Journal of Pediatric Gastroenterology & Nutrition:
Original Articles: Gastroenterology
High Helicobacter pylori Resistance to Metronidazole and Clarithromycin in Brazilian Children and Adolescents
Ogata, Silvio K.*; Godoy, Anita P. Ortiz*; da Silva Patricio, Francy R.†; Kawakami, Elisabete*
*Division of Pediatric Gastroenterology, Hepatology, and Nutrition
†Department of Clinical Pathology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Address correspondence and reprint requests to Silvio K. Ogata, Rua Pedro de Toledo, 443 Vila Clementino, São Paulo 04039-031, Brazil (e-mail: firstname.lastname@example.orgemail@example.com).
Received 20 March, 2012
Accepted 1 February, 2013
The study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP.
The authors report no conflicts of interest.
Objective: The aim of the present study was to assess the primary and secondary resistance of Helicobacter pylori strains to clarithromycin, amoxicillin, furazolidone, tetracycline, and metronidazole, the conventional antibiotics presently used in Brazilian children and adolescents.
Methods: Seventy-seven consecutive H pylori strains, 71 of 77 strains obtained from patients without previous eradication treatment for H pylori infection, and 6 strains from patients in whom previous eradication treatment had failed.
Results: Global rate of resistance was 49.3% (38/77): 40% of strains were resistant to metronidazole, 19.5% to clarithromycin, and 10.4% to amoxicillin. All of the tested H pylori strains were susceptible to furazolidone and tetracycline. Multiple resistance were detected in 18.2% (14/77 patients) of the strains: 6 of 14 (43%) simultaneously resistant to clarithromycin and metronidazole; 5 of 14 (36%) to amoxicillin and metronidazole; 2 of 14 (14%) to amoxicillin, clarithromycin, and metronidazole; and 1 of 14 (7%) to clarithromycin and amoxicillin.
Conclusions: The high resistance rate to metronidazole and clarithromycin observed in clinical H pylori isolates can exclude these antimicrobials in empirical eradication treatment in Brazil. Otherwise, furazolidone and tetracycline presented no resistance. Properly assessing the risks and benefits, these 2 antimicrobials and their derivatives could be used in empirical eradication schedules, both associated with amoxicillin, which showed a low resistance rate despite its wide use in pediatric patients.
Helicobacter pylori eradication is the most important target in the treatment of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and prevention of gastric cancer development (1). Antimicrobial resistance, specially to clarithromycin, is the main cause of the eradication failure (2). H pylori infection is acquired during the first 5 years of age, but there are far fewer indications for eradication in infected children than in adult patients (3). As a consequence, there is little consensus on testing H pylori susceptibility to antimicrobials, mainly when retreatment is required because of failure in the first-line regimen (1).
Standard triple therapy (clarithromycin or metronidazole and amoxicillin) based on susceptibility test increases eradication rates (4), and it remains the first choice in regions with proven low clarithromycin resistance rates (1). An effective anti—H pylori therapy with susceptible strains should always reach 100% eradication per protocol (5), but in developing countries, even an efficient eradication regimen shows lower rates and children usually present rates 10% lower than adults using the same triple therapy (3).
The efficacy of H pylori treatment differs according to the geographical region (6). In developing countries, the resistance rate to antimicrobials is higher because of an increased use of these drugs. Then, antimicrobial choice should be determined according to resistance pattern to predict the range of possible outcomes (5,7). Thus, the growing emergence of resistant strains shows the need to know primary and secondary resistance rates in each region by routine susceptibility tests (8).
H pylori eradication requires an approach similar to those used in other infectious diseases, which is based on susceptibility tests, regional susceptibility, or both. Nevertheless, antimicrobials are empirically prescribed (9).
In Brazilian adults, metronidazole resistance ranges from 52.9% to 55% (10,11), resistance to furazolidone occurs in 4% and resistance to amoxicillin occurs in 29% (12). Tetracycline presented resistance rates of 9% (11) and clarithromycin of 9.8% (10). No studies exist regarding antimicrobial susceptibility in infected children, and the resistance pattern must be known to assist in choosing the antimicrobial schedule and to predict the probability of eradication failure (8). This is mainly because unlike American children, studies show low eradication rates using triple therapy in Brazilian children (13,14)
The aim of the present study was to assess the resistance rate of H pylori strains isolated in gastric biopsies from children and adolescents to clarithromycin, amoxicillin, furazolidone, tetracycline, and metronidazole, the most commonly used antimicrobials in eradication treatment.
From February 2008 to August 2009, 77 consecutive H pylori isolates, 71 strains obtained from patients without previous eradication treatment for H pylori infection, and 6 strains from patients in whom previous eradication treatment had failed were obtained from children and adolescents (range 3–20 years, mean age 11.1 ± 3.9 years, median 10.8 years; M/F: 1:1.08), 42 (54.5%) whites, and 35 (45.5%) nonwhite, who were subject to endoscopic examination to evaluate dyspeptic symptoms at Hospital São Paulo, Universidade Federal de São Paulo, and Cândido Fontoura Children's Hospital (Table 1). The findings were antral nodularity in 62 (80.5%) patients, duodenal ulcer in 5 (6.5%), antral erosive gastritis in 4 (5.2%), normal endoscopy in 3 (3.8%), gastric erythema in 3 (3.8%), and erosive esophagitis and gastric polyp (1.3%).
Previous use of proton pump inhibitors or antimicrobials, at least for 1 month, or associated chronic disease, were exclusion criteria. The study was approved by the Federal University of São Paulo ethics committee, and informed consents were signed.
Biopsy Sampling and Bacterial Strains
Two antral biopsy specimens were collected and transported to the microbiology laboratory in brain-heart infusion (BHI) broth plus glycerol (10%) preserved at a temperature of 4°C. Gastric biopsy specimens were homogenized and 5-μL solution was inoculated into selective BHI agar base (DIFCO, Lawrence, KS) containing 7% to 10% defibrinated sheep blood, vancomycin (10 mg/L), trimethoprim (5 mg/L), cefsulodin (5 mg/L), and amphotericin B (5 mg/L) (H pylori selective medium, Dent Supplement Oxoid, Basingstoke, Hampshire, UK). All of the plates were incubated for 10 days at 37°C in a microaerobic atmosphere (10% CO2, 85% N2, 5% O2) at 95% humidity (Microaerobac, Probac do Brasil, São Paulo, Brazil). H pylori INCQS 00380—ATCC 43504 strain was used as control.
Isolates that produced a bacterial growth demonstrating typical morphologic features by dark-field microscopy and produced urease, oxidase, and catalase were stored at −70°C in BHI broth containing 30% glycerol.
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed by the agar dilution method in accordance with Clinical Laboratory Standards Institute protocols (2006, M7-A5). For antimicrobial agents without a Clinical Laboratory Standards Institute–recommended breakpoint, cutoff value was selected based on the literature. Five milliliters of frozen isolates were subcultured onto BHI agar containing 10% defibrinated sheep blood and incubated for 3 days at 37°C under microaerophilic conditions. The colonies were suspended in BHI broth and adjusted to McFarland 4 turbidity standard (approximately 1 × 108 cfu/mL), then inoculated onto Mueller-Hinton agar plates using a multipoint replicating device delivering 2 μL of inoculum.
The final concentrations of clarithromycin (Abbott Laboratories, Chicago, IL), tetracycline, furazolidone, and amoxicillin (Sigma Aldrich Chemie, Steinheim, Germany) ranged from 0.015 to 64 μg/mL. Metronidazole (Sigma Aldrich Chemie) concentration ranged from 0.015 to 256 μg/mL. The plates were incubated at 37°C under microaerophilic conditions (5% O2, 10% CO2, and 85% N2 at 95% humidity) for 72 hours. Isolates were considered resistant if the minimum inhibitory concentration (MIC) was ≥2 μg/mL to amoxicillin and furazolidone, ≥4 μg/mL to tetracycline, and ≥8 μg/mL to metronidazole. Clarithromycin isolates were considered resistant with MIC ≥2 μg/mL, and intermediary if MIC was 0.5 μg/mL.
Statistical analysis of categorical data from resistant and sensible H pylori isolates was performed by χ2 test. Differences with P < 0.05 were considered statistically significant. Susceptibility and resistance rates were calculated by proportion and expressed as percentage. MIC 50 and MIC 90 were determined as minimum concentration of antimicrobial that is capable to inhibit the development of 50% and 90% of bacteria isolates, respectively.
Resistance was observed in 49.3% (38/77) of H pylori strains; almost all of them (40.2%) presented resistance to metronidazole, 19.5% to clarithromycin, and 10.4% to amoxicillin. Furazolidone and tetracycline showed no resistance (0%). Fourteen (18.2%) H pylori isolates showed multiple resistance: 6 of 14 (43%) to clarithromycin and metronidazole; 5 of 14 (36%) to amoxicillin and metronidazole; 2 of 14 (14%) to amoxicillin, clarithromycin, and metronidazole; and 1 of 14 (7%) to clarithromycin and amoxicillin. All strains resistant to amoxicillin presented multiple resistance.
Resistance rates, MIC range, MIC 50, and MIC 90 are shown in Table 2. MIC 90 was near the cutoff value (≤1 μg/mL) to clarithromycin and 3 log2 over recommended cutoff (64 μg/mL) to metronidazole; however, MIC 50 was within the antimicrobial susceptibility patterns of tested antibiotics (Table 2).
Secondary resistance was observed in 3 of 6 (50%) patients who had been treated previously: 2 had used clarithromycin, amoxicillin, and proton pump inhibitor; one of them showed resistance to clarithromycin and another to amoxicillin and metronidazole; and the third patient had used doxycycline and furazolidone and was resistant to clarithromycin and metronidazole. There were no association of resistant H pylori strain with race, sex, age (cutoff 12 years), and familial peptic disease (P > 0.05) (Table 1).
In the present study, 5 of the most used antimicrobials for H pylori eradication in Brazilian children were evaluated (13–15). Our results showed resistance to 3 antimicrobials: metronidazole, clarithromycin, and amoxicillin. As expected in a developing country, metronidazole presented the highest resistance rate, that is, 40.2%, probably caused by disseminated consumption to intestinal parasitic and gynecological infections. This result is similar to Brazilian adults (10,11) and children in other developing countries (16). Unfortunately, metronidazole should not be prescribed empirically when resistance is >40% (1); however, metronidazole is a prodrug that needs activation by nitroreductase enzymes, and the antimicrobial action depends on the oxireduction process; H pylori has several nitroreductases that can activate the drugs (17). Then, in vitro resistance cannot impede the use of metronidazole because resistance could be beaten by a higher dosage (17), but it needs further study.
Clarithromycin is one of the most widely used antimicrobials to eradicate H pylori infection, and resistance is the main reason for eradication failure in adults (18) and children (19). Previous local studies using clarithromycin in empirical antimicrobial treatment showed lower eradication rates comparing with triple therapy without clarithromycin (13–15). Our results showed a high resistance rate of H pylori to clarithromycin (19.5%), similar to European children. Megraud (20) observed primary resistance rate of 12.4% to 23.5% and Koletzko et al (21) reported a global resistance to clarithromycin of 24% (primary resistance in 20% and secondary in 42%) in multicenter studies. Our rate of resistance to clarithromycin probably results from the disseminated use of this antimicrobial for respiratory infectious diseases. Considering that clarithromycin should not be prescribed empirically if resistance rate ranges from 15% to 20% (1), and 1 study showed that no eradication was achieved when strains are resistant (8), clarithromycin should be precluded in empirical eradication therapy in Brazil; however, the rate of primary resistance to clarithromycin depends on the assessed population and the period of the study. A recent study showed that resistance rates to clarithromycin were stable in Belgian children, probably because of the decreasing prescription of macrolides (22).
Amoxicillin is another key antimicrobial in the standard triple therapy to eradicate H pylori. In the literature, the resistance rate ranged from 0% to 2% (20–27). Our study showed a resistance rate of 10.4%, which surprisingly is not higher considering its wide use in pediatric practice. Moreover, in vitro resistance does not necessarily affect eradication rates; even when genetic markers, for example, pbp 1A (“penicillin-binding protein”) is present, mutation is present. There are some possible reasons: the low frequency of responsible mutations for resistance, wide variations in pbp 1A mutation, and the necessity of “cooperative” mutations to express amoxicillin resistance (28–30); therefore, even when susceptibility tests demonstrate resistance, amoxicillin can be used in the eradication schedule. Amoxicillin, however, demonstrates other difficulties in susceptibility tests. The amoxicillin susceptibility test should be performed rapidly after obtaining the biopsy because amoxicillin-resistant strains often change to susceptible strains by freezing at −80°C. The 10.4% rate could, therefore, be higher than observed. Another phenomenon that needs attention is β-lactam antimicrobial tolerance, which occurs because there is a difference between the MIC and the minimum bactericidal concentration (MBC). In this situation, a serum concentration of antimicrobial can inhibit the bacteria but cannot kill them. Considering this, the MBC also should be evaluated in amoxicillin (31).
The H pylori strains in our study showed total susceptibility to tetracycline and furazolidone, but these antimicrobials are not generally prescribed for children. No resistance to tetracycline was observed in some studies (23,24,32), and low rates were observed in others (26,27,33); however, tetracycline presents adverse events mainly in tooth calcification. The degree of exposure, number of courses, total dosage, and timing of tooth development may affect the risk of adverse event occurrence (34); however, doxycycline can replace tetracycline (15) with less occurrence of tooth discoloration, even in children (mean age 4 years) (35).
Studies also presented low resistance rate to furazolidone in Brazilian (4%) (12), Spanish (1.8%) (36), and Korean adults (2%) (37); however, there are no studies on furazolidone resistance in children, probably because it is not recommended for children younger than 8 years. Furazolidone is commonly used to replace nitroimidazoles, and it is commercially available in some developing countries such as Iran, Pakistan, Mexico, and Brazil, and in some Asian countries such as China (38). European countries (European Medicines Agency) and the United States (FDA) had banned furazolidone because of the adverse effects. Unfortunately, furazolidone seems to be an option to retreatment, mainly in developing countries where resistance to metronidazole is too high. More studies are needed to link the adverse effects (genotoxic and carcinogenic effects) to the dose and time prescribed to H pylori infection; furazolidone presents a higher risk of complications in long-term use (39). Furthermore, the risks of genotoxicity and carcinogenic effects need to be evaluated against the risk of carcinogenic effects of H pylori infection in high-risk patients. Despite the adverse effects, and considering the cost-benefit ratio compared with conventional triple therapy, furazolidone should be an option in developing countries, where this antimicrobial could expand the range of “intention-to-treat” treatments.
In this study, there was no clinical difference between the group of patients infected by resistant strains and the group infected by susceptible strains, but differences are usually observed between these groups when sex, age, and region of study were evaluated (20,40,41). It is probable that the small sample in this study has confounded the statistical analysis. The present study was restricted to 2 centers in São Paulo City, so clinical condition and antimicrobial resistance status may not be representative of the Brazilian population. Further prospective surveillance of H pylori resistance is essential to ensure that appropriate data are available to guide the choice of therapy, particularly in high-risk populations.
In summary, we have observed high resistance to clarithromycin and metronidazole in clinical H pylori isolates and this resistance can exclude these antimicrobials in empirical eradication treatment in Brazil. Furazolidone and tetracycline presented no resistance. These antimicrobials usually are not prescribed to children, mainly because of adverse effects. When the risks and benefits are properly assessed, these 2 antimicrobials and their derivatives can be used in empirical eradication schedules in developing countries where resistance rates are high. Amoxicillin is a good choice in standard triple therapy because of its low resistance rate despite its wide use in pediatric patients.
1. Malfertheiner P, Megraud F, O’Morain C, et al. Current concepts in the management of Helicobacter pylori
infection: the Maastricht III Consensus Report. Gut
2. Bytzer P, O’Morain C. Treatment of Helicobacter pylori
3. Oderda G, Scherbakov P, Bontems P, et al. Results from the pediatric European register for treatment of Helicobacter pylori
4. Cavallaro LG, Egan B, O’Morain C, et al. Treatment of Helicobacter pylori
5. Graham DY, Fischbach L. Helicobacter pylori
treatment in the era of increasing antibiotic resistance. Gut
6. Mégraud F, Lehours P. Helicobacter pylori
detection and antimicrobial susceptibility testing. Clin Microbiol Reviews
7. Sun Q-J, Liang X, Zheng Q, et al. Resistance of Helicobacter pylori
to antibiotics from 200 to 2009 in Shanghai. World J Gastroenterol
8. Kalach N, Benhamou PH, Bergeret M, et al. Acquisition of secondary resistance after failure of a first treatment of Helicobacter pylori
infection in children. Arch Pediatr
9. Fischbach LA, Goodman KJ, Feldman M, et al. Sources of variation of Helicobacter pylori
treatment success in adults worldwide: a meta-analysis. Int J Epidemiol
10. Magalhães PP, Queiroz DMM, Barbosa DVC, et al. Helicobacter pylori
primary resistance to metronidazole and clarithromycin in Brazil. Antimicrobial Agents Chemother
11. Godoy APO, Ribeiro ML, Benvengo YHB, et al. Analysis of antimicrobial susceptibility and virulence factors in Helicobacter pylori
clinical isolates. BMC Gastroenterology
12. Mendonça S, Ecclissato C, Sartori MS, et al. Prevalence of Helicobacter pylori
resistance to metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone in Brazil. Helicobacter
13. Kawakami E, Ogata SK, Portorreal ACM, et al. Triple therapy with clarithromycin, amoxicillin and omeprazole for Helicobacter pylori
eradication in children and adolescents. Arq Gastroenterol
14. Kawakami E, Machado RS, Ogata SK, et al. Furazolidone based triple therapy for Helicobacter pylori
gastritis in children. World J Gastroenterol
15. Machado RS, Silva MR, Viriato A. Furazolidone, tetracycline and omeprazole: a low-cost alternative for Helicobacter pylori
eradication in children. J Pediatr (Rio J)
16. Cuadrado-Lavin A, Salcines-Caviedes JR, Carrascosa MF, et al. Antimicrobial susceptibility of Helicobacter pylori
to six antibiotics currently used in Spain. J Antimicrob Chemother
17. Graham DY, Shiotani A. New concepts of resistance in the treatment of Helicobacter pylori
infections. Nature Clin Practice
18. Mégraud F, Occhialini A, Doermann HP. Resistance of Helicobacter pylori
to macrolides and nitroimidazole compounds. The current situation. J Physiol Pharmacol
1997; 48 (Suppl 4):25–38.
19. Kalach N, Benhamou PH, Campeotto F, et al. Clarithromycin resistance and eradication of Helicobacter pylori
in children. Antimicrobial Agents Chemother
20. Megraud F. H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut
21. Koletzko S, Richy F, Bontems P, et al. Prospective multicentre study on antibiotic resistance of Helicobacter pylori
strains obtained from children living in Europe. Gut
22. Deyi VYM, Bontems P, Vandenplas J, et al. Routine-based multicentre survey of antimicrobials resistance in Helicobacter pylori
over last twenty years (1990–2009) in Belgium. J Clin Microbiol
23. Rerksuppaphol S, Hardikar W, Midolo PD, et al. Antimicrobial resistance in Helicobacter pylori
isolates from children. J Paediatr Child Health
24. Liu G, Xu X, He L, et al. Primary antibiotic resistance of Helicobacter pylori
isolated from Beijing children. Helicobacter
25. Karczewska E, Wojtas-Bonior I, Sito E, et al. Primary and secondary clarithromycin, metronidazole, amoxicillin and levofloxacin resistance to Helicobacter pylori
in southern Poland. Pharmacol Rep
26. Boyanova L, Nikolov R, Lazarova E, et al. Antibacterial resistance in Helicobacter pylori
strains isolated from Bulgarian children and adult patients over 9 years. J Med Microbiol
27. Boyanova L, Koumanova R, Gergova G, et al. Prevalence of resistant Helicobacter pylori
isolates in Bulgarian children. J Med Microbiol
28. Tseng YS, Wu DC, Chang CY, et al. Amoxicillin resistance with beta-lactamase production in Helicobacter pylori
. Eur J Clin Invest
29. Qureshi NN, Morikis D, Schiller NL. Contribution of specific amino acid changes in penicillin binding protein 1 to amoxicillin resistance in clinical Helicobacter pylori
isolates. Antimicrobial Agents Chemother
30. Gerrits MM, Godoy APO, Kuipers EJ, et al. Multiple mutations in or adjacent to the conserved penicillin-binding protein motifs of the penicillin-binding protein 1A confer amoxicillin resistance to Helicobacter pylori
31. Dore MP, Osato MS, Realdi G, et al. Amoxycillin tolerance in Helicobacter pylori
. J Antimicrobial Chemother
32. Oleastro M, Cabral J, Ramalho PM, et al. Primary antibiotic resistance of Helicobacter pylori
strains isolated from Portuguese children: a prospective multicentre study over a 10 years period. J Antimicrobial Chemother
33. Street ME, Caruana P, Cafarelli C, et al. Antibiotic resistance and antibiotic sensitivity based treatment in Helicobacter pylori
infection: advantages and outcome. Arch Dis Child
34. Kline JM, Wietholter JP, Klilne VT, et al. Pediatric antibiotic use: a focused review of fluoroquinolones and tetracyclines. US Pharm
35. Volovitz B, Shkap R, Amir J, et al. Absence of tooth staining with doxycycline treatment in young children. Clin Pediatr (Phila)
36. Alarcon T, de la Obra P, Domingo D, et al. Actividad in vitro de la furazolidona y la nitrofurantoína en aislamientos clínicos de Helicobacter pylori
y estudio de la tasa de mutación. Rev Esp Quimioterap
37. Kwon DH, Lee M, Kim JJ, et al. Furazolidone and nitrofurantoin-resistant Helicobacter pylori
: prevalence and role of genes involved in metronidazole resistance. Antimicrob Agents Chemother
38. De Francesco V, Ierardi E, Hassan C, et al. Is furazolidone therapy for Helicobacter pylori
effective and safe? Dig Dis Sci
39. XiaoMing Y, JianQiao F, FangFang Z, et al. Loss of menstruation associated with furazolidone: a case report and review of literature. African J Pharmacy Pharmacol
40. Falsafi T, Mobasheri F, Nariman F, et al. Susceptibilities to different antibiotics of Helicobacter pylori
strains isolated from patients at the Pediatric Medical Center of Tehran, Iran. J Clin Microbiol
41. De Francesco V, Giorgio F, Ierardi E, et al. Primary clarithromycin resistance in Helicobacter pylori
: the Multicentric Italian Clarithromycin Resistance Observational (MICRO) Study. J Gastrointestin Liver Dis
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amoxicillin; clarithromycin; furazolidone; Helicobacter pylori; metronidazole; resistance; susceptibility; tetracycline
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