1.5) 2 (3.0) 1 (1.5) 21 (31.3) 1 (1.5) – – 6 (9.0) – 1 (1.5) – – – 2 (3.0) 1 (1.5) – – 1 (1.5) 4 (6.0) 1 (1.5) – 1 (1.5) 3 (4.5) 1 (1.5) 7 (10.4) 9 (13.4)

1 (1.5) 1 (1.5) 2 (3.1) 1 (1.5) 3 (4.6) 1 (1.5) – 1 (1.5) – 5 (7.7) – 1 (1.5) 1 (1.5) 2 (3.1) 1 (1.5) – 1 (1.5) 1 (1.5) 3 (4.6) – – 2 (3.1) 1 (1.5) – – – 3 (4.6) – 1 (1.5) – 1 (1.5) 1 (1.5)

AMX, amoxicillin; MT, metronidazole; CIP, ciprofloxacin; CLR, clarithromycin; AZM, azithromycin; TET, tetracycline; LVX, levofloxacin; MXF, moxifloxacin. a Minimum inhibitory concentrations were determined by the agar dilution method. Resistance breakpoints for amoxicillin and metronidazole were defined as ≥0.5 ␮g/mL and >8 ␮g/mL, respectively. The breakpoints for clarithromycin, azithromycin, ciprofloxacin, levofloxacin and moxifloxacin were defined as >1.0 ␮g/mL. b Nine of 67 patients were infected with at least two H. pylori strains showing two or more different multidrug resistance patterns.

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resistant strain in any site of the stomach would be designated resistance. [4]

3.4. Multidrug resistance (MDR) of H. pylori In the present study, 60 (89.6%) of 67 patients with treatment failure had H. pylori strains resistant to two or more antimicrobial agents (i.e. multidrug resistance (MDR)). Nine patients (13.4%) were infected with at least two H. pylori strains showing two or more different MDR patterns. As shown in Table 4, the most common pattern of MDR was clarithromycin, metronidazole and azithromycin. In secondary isolates, the resistance rate to both amoxicillin and clarithromycin was 34.3% (23 of 67 patients) and the resistance rate to both clarithromycin and metronidazole was 73.1% (49 of 67 patients). In comparison, 34 (52.3%) of 65 primary patients (i.e. not taking antibiotics, PPIs or non-steroidal anti-inflammatory drugs during the preceding 3 months) showed MDR. The resistance rate to both amoxicillin and clarithromycin was 6.2% in primary isolates. Interestingly, all clarithromycin-resistant strains were also azithromycin resistant. This result is consistent with a Japanese report showing 92% of clarithromycin-resistant strains were also azithromycin resistant [34]. Based on cross-resistance among macrolides [35], there is a possibility that the rise in prevalence of clarithromycin resistance may be associated with the increasing use of macrolides in clinical practice. In conclusion, we found that the MIC values of secondary isolates were higher than those of primary isolates and that resistance to amoxicillin is probably mediated through mutations in pbp1A. In addition, this study showed that mixed infection with antibiotic-susceptible and -resistant H. pylori strains may exist within the stomach of an individual patient.

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Acknowledgments We thank Mi-Soon Kim and Han-Jin Lee for their excellent technical assistance. We also thank Abbott Laboratories (Abbott Park, IL) for the gift of clarithromycin, Groton Laboratories (Pfizer Inc., Groton, CT) for the gift of azithromycin and Bayer AG Pharmaceuticals (Germany) for the gift of moxifloxacin. This work was supported by a grant from the National Research Lab programme (NRL M1040000001904J0000-01910).

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