EPIDEMIOLOGY
MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2014.0081
Prevalence of Multiple Drug-Resistant Helicobacter pylori Strains Among Patients with Different Gastric Disorders in Iran Leila Shokrzadeh,1,2 Masoud Alebouyeh,1,2,* Tabassom Mirzaei,1 Nastaran Farzi,1,2 and Mohammad Reza Zali1,2,*
Emergence of multidrug-resistant (MDR) strains of Helicobacter pylori is a global health concern. This study was aimed to determine the frequency of MDR H. pylori strains in Iran. H. pylori isolates were obtained from cultured gastric biopsy samples on selective culture media after their characterization by PCR and conventional biochemical methods. The minimal inhibitory concentrations of rifampicin, ciprofloxacin, levofloxacin, ampicillin, clarithromycin, erythromycin, metronidazole, and tetracycline were determined for 111 strains that were isolated from 197 dyspeptic patients by the agar dilution method. The primary resistance rates were 61.3% (68/111) for metronidazole, 15.3% (17/111) for ampicillin, and 14.4% (16/111) for rifampicin. Resistance rates for other antimicrobials were as follows: macrolides (erythromycin or clarithromycin) 32.4% (36/111) and quinolones (levofloxacin or ciprofloxacin) 30.6% (34/111). Among the resistant strains, the rates of double and multiple drug resistance phenotypes were 22.6% (19/84) and 34.5% (29/84), respectively. The quadruple drug resistance phenotype encompasses 37.9% of the MDR strains, of which 90% of them was resistant to metronidazole. In conclusion, these results showed a high frequency of MDR phenotypes among the studied H. pylori strains in Iran. The eradication of the H. pylori strains presenting high resistance rates to macrolides, fluoroquinolones, or metronidazole could be achieved by approved tetracycline- or amoxicillin-containing regimens as alternative regimens to standard triple therapy.
of treatment failures, several other antibiotics such as fluoroquinolones (ciprofloxacin or levofloxacin) and tetracycline are used as secondary options. The traditional triple therapy is only effective if local prevalence of resistance is relatively low.21 In recent years, the efficacy of the first-line triple therapy regimens has been compromised by the emergence of resistance strains. The rate of resistance varies according to the geographic area and is thought to be increasing worldwide.30,35 There are some reports about primary H. pylori resistance to clarithromycin (up to 25%),29 metronidazole (20–40%),25,36 amoxicillin (0–2.4%), and macrolides (erythromycin or clarithromycin) (33–57%) in untreated and treated adult patients.32 In the case of tetracycline, the reported rate of resistance is low (1.4–7%),9,42 whereas the resistance to fluoroquinolones (ciprofloxacin and levofloxacin) is increasing in many countries (11.6–24.1%).26,31 The success of common therapeutic strategies for treatment of H. pylori infection could mainly be affected by the
Introduction
H
elicobacter pylori is an important bacterial pathogen responsible for several gastrointestinal diseases, including peptic ulcer disease (PUD), gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. More than 50% of the world’s population and nearly 40–80% of the Iranian populations are infected with this bacterium.3,28,30,40 The eradication of H. pylori infection with most effective antibiotic regimens plays an important role in the prevention of resistance development and disease progression.7 The successful treatment of H. pylori infections requires multidrug regimens typically containing an antisecretory drug and two or three antimicrobials. Triple therapy regimens, containing combinations of a proton pump inhibitor or bismuth citrate and two antibiotics, such as amoxicillin, clarithromycin, or metronidazole, are considered the first-line treatment for H. pylori eradication in patients with gastric disorders. In cases
1 Gastroenterology and Liver Diseases Research Center and 2Basic Science and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. *These authors contributed equally to this work.
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emergence of multidrug-resistant (MDR) strains in each country. Although the double drug resistance rate of H. pylori was determined as 10% in Europe, triple resistance to amoxicillin, metronidazole, and clarithromycin was reported only occasionally (3.5% of the untreated and 13.6% of the treated adults).4,31 Regarding the less controlled administration of these antibiotics in countries with higher rates of H. pylori infection, the existence of higher levels of multiresistance phenotypes seems plausible. In the present study, we analyzed the frequency of in vitro resistance, MDR including quadruple drug resistance (QDR) phenotypes, and minimum inhibitory concentration (MIC) values of H. pylori isolates to different antimicrobial agents during 2010–2011 in Tehran, Iran. Materials and Methods Patients and specimens
A total of 111 clinical H. pylori isolates were obtained from 197 (56.3%) patients who were referred to one endoscopy unit in Tehran, Iran, between April 2010 and February 2011. The patients included were 75 (38%) men and 122 (62%) women (mean age, 46 years). These patients had clinical presentations of gastritis (147/197), PUD (42/197), and gastric cancer (8/197). The patients’ demographic data, including age, sex, treatment history for H. pylori infection, and their related endoscopic findings, were recorded. H. pylori infection was proven by culture of biopsy samples, as described below. The patients gave an informed consent and agreed to complete a standardized data collection form. This study was approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences. Isolation and confirmation of H. pylori strains
Two gastric antral and body biopsy specimens were taken from each patient. One of the specimens was kept in a transport medium, consisting of thioglycolate with 1.3 g/L agar (Merck Co., Darmstadt, Germany) and 3% yeast extract (Oxoid, Hampshire, United Kingdom), for culture and the other one was placed in the Rapid Urease Test medium. Isolation and biochemical identification of the isolates were performed, as described previously.36 In brief, all the biopsy samples were homogenized and smeared on the surface of Brucella agar plates supplemented with 7% horse blood, Campylobacter selective supplement (vancomycin 2.0 mg, polymyxin 0.05 mg, trimethoprim 1.0 mg), and amphotericin B (2.5 mg/L) (Merck Co.). Incubation was performed in microaerophilic conditions at 37C for 5–7 days. Suspicious colonies were analyzed for cell morphology and positive reactions of oxidase, catalase, and urease tests. The confirmed bacterial colonies were harvested and their genomic DNAs were extracted using the QIAamp tissue DNA extraction kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The glmM gene was identified by PCR using specific forward and reverse primer pairs 5¢-GGATAAGCTTTTAGGGGTGTTAGGGG-3¢ and 5¢-GCTTACTTTCTAACACTAACGCGC-3¢ with a 296bp size product, respectively.36 Antibiotic susceptibility testing
The MIC was determined by the agar dilution method for each isolate based on the guidelines established by the
SHOKRZADEH ET AL.
Clinical and Laboratory Standards Institute (CLSI).8 Accordingly, various concentrations of antibiotics from 0.016 to 32 mg/ml were added to the Mueller-Hinton agar medium (Merck Co.) containing 5% defibrinated sheep blood. H. pylori suspensions were prepared equivalent to a No. 2 McFarland standard, and 10 ml of them was inoculated on the prepared Mueller-Hinton agar plates. The MIC values were determined after 72 hr of incubation at 37C under microaerophilic conditions in a CO2 incubator (Innova-Co 170; New Brunswick Scientific, Edison, NJ). The resistance rates for ampicillin, metronidazole, ciprofloxacin, levofloxacin, erythromycin, tetracycline, rifampicin (MAST, London, United Kingdom), and clarithromycin (Sigma, St. Louis, MO) were defined at the following ranges: > 0.5, > 8, > 2.0, > 1.0, > 0.5, > 4, > 4.0, and ‡ 1 mg/ml, respectively.5,29 H. pylori strains with resistance to at least three families of potentially effective antimicrobial agents were considered as isolates with the MDR phenotype.13 A clinical isolate of H. pylori with known MIC values was used as a control strain in susceptibility tests. Susceptibility and MIC values of this strain to noted antibiotics were studied by three independent experiments using antibiotics from different companies. Data statistical analyses
Chi-squared and Fisher’s exact tests were used for the analysis of the association between the patients’ antibiotic consumption and the resistance patterns. The analyses were done using Sigma Stat for Windows V2.03 (SPSS, Chicago, IL). A probability value less than 0.05 was accepted as statistically significant. Results
The results of this study showed a history of medications, including omeprazole, metronidazole, amoxicillin, and bismuth, among 16% (18/111) of the infected patients. The resistance patterns of the H. pylori strains to the tested antibiotics are shown in Table 1. As shown in Table 2, the highest and lowest resistance rates were detected as 61.3% (68/111) to metronidazole and 2.7% (3/111) to tetracycline. Twenty-four percent (27/111) of the isolates were susceptible to all the tested antibiotics and 32.4% (36/111) were resistant to only one antibiotic family. In total, 84 of the 111 strains were resistant to at least one antimicrobial agent. Double drug resistance and MDR phenotypes were detected in 22.6% (19/84) and 34.5% (29/84) of the resistant strains, respectively. QDR strains were detected in 11 (37.9%) of the 29 examined MDR strains (Table 1). Simultaneous resistance to metronidazole, quinolones, and macrolides was determined as the more frequent MDR phenotype among the studied strains. The distribution of MICs and percentage of resistance to different antibiotics among the H. pylori strains are presented in Table 2. Of the 76.4% (52/68) metronidazoleresistant H. pylori strains with MIC of ‡ 32 mg/ml, 67.3% (35/52) belonged to female patients and 32.7% (17/52) to male patients. A total of 20.7% (23/111) of the strains showed single resistance to this compound, whereas 25.2% (29/111) of them belonged to the strains with multiple drugresistant phenotypes (Table 1). Total resistance to quinolones (ciprofloxacin and/or levofloxacin) was detected in
MULTIPLE DRUG-RESISTANT HELICOBACTER PYLORI
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Table 1. Frequency of Multidrug Resistance Phenotype in Clinical Isolates of Helicobacter pylori MDR patterns (%) Double resistance (n)a
MDR phenotypes CIP/LEV + ERY CLA + AMP CLA/ERY + CIP/LEV + MET CLA/ERY + TET + CIP + MET CIP/LEV + MET CLA + TET + MET AMP + CIP + MET + RIF CLA + CIP/LEV + MET + RIF MET + RIF TET + CIP/LEV + MET AMP + CIP/LEV + MET CLA/ERY + AMP + MET CLA/ERY + MET CLA/ERY + MET + RIF AMP + MET CIP/LEV + MET + RIF AMP + MET + RIF CLA/ERY + AMP + MET + RIF CLA/ERY + AMP + MET + LEV CLA + AMP + CIP/LEV + RIF AMP + CIP/LEV CLA/ERY + CIP/LEV + AMP + MET CIP/LEV + AMP + MET + RIF CLA/ERY + CIP/LEV + MET + RIF Totalb
Triple resistance, n (%)
Quadruple resistance, n (%)
2 1 6 (11.5) 1 (1.9) 5 1 (1.9) 1 (1.9) 1 (1.9) 3 1 (1.9) 2 (3.8) 3 (5.7) 6 2 (3.8) 1 2 (3.8) 1 (1.9) 2 (3.8) 1 (1.9) 1 (1.9) 1
19
1 2 1 11
18 (62)
(1.9) (3.8) (1.9) (37.9)
a
Number of strains with defined resistance patterns. The overall percentage of strains with triple and quadruple resistance phenotypes were presented among the isolates with MDR phenotype. AMP, ampicillin; CIP, ciprofloxacin; CIP/LEV, resistance to levofloxacin and/or ciprofloxacin; CLA, clarithromycin; CLA/ERY, resistance to clarithromycin and/or erythromycin; ERY, erythromycin; LEV, levofloxacin; MDR, multidrug-resistant; MET, metronidazole; RIF, rifampicin; TET, tetracycline. b
30.6% (34/111) of the isolates [27% (30/111) to ciprofloxacin and 24.3% (27/111) to levofloxacin]. In the case of resistance to macrolides, erythromycin and/or clarithromycin (32.4%, 36/111), resistance to clarithromycin and erythromycin was detected in 26% (29/111) and 22.5% (25/111) strains, respectively. The MIC values for these two antibiotics were higher than those observed for ampicillin (Table 2). While a low rate of ampicillin-resistant strains was detected
(17/111, 15.3%), all these strains showed cross-resistance to one or more of the other tested antibiotics. The statistical analyses showed no significant differences for resistance rates between the categorized patients at different age and sex groups ( p > 0.05). A relationship between previous exposure to metronidazole and development of resistance to metronidazole in H. pylori strains was documented in three patients. However, no association was
Table 2. Minimum Inhibitory Concentrations (mg/ml) of Antibacterial Agents Against H. pylori Isolates Antimicrobial agenta Clarithromycin Metronidazole Ampicillin Tetracycline Rifampicin Levofloxacin Ciprofloxacin Erythromycin a
Resistance rates, n (%) 29 68 17 3 16 27 30 25
(26) (61.3) (15.3) (2.7) (14.4) (24.3) (27) (22.5)
MIC50 (mg/ml)b 0.25 16 0.25 0.5 2 1 1 0.25
(65/111) (61/111) (68/111) (56) (77/111) (84/111) (60/111) (65/111)
MIC90 (mg/ml)c 8 > 32 1 4 >8 4 >8 4
(99/111) (111/111) (100/111) (98/111) (111/111) (109/111) (111/111) (106/111)
Range 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06
to to to to to to to to
16 > 32 4 8 >8 16 >8 16
The MIC value of each antimicrobial agent was determined by the agar dilution method. The MIC value at which 50% of the tested strains were inhibited. Number of the isolates that displayed the given MIC50 is shown in parenthesis for each antibiotic. c The MIC value at which 90% of the tested strains were inhibited. Number of the isolates that displayed the given MIC90 is shown in parenthesis for each antibiotic. MIC, minimum inhibitory concentration. b
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detected between the determined MDR patterns and the documented therapeutic regimens. In addition, there were no significant associations between age, gender, medication, and the studied clinical outcomes. Discussion
The infection rate of H. pylori varies among the developed and developing countries.1,11,39 H. pylori infection has decreased in most of the developed countries, due to the success of combination therapies, improved personal hygiene, and community sanitation.2,39 However, it seems that in developing countries, due to the failure of effective treatment regimens and emergence of drug-resistant strains, the disease situation is not improving.21 As a result of this increased rate of resistance, all the patients who are infected with H. pylori should be considered as carriers of resistant strains and should be treated based on in vitro antibiotic susceptibility testing results.20 Despite the increasing levels of resistant strains among the treated patients with common therapeutic regimens, unfortunately, few studies have reported the rate of emerging MDR H. pylori isolates in the world. The primary resistance rates of the H. pylori isolates in our study were higher than those reported in other countries. The resistance rate to at least two antimicrobial agents was detected in 43% of our isolates (48/111), whereas it was reported to be 33.8% in Taiwan and 31% in Mexican adults.22,38 Resistance in these strains was related to metronidazole (15/19, 78.9%), quinolones (8/19, 42.1%), and macrolides (9/19, 47.3%). The observed high prevalence rate of the metronidazole resistance phenotype in our strains (61.3%) could be explained by its exhaustive use in Iran, as this drug is a cornerstone of many triple therapy regimens prescribed for the treatment of H. pylori infection. However, our results showed no association between resistance to metronidazole and other antibiotics and the reported medications in our patients. The resistance rate to metronidazole was higher than those reported in last years in Iran (40.5%).36 Nevertheless, higher rates of resistance to this antibiotic were reported up to 90% in other developing countries.14,15,33,34 The increased resistance might be also related to frequent usage of this antibiotic in protozoan, genital, and dental infections.12 In the present study, all the MDR (18/18) and most of the QDR (10/11) strains were resistant to metronidazole (Table 1). Of the 68 metronidazole-resistant and 29 MDR strains, 47% (32/68) and 86.7% (59/68) showed MICs of ‡ 32 mg/ml. This resistance rate can hinder the success of its usage in common eradication regimens. Amoxicillin is often implemented in H. pylori treatment protocols. Although in most studies it was shown that H. pylori resistance to either amoxicillin or ampicillin is very rare, the rate of resistance is increasing now.16 The resistance rate in Iran changed from 1.4% in 2000 to 3.7% in 2004 and 7.3% in 2008.37 This increase was also observed in the present study compared with our previous report (15% vs. 2.5%). The highest resistance rate to amoxicillin has ranged 18.5% in South Korea, 19.4% in Indonesia, 32.8% in India, and 38% in Brasilia.19,22,23,27 A total of 48.2% (14/ 29) of our MDR isolates showed resistance to ampicillin, of which 14.5% showed simultaneous resistance to ampicillin and metronidazole. As metronidazole and amoxicillin are
SHOKRZADEH ET AL.
among the most commonly administered antibiotics in the treatment of H. pylori infection in Iran, simultaneous resistance to these antibiotics could be problematic in clinical settings (Table 1). The rate of clarithromycin resistance was reported to be higher among developing countries (25–50%) compared with developed countries (10%).5,23 This resistance was observed in 26% (29/111) of our isolates, which was higher than our previous results (14%).36 Similar to our results, an increasing trend in resistance rates to clarithromycin (8.6%, 9.0%, and 20.7%) was reported during 2000–2009 in Shanghai.6 Among the clarithromycin-resistant isolates, 69% and 27.5% (20/29 and 8/29, respectively) were found to have the MDR and QDR patterns, respectively (Table 1). Owing to the failure of clarithromycin-based standard triple therapy, the use of levofloxacin for H. pylori eradication is increasing worldwide. The rate of resistance to levofloxacin and ciprofloxacin in the studied strains was 24.3% (27/111) and 27% (30/111), respectively, which was higher than that reported in our previous study (7%).36 In Germany, the ciprofloxacin resistance rate was reported to be 11% in 2003, 16.6% in 2004, and 22% in 2005.18 Although low rates of resistance to quinolones were reported in most countries, the frequency of resistance increased up to > 26%.43 The widespread use of fluoroquinolones for H. pylori infection has led to an elevated resistance rate in some areas.9,41 However, our results showed that this increased rate of resistance was not associated with the history of its usage among the studied patients in Iran. Resistance against rifamycins and rifabutin was also very rare until a few years back; however, the incidence of resistance to these antibiotics is increasing.24 In the present study, 14.40% (16/111) of our isolates showed phenotypic resistance to rifampicin (MIC > 4 mg/L); however, different rates of resistance were reported in other investigations. In Germany, rifampicin-resistant clinical H. pylori isolates were detected in 1.4% of cases, which was significantly lower than our isolates.17 Most of the rifampicin-resistant strains in our study were related to MDR (13/29, 44.8%) or QDR (8/11, 72.2%) strains. In conclusion, an increased rate of resistance to the mainly prescribed antibiotics against H. pylori strains was detected in Iran. The findings showed a high frequency of MDR phenotypes, especially triple and quadruple drug resistance profiles, among these strains, which is a case of major concern. According to the low rate of resistance to amoxicillin and tetracycline among the studied strains, treatment of the infected patients with H. pylori strains harboring dual resistance to clarithromycin and metronidazole could be achieved due to the administration of tetracycline-containing (e.g., bismuth-containing quadruple therapy) or amoxicillincontaining (e.g., hybrid therapy) regimens. Further studies are needed to assess the efficacy of these regimens in these patients. Acknowledgments
This study was supported by a grant from the Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. The authors thank Dr. Mohammad Mehdi Aslani, Dariush
MULTIPLE DRUG-RESISTANT HELICOBACTER PYLORI
Mirsattari, Sara Sayyadi, and Babak Yadegar for their kind cooperation in the recruitment of this study. Disclosure Statement
No competing financial interests exist. References
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39. 40.
41. 42. 43.
children and adults in Mexico. J. Clin. Microbiol. 39:2677– 2680. Van Belkum, A. 2003. High-throughput epidemiologic typing in clinical microbiology. Clin. Microbiol. Infect. 9:86–100. Vaziri F., S. Najar Peerayeh, M. Alebouyeh, T. Mirzaei, Y. Yamaoka, M. Molaei, N. Maghsoudi, and M.R. Zali. 2013. Diversity of Helicobacter pylori genotypes in Iranian patients with different gastroduodenal disorders. World J. Gastroenterol. 19:5685–5692. Wang, L.H., H. Cheng, F.L. Hu, and J. Li. 2010. Distribution of gyrA mutations in fluoroquinolone-resistant Helicobacter pylori strains. World J. Gastroenterol. 16:2272. Wu, W., Y. Yang, and G. Sun. 2012. Recent insights into antibiotic resistance in Helicobacter pylori eradication. Gastroenterol. Res. Pract. 72:3183. Yoon, H., N. Kim, B.H. Lee, T.J. Hwang, D.H. Lee, Y.S. Park, R.H. Nam, H.C. Jung, and I.S. Song. 2009. Moxifloxacin-containing triple therapy as second-line treatment for Helicobacter pylori infection: effect of treatment duration and antibiotic resistance on the eradication rate. Helicobacter 14:429–437.
Address correspondence to: Masoud Alebouyeh, PhD Gastroenterology and Liver Diseases Research Center and Basic Science and Molecular Epidemiology of Gastrointestinal Disorders Research Center Shahid Beheshti University of Medical Sciences 7th Floor of Taleghani Hospital Velenjak Shahid Aerabi Street Yemen Street Tehran 1985717413 Iran E-mail: [email protected] Mohammad Reza Zali, MD, FACG Gastroenterology and Liver Diseases Research Center Shahid Beheshti University of Medical Sciences 7th Floor of Taleghani Hospital Velenjak Shahid Aerabi Street Yemen Street Tehran 1985717413 Iran E-mail: [email protected]
MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2014.0081
Prevalence of Multiple Drug-Resistant Helicobacter pylori Strains Among Patients with Different Gastric Disorders in Iran Leila Shokrzadeh,1,2 Masoud Alebouyeh,1,2,* Tabassom Mirzaei,1 Nastaran Farzi,1,2 and Mohammad Reza Zali1,2,*
Emergence of multidrug-resistant (MDR) strains of Helicobacter pylori is a global health concern. This study was aimed to determine the frequency of MDR H. pylori strains in Iran. H. pylori isolates were obtained from cultured gastric biopsy samples on selective culture media after their characterization by PCR and conventional biochemical methods. The minimal inhibitory concentrations of rifampicin, ciprofloxacin, levofloxacin, ampicillin, clarithromycin, erythromycin, metronidazole, and tetracycline were determined for 111 strains that were isolated from 197 dyspeptic patients by the agar dilution method. The primary resistance rates were 61.3% (68/111) for metronidazole, 15.3% (17/111) for ampicillin, and 14.4% (16/111) for rifampicin. Resistance rates for other antimicrobials were as follows: macrolides (erythromycin or clarithromycin) 32.4% (36/111) and quinolones (levofloxacin or ciprofloxacin) 30.6% (34/111). Among the resistant strains, the rates of double and multiple drug resistance phenotypes were 22.6% (19/84) and 34.5% (29/84), respectively. The quadruple drug resistance phenotype encompasses 37.9% of the MDR strains, of which 90% of them was resistant to metronidazole. In conclusion, these results showed a high frequency of MDR phenotypes among the studied H. pylori strains in Iran. The eradication of the H. pylori strains presenting high resistance rates to macrolides, fluoroquinolones, or metronidazole could be achieved by approved tetracycline- or amoxicillin-containing regimens as alternative regimens to standard triple therapy.
of treatment failures, several other antibiotics such as fluoroquinolones (ciprofloxacin or levofloxacin) and tetracycline are used as secondary options. The traditional triple therapy is only effective if local prevalence of resistance is relatively low.21 In recent years, the efficacy of the first-line triple therapy regimens has been compromised by the emergence of resistance strains. The rate of resistance varies according to the geographic area and is thought to be increasing worldwide.30,35 There are some reports about primary H. pylori resistance to clarithromycin (up to 25%),29 metronidazole (20–40%),25,36 amoxicillin (0–2.4%), and macrolides (erythromycin or clarithromycin) (33–57%) in untreated and treated adult patients.32 In the case of tetracycline, the reported rate of resistance is low (1.4–7%),9,42 whereas the resistance to fluoroquinolones (ciprofloxacin and levofloxacin) is increasing in many countries (11.6–24.1%).26,31 The success of common therapeutic strategies for treatment of H. pylori infection could mainly be affected by the
Introduction
H
elicobacter pylori is an important bacterial pathogen responsible for several gastrointestinal diseases, including peptic ulcer disease (PUD), gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. More than 50% of the world’s population and nearly 40–80% of the Iranian populations are infected with this bacterium.3,28,30,40 The eradication of H. pylori infection with most effective antibiotic regimens plays an important role in the prevention of resistance development and disease progression.7 The successful treatment of H. pylori infections requires multidrug regimens typically containing an antisecretory drug and two or three antimicrobials. Triple therapy regimens, containing combinations of a proton pump inhibitor or bismuth citrate and two antibiotics, such as amoxicillin, clarithromycin, or metronidazole, are considered the first-line treatment for H. pylori eradication in patients with gastric disorders. In cases
1 Gastroenterology and Liver Diseases Research Center and 2Basic Science and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. *These authors contributed equally to this work.
1
2
emergence of multidrug-resistant (MDR) strains in each country. Although the double drug resistance rate of H. pylori was determined as 10% in Europe, triple resistance to amoxicillin, metronidazole, and clarithromycin was reported only occasionally (3.5% of the untreated and 13.6% of the treated adults).4,31 Regarding the less controlled administration of these antibiotics in countries with higher rates of H. pylori infection, the existence of higher levels of multiresistance phenotypes seems plausible. In the present study, we analyzed the frequency of in vitro resistance, MDR including quadruple drug resistance (QDR) phenotypes, and minimum inhibitory concentration (MIC) values of H. pylori isolates to different antimicrobial agents during 2010–2011 in Tehran, Iran. Materials and Methods Patients and specimens
A total of 111 clinical H. pylori isolates were obtained from 197 (56.3%) patients who were referred to one endoscopy unit in Tehran, Iran, between April 2010 and February 2011. The patients included were 75 (38%) men and 122 (62%) women (mean age, 46 years). These patients had clinical presentations of gastritis (147/197), PUD (42/197), and gastric cancer (8/197). The patients’ demographic data, including age, sex, treatment history for H. pylori infection, and their related endoscopic findings, were recorded. H. pylori infection was proven by culture of biopsy samples, as described below. The patients gave an informed consent and agreed to complete a standardized data collection form. This study was approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences. Isolation and confirmation of H. pylori strains
Two gastric antral and body biopsy specimens were taken from each patient. One of the specimens was kept in a transport medium, consisting of thioglycolate with 1.3 g/L agar (Merck Co., Darmstadt, Germany) and 3% yeast extract (Oxoid, Hampshire, United Kingdom), for culture and the other one was placed in the Rapid Urease Test medium. Isolation and biochemical identification of the isolates were performed, as described previously.36 In brief, all the biopsy samples were homogenized and smeared on the surface of Brucella agar plates supplemented with 7% horse blood, Campylobacter selective supplement (vancomycin 2.0 mg, polymyxin 0.05 mg, trimethoprim 1.0 mg), and amphotericin B (2.5 mg/L) (Merck Co.). Incubation was performed in microaerophilic conditions at 37C for 5–7 days. Suspicious colonies were analyzed for cell morphology and positive reactions of oxidase, catalase, and urease tests. The confirmed bacterial colonies were harvested and their genomic DNAs were extracted using the QIAamp tissue DNA extraction kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The glmM gene was identified by PCR using specific forward and reverse primer pairs 5¢-GGATAAGCTTTTAGGGGTGTTAGGGG-3¢ and 5¢-GCTTACTTTCTAACACTAACGCGC-3¢ with a 296bp size product, respectively.36 Antibiotic susceptibility testing
The MIC was determined by the agar dilution method for each isolate based on the guidelines established by the
SHOKRZADEH ET AL.
Clinical and Laboratory Standards Institute (CLSI).8 Accordingly, various concentrations of antibiotics from 0.016 to 32 mg/ml were added to the Mueller-Hinton agar medium (Merck Co.) containing 5% defibrinated sheep blood. H. pylori suspensions were prepared equivalent to a No. 2 McFarland standard, and 10 ml of them was inoculated on the prepared Mueller-Hinton agar plates. The MIC values were determined after 72 hr of incubation at 37C under microaerophilic conditions in a CO2 incubator (Innova-Co 170; New Brunswick Scientific, Edison, NJ). The resistance rates for ampicillin, metronidazole, ciprofloxacin, levofloxacin, erythromycin, tetracycline, rifampicin (MAST, London, United Kingdom), and clarithromycin (Sigma, St. Louis, MO) were defined at the following ranges: > 0.5, > 8, > 2.0, > 1.0, > 0.5, > 4, > 4.0, and ‡ 1 mg/ml, respectively.5,29 H. pylori strains with resistance to at least three families of potentially effective antimicrobial agents were considered as isolates with the MDR phenotype.13 A clinical isolate of H. pylori with known MIC values was used as a control strain in susceptibility tests. Susceptibility and MIC values of this strain to noted antibiotics were studied by three independent experiments using antibiotics from different companies. Data statistical analyses
Chi-squared and Fisher’s exact tests were used for the analysis of the association between the patients’ antibiotic consumption and the resistance patterns. The analyses were done using Sigma Stat for Windows V2.03 (SPSS, Chicago, IL). A probability value less than 0.05 was accepted as statistically significant. Results
The results of this study showed a history of medications, including omeprazole, metronidazole, amoxicillin, and bismuth, among 16% (18/111) of the infected patients. The resistance patterns of the H. pylori strains to the tested antibiotics are shown in Table 1. As shown in Table 2, the highest and lowest resistance rates were detected as 61.3% (68/111) to metronidazole and 2.7% (3/111) to tetracycline. Twenty-four percent (27/111) of the isolates were susceptible to all the tested antibiotics and 32.4% (36/111) were resistant to only one antibiotic family. In total, 84 of the 111 strains were resistant to at least one antimicrobial agent. Double drug resistance and MDR phenotypes were detected in 22.6% (19/84) and 34.5% (29/84) of the resistant strains, respectively. QDR strains were detected in 11 (37.9%) of the 29 examined MDR strains (Table 1). Simultaneous resistance to metronidazole, quinolones, and macrolides was determined as the more frequent MDR phenotype among the studied strains. The distribution of MICs and percentage of resistance to different antibiotics among the H. pylori strains are presented in Table 2. Of the 76.4% (52/68) metronidazoleresistant H. pylori strains with MIC of ‡ 32 mg/ml, 67.3% (35/52) belonged to female patients and 32.7% (17/52) to male patients. A total of 20.7% (23/111) of the strains showed single resistance to this compound, whereas 25.2% (29/111) of them belonged to the strains with multiple drugresistant phenotypes (Table 1). Total resistance to quinolones (ciprofloxacin and/or levofloxacin) was detected in
MULTIPLE DRUG-RESISTANT HELICOBACTER PYLORI
3
Table 1. Frequency of Multidrug Resistance Phenotype in Clinical Isolates of Helicobacter pylori MDR patterns (%) Double resistance (n)a
MDR phenotypes CIP/LEV + ERY CLA + AMP CLA/ERY + CIP/LEV + MET CLA/ERY + TET + CIP + MET CIP/LEV + MET CLA + TET + MET AMP + CIP + MET + RIF CLA + CIP/LEV + MET + RIF MET + RIF TET + CIP/LEV + MET AMP + CIP/LEV + MET CLA/ERY + AMP + MET CLA/ERY + MET CLA/ERY + MET + RIF AMP + MET CIP/LEV + MET + RIF AMP + MET + RIF CLA/ERY + AMP + MET + RIF CLA/ERY + AMP + MET + LEV CLA + AMP + CIP/LEV + RIF AMP + CIP/LEV CLA/ERY + CIP/LEV + AMP + MET CIP/LEV + AMP + MET + RIF CLA/ERY + CIP/LEV + MET + RIF Totalb
Triple resistance, n (%)
Quadruple resistance, n (%)
2 1 6 (11.5) 1 (1.9) 5 1 (1.9) 1 (1.9) 1 (1.9) 3 1 (1.9) 2 (3.8) 3 (5.7) 6 2 (3.8) 1 2 (3.8) 1 (1.9) 2 (3.8) 1 (1.9) 1 (1.9) 1
19
1 2 1 11
18 (62)
(1.9) (3.8) (1.9) (37.9)
a
Number of strains with defined resistance patterns. The overall percentage of strains with triple and quadruple resistance phenotypes were presented among the isolates with MDR phenotype. AMP, ampicillin; CIP, ciprofloxacin; CIP/LEV, resistance to levofloxacin and/or ciprofloxacin; CLA, clarithromycin; CLA/ERY, resistance to clarithromycin and/or erythromycin; ERY, erythromycin; LEV, levofloxacin; MDR, multidrug-resistant; MET, metronidazole; RIF, rifampicin; TET, tetracycline. b
30.6% (34/111) of the isolates [27% (30/111) to ciprofloxacin and 24.3% (27/111) to levofloxacin]. In the case of resistance to macrolides, erythromycin and/or clarithromycin (32.4%, 36/111), resistance to clarithromycin and erythromycin was detected in 26% (29/111) and 22.5% (25/111) strains, respectively. The MIC values for these two antibiotics were higher than those observed for ampicillin (Table 2). While a low rate of ampicillin-resistant strains was detected
(17/111, 15.3%), all these strains showed cross-resistance to one or more of the other tested antibiotics. The statistical analyses showed no significant differences for resistance rates between the categorized patients at different age and sex groups ( p > 0.05). A relationship between previous exposure to metronidazole and development of resistance to metronidazole in H. pylori strains was documented in three patients. However, no association was
Table 2. Minimum Inhibitory Concentrations (mg/ml) of Antibacterial Agents Against H. pylori Isolates Antimicrobial agenta Clarithromycin Metronidazole Ampicillin Tetracycline Rifampicin Levofloxacin Ciprofloxacin Erythromycin a
Resistance rates, n (%) 29 68 17 3 16 27 30 25
(26) (61.3) (15.3) (2.7) (14.4) (24.3) (27) (22.5)
MIC50 (mg/ml)b 0.25 16 0.25 0.5 2 1 1 0.25
(65/111) (61/111) (68/111) (56) (77/111) (84/111) (60/111) (65/111)
MIC90 (mg/ml)c 8 > 32 1 4 >8 4 >8 4
(99/111) (111/111) (100/111) (98/111) (111/111) (109/111) (111/111) (106/111)
Range 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06
to to to to to to to to
16 > 32 4 8 >8 16 >8 16
The MIC value of each antimicrobial agent was determined by the agar dilution method. The MIC value at which 50% of the tested strains were inhibited. Number of the isolates that displayed the given MIC50 is shown in parenthesis for each antibiotic. c The MIC value at which 90% of the tested strains were inhibited. Number of the isolates that displayed the given MIC90 is shown in parenthesis for each antibiotic. MIC, minimum inhibitory concentration. b
4
detected between the determined MDR patterns and the documented therapeutic regimens. In addition, there were no significant associations between age, gender, medication, and the studied clinical outcomes. Discussion
The infection rate of H. pylori varies among the developed and developing countries.1,11,39 H. pylori infection has decreased in most of the developed countries, due to the success of combination therapies, improved personal hygiene, and community sanitation.2,39 However, it seems that in developing countries, due to the failure of effective treatment regimens and emergence of drug-resistant strains, the disease situation is not improving.21 As a result of this increased rate of resistance, all the patients who are infected with H. pylori should be considered as carriers of resistant strains and should be treated based on in vitro antibiotic susceptibility testing results.20 Despite the increasing levels of resistant strains among the treated patients with common therapeutic regimens, unfortunately, few studies have reported the rate of emerging MDR H. pylori isolates in the world. The primary resistance rates of the H. pylori isolates in our study were higher than those reported in other countries. The resistance rate to at least two antimicrobial agents was detected in 43% of our isolates (48/111), whereas it was reported to be 33.8% in Taiwan and 31% in Mexican adults.22,38 Resistance in these strains was related to metronidazole (15/19, 78.9%), quinolones (8/19, 42.1%), and macrolides (9/19, 47.3%). The observed high prevalence rate of the metronidazole resistance phenotype in our strains (61.3%) could be explained by its exhaustive use in Iran, as this drug is a cornerstone of many triple therapy regimens prescribed for the treatment of H. pylori infection. However, our results showed no association between resistance to metronidazole and other antibiotics and the reported medications in our patients. The resistance rate to metronidazole was higher than those reported in last years in Iran (40.5%).36 Nevertheless, higher rates of resistance to this antibiotic were reported up to 90% in other developing countries.14,15,33,34 The increased resistance might be also related to frequent usage of this antibiotic in protozoan, genital, and dental infections.12 In the present study, all the MDR (18/18) and most of the QDR (10/11) strains were resistant to metronidazole (Table 1). Of the 68 metronidazole-resistant and 29 MDR strains, 47% (32/68) and 86.7% (59/68) showed MICs of ‡ 32 mg/ml. This resistance rate can hinder the success of its usage in common eradication regimens. Amoxicillin is often implemented in H. pylori treatment protocols. Although in most studies it was shown that H. pylori resistance to either amoxicillin or ampicillin is very rare, the rate of resistance is increasing now.16 The resistance rate in Iran changed from 1.4% in 2000 to 3.7% in 2004 and 7.3% in 2008.37 This increase was also observed in the present study compared with our previous report (15% vs. 2.5%). The highest resistance rate to amoxicillin has ranged 18.5% in South Korea, 19.4% in Indonesia, 32.8% in India, and 38% in Brasilia.19,22,23,27 A total of 48.2% (14/ 29) of our MDR isolates showed resistance to ampicillin, of which 14.5% showed simultaneous resistance to ampicillin and metronidazole. As metronidazole and amoxicillin are
SHOKRZADEH ET AL.
among the most commonly administered antibiotics in the treatment of H. pylori infection in Iran, simultaneous resistance to these antibiotics could be problematic in clinical settings (Table 1). The rate of clarithromycin resistance was reported to be higher among developing countries (25–50%) compared with developed countries (10%).5,23 This resistance was observed in 26% (29/111) of our isolates, which was higher than our previous results (14%).36 Similar to our results, an increasing trend in resistance rates to clarithromycin (8.6%, 9.0%, and 20.7%) was reported during 2000–2009 in Shanghai.6 Among the clarithromycin-resistant isolates, 69% and 27.5% (20/29 and 8/29, respectively) were found to have the MDR and QDR patterns, respectively (Table 1). Owing to the failure of clarithromycin-based standard triple therapy, the use of levofloxacin for H. pylori eradication is increasing worldwide. The rate of resistance to levofloxacin and ciprofloxacin in the studied strains was 24.3% (27/111) and 27% (30/111), respectively, which was higher than that reported in our previous study (7%).36 In Germany, the ciprofloxacin resistance rate was reported to be 11% in 2003, 16.6% in 2004, and 22% in 2005.18 Although low rates of resistance to quinolones were reported in most countries, the frequency of resistance increased up to > 26%.43 The widespread use of fluoroquinolones for H. pylori infection has led to an elevated resistance rate in some areas.9,41 However, our results showed that this increased rate of resistance was not associated with the history of its usage among the studied patients in Iran. Resistance against rifamycins and rifabutin was also very rare until a few years back; however, the incidence of resistance to these antibiotics is increasing.24 In the present study, 14.40% (16/111) of our isolates showed phenotypic resistance to rifampicin (MIC > 4 mg/L); however, different rates of resistance were reported in other investigations. In Germany, rifampicin-resistant clinical H. pylori isolates were detected in 1.4% of cases, which was significantly lower than our isolates.17 Most of the rifampicin-resistant strains in our study were related to MDR (13/29, 44.8%) or QDR (8/11, 72.2%) strains. In conclusion, an increased rate of resistance to the mainly prescribed antibiotics against H. pylori strains was detected in Iran. The findings showed a high frequency of MDR phenotypes, especially triple and quadruple drug resistance profiles, among these strains, which is a case of major concern. According to the low rate of resistance to amoxicillin and tetracycline among the studied strains, treatment of the infected patients with H. pylori strains harboring dual resistance to clarithromycin and metronidazole could be achieved due to the administration of tetracycline-containing (e.g., bismuth-containing quadruple therapy) or amoxicillincontaining (e.g., hybrid therapy) regimens. Further studies are needed to assess the efficacy of these regimens in these patients. Acknowledgments
This study was supported by a grant from the Gastroenterology and Liver Diseases Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. The authors thank Dr. Mohammad Mehdi Aslani, Dariush
MULTIPLE DRUG-RESISTANT HELICOBACTER PYLORI
Mirsattari, Sara Sayyadi, and Babak Yadegar for their kind cooperation in the recruitment of this study. Disclosure Statement
No competing financial interests exist. References
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Address correspondence to: Masoud Alebouyeh, PhD Gastroenterology and Liver Diseases Research Center and Basic Science and Molecular Epidemiology of Gastrointestinal Disorders Research Center Shahid Beheshti University of Medical Sciences 7th Floor of Taleghani Hospital Velenjak Shahid Aerabi Street Yemen Street Tehran 1985717413 Iran E-mail: [email protected] Mohammad Reza Zali, MD, FACG Gastroenterology and Liver Diseases Research Center Shahid Beheshti University of Medical Sciences 7th Floor of Taleghani Hospital Velenjak Shahid Aerabi Street Yemen Street Tehran 1985717413 Iran E-mail: [email protected]
