الخلاصـة: إن ظهور الذراري المقاوِمة للمتـرونيدازول، يؤدِّي إلى إخفاق نظام استئصال الـمَلْوِيَّة البوابيَّة Helicobacter pylori بالأدوية الأربعة، في جمهورية إيران الإسلامية. وقد تم في هذه الدراسة مقارنة النجاعة المختبرية للفورازوليدون مع نجاعة كل من المتـرونيدازول، والكلاريثروميسين، والأموكسيسيلين، والتـتـراسيكلين، في معالجة 70 مستفرَدة من الـمَلْوِيَّة البوابيَّة، من مرضى يعانون من عسر الهضم. وقد كان ثلث (33%) المستفرَدات مقاوِمة للمتـرونيدازول، في حين كانت جميعاً شديدة الحساسية للفورازوليدون. ومن ثـَمَّ يمكن اعتبار الفورازوليدون بديلاً ملائماً للمتـرونيدازول في معالجة العدوى بالـمَلْوِيَّة البوابيَّة.

ABSTRACT: The occurrence of strains resistant to metronidazole is causing failure of the 4-drug regimen for eradication of Helicobacter pylori in the Islamic Republic of Iran. This study compared the in vitro efficacy of furazolidone with metronidazole, clarithromycin, amoxicillin and tetracycline in 70 H. pylori isolates from dyspeptic patients. Of the isolates, 33% were resistant to metronidazole but all were susceptible to furazolidone. Furazolidone could be considered as an appropriate substitute for metronidazole for H. pylori infections.

Efficacité antimicrobienne de la furazolidone contre les souches d’Helicobacter pylori résistantes au métronidazole

RÉSUMÉ: L’apparition de souches résistantes au métronidazole cause l’échec du schéma associant quatre médicaments pour un traitement d’éradication d’Helicobacter pylori en République islamique d’Iran. La présente étude a comparé l’efficacité in vitro de la furazolidone avec le métronidazole, la clarithromycine, l’amoxicilline et la tétracycline pour 70 isolats de H. pylori provenant de patients dyspeptiques. Parmi ces isolats, 33 % présentaient une résistance au métronizadole, mais tous ont montré une sensibilité à la furazolidone. La furazolidone pourrait être considérée comme substitut approprié du métronidazole pour les infections à H. pylori.

¹Immunology, Asthma and Allergy Research Institute; 3Digestive Disease Research Centre, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran.
²Faculty of Sciences, University of Tehran, Tehran, Islamic Republic of Iran (Correspondence to F. Siavoshi: Siavoshi@Khayam.ut.ac.ir).
Received: 25/12/03; accepted: 11/11/04
 

Introduction
Many studies on the chemotherapy of Helicobacter pylori infections have indicated that eradication of H. pylori from the human stomach is hard to achieve and recrudescence occurs as a result of a persistent residual population that survived the therapy or re-infection with new strains. The low efficacy of currently used antimicrobials, even as quadruple regimens, urges researchers to look for novel antimicrobials with higher efficacy.
Metronidazole has been included in quadruple therapies in the Islamic Republic of Iran, but due to suboptimal eradication rates, investigators have attempted to replace it with other antimicrobials such as furazolidone [1]. However, resistance to metronidazole has been reported with varying degrees (20%–30%), reaching up to 70% in some European countries [2]. There is evidence that most metronidazole-resistant strains have a mutation in the RDXA gene, which leads to an inability to reduce the active nitro- group in metronidazole [3].
Although clarithromycin is effective when used in combination with a bismuth salt, a proton pump inhibitor (PPI), and amoxicillin or metronidazole [4,5], worldwide reports also describe an increasing emergence of resistant strains [6,7], reaching up to 14% in France [8]. Clarithromycin resistance is believed to result from clonal selection of resistant variants rather than from reinfection with exogenous clarithromycin-resistant strains [9]. Resistance appears to be due to a single nucleotide mutation [10] or post-transcriptional methylation of the 23S rRNA [11]. Amoxicillin and tetracycline are the 2 most highly effective antimicrobials against H. pylori in vitro and there are very few reports of resistant strains emerging [12–14]. Furazolidone appears to be an effective antimicrobial agent against H. pylori, particularly in combination with other antimicrobials such as clarithromycin [15,16] or tetracycline [17]. Furthermore, there are few reports of H. pylori resistance to furazolidone [18,19].
H. pylori infection is highly prevalent in the population of the Islamic Republic of Iran (> 80% in one study [20]) and a considerable proportion of individuals suffer from dyspeptic diseases such as gastric ulcer [20] or cancer [21]. Furthermore, a high frequency (37%) of metronidazole-resistant strains in the country increases the risk of persistence of H. pylori infection [22]. Accordingly, this might be a plausible reason for incorporating furazolidone in quadruple regimens for the eradication of metronidazole-resistant strains of H. pylori.
In this study, epsilometer (E-test) and disk diffusion methods were used to compare the susceptibility of H. pylori isolates from Iranian patients to a range of antimicrobials including furazolidone. The minimum inhibitory concentration (MIC) of the antimicrobials was also determined.

Methods
The study group was 70 dyspeptic patients referred to the endoscopy unit at Shariaty Hospital in Tehran, Islamic Republic of Iran between 2001–03. The patients were diagnosed with ulcer (9), oesophagitis (18), Barrett’s oesophagus (15) and gastritis (28).
Antral biopsies were cultured on selective Brucella blood agar (Merck), and plates were incubated under microaerobic conditions (5% CO2) at 37 °C. The identity of bacterial isolates was confirmed by microscopy and positive catalase, oxidase and urease reactions. Three-day cultures were used to prepare bacterial suspensions in normal saline, with the turbidity equivalent to McFarland standard no. 1. Volumes of 100 μL of bacterial suspensions were spread evenly over the Mueller–Hinton agar containing 7% defibrinated blood. E-test strips (AB Biodisk, Solna, Sweden) or blank paper disks were then deposited on the surface of the inoculated plates. Plates were incubated as mentioned earlier and examined after 2–5 days.
The E-test was used to assess the susceptibility of H. pylori isolates to metronidazole, clarithromycin, amoxicillin and tetracycline. The MIC obtained for amoxicillin, tetracycline and clarithromycin were within the same range of 0.016–0.25 μg/mL. Highly susceptible strains produced inhibition zones at MIC ≤ 0.016 mμg/mL. Susceptible isolates had MIC ranging from 0.016–0.25 μg/mL. Those H. pylori strains which were not inhibited by antimicrobial concentrations of  0.25 μg/mL were considered as resistant or highly resistant. For metronidazole, however, the MIC was different, ranging from 8–32 μg/mL. Highly resistant strains were not inhibited by concentrations of ≥ 32 μg/mL and resistant strains grew at metronidazole concentrations between 8–32 μg/mL. Susceptible strains produced inhibition zones at metronidazole concentrations of < 8 μg/mL.
The antimicrobial efficacy of furazolidone against H. pylori was assessed by the disk diffusion method. Serial dilutions of furazolidone (Sigma): 1, 0.75, 0.5, 0.25, 0.12 and 0.06 μg/mL were prepared in dimethylformamide. Then 10 μL volumes of furazolidone dilutions were introduced into paper disks on the surface-inoculated blood agar. The plates were examined after 2–5 days of microaerobic incubation. The MIC for furazolidone was determined as 0.12 μg/mL and susceptibility of H. pylori isolates was determined on the basis of the diameter of inhibition zones. Strains of H. pylori exhibiting the inhibition zones of 13–16 mm were considered as susceptible, and those producing inhibition zones of > 16 mm were considered as highly susceptible. Growth inhibition was not observed on plates deposited with blank discs containing dimethylformamide only.

Results
From 70 H. pylori isolates tested for susceptibility to amoxicillin, 61.4% were highly susceptible, 37.1% susceptible, and only 1 strain (1.4%) exhibited resistance (Table 1). The latter was highly susceptible to other antimicrobials. The majority of H. pylori isolates (72.8%) were highly susceptible to low concentrations of tetracycline, but 27.1% were inhibited by higher concentrations (0.016–0.25 mμg/mL), and are thus considered as susceptible. Resistance to tetracycline was not observed among H. pylori isolates (Table 1).

Clarithromycin showed a considerable efficacy in inhibiting H. pylori. Sixty-five out of 70 (92.9%) strains were highly susceptible, 5.7% susceptible, and only 1 strain (1.4%) showed resistance to clarithromycin (Table 1).
H. pylori isolates were also resistant to metronidazole: 21.4% were highly resistant, 11.4% resistant and 67.1% susceptible to metronidazole (Table 2).

The frequency of metronidazole resistance in isolates from patients with Barrett’s oesophagus (20.0%), ulcer (22.2%), and oesophagitis (27.8%) was higher than those from gastritis patients (17.9%), but t-test analysis showed it was not significant (P = 0.56) (Table 3).

Among 70 H. pylori isolates, 7 (10.0%; 95% CI: 4.1–19.5%) were susceptible and 63 (90.0%; 95% CI: 80.5–95.9%) were highly susceptible to furazolidone. None of the isolates exhibited resistance to furazolidone (Table 4). The number of highly susceptible H. pylori isolates to furazolidone was significantly more than to amoxicillin (P < 0.001; t-test), tetracycline (P < 0.01; t-test), and metronidazole (P < 0.001; t-test).

Discussion
Quadruple therapies have been proved to be the most effective antimicrobial regimens against H. pylori in the Islamic Republic of Iran [1,4,23], and recrudescence of infection occurs mainly due to the occurrence of strains resistant to metronidazole [1]. Metronidazole, although considered as one of the most suitable antimicrobials, induces the highest rate of resistance (15%–90%) in H. pylori populations [24]. In this study, resistance to metronidazole was 33%. It appeared that patients with ulcer, Barrett’s oesophagus and oesophagitis were more often infected with resistant strains compared with gastritis patients. Previous study in the Islamic Republic of Iran showed that 37% of isolates were resistant to metronidazole [22]. Resistance to metronidazole is also prevalent in Japan with frequencies of 54.5% [25] and 26.5% [26], and in Netherlands with a rate of 24% [27]. Resistance as high as 61% is reported from Peru [28].

Clarithromycin efficacy, when used in combination therapies, has been reported [4,5], but the emergence of resistant strains plus its high cost, make its application limited. The frequency of resistance to clarithromycin in this study was 1.4%. This was close to the lower range of resistance (1%–13%) reported from different regions of the world, including Sweden, Poland and Spain [2].

Amoxicillin and tetracycline have applications against a wide range of pathogenic microorganisms and there are very few reports on the emergence of resistant strains. These 2 antimicrobials thus continue to be successfully used in combination therapies against H. pylori [29,30]. In this study, resistance to tetracycline was not observed, indicating its high efficacy against.
H. pylori. Bacterial isolates from Peruvian patients did not show resistance to tetracycline [28], although 4.9% resistant strains occurred in Korea and 6.7% in Japan [31], and 58% in China [32]. Among 70 H. pylori isolates, 1 (1.43%) exhibited resistance to amoxicillin. The majority of studies reported no resistance to amoxicillin [13,14]; however, resistance as high as 71.9% was found in China [32].

Furazolidone with MIC of 0.12 μg/mL was comparable to amoxicillin, tetracycline and clarithromycin and showed a remarkable efficacy against H. pylori. None of the isolates exhibited resistance to this antimicrobial. Reports from different regions of the world also describe the high efficacy of furazolidone in eradication of H. pylori [33]. Furazolidone was effective in eradication of H. pylori when used in triple [15,34] or quadruple therapies [35]. Different MIC have been obtained in various laboratories, e.g. from < 0.006–0.2 μg/mL in China [36,37]. However, 4% furazolidone resistance and 42% metronidazole resistance were reported for H. pylori in Brazil [19]. A similar report from South Korea described 2% furazolidone and 52% metronidazole-resistant strains of H. pylori [18]. It was also found that metronidazole-resistant and -susceptible strains were both similarly inhibited by furazolidone [33,38]. These data are confirmed by other reports, indicating that there is no cross-resistance between metronidazole and furazolidone among H. pylori strains [33].

Furazolidone is an antimicrobial from the nitrofuran group. Like that of metronidazole, the bactericidal mechanism of action of this group of antimicrobials involves enzymatic reduction of the parent compound to electrophilic radicals [39,40]. In spite of the similarity in the mechanisms of action, it appears that development of bacterial resistance to metronidazole [41,42] is different from that of nitrofurans [43]. Furthermore, H. pylori does not appear to readily acquire resistance to nitrofurans [44]. Prescription of furazolidone in combination with amoxicillin or tetracycline, plus ranitidine and a bismuth salt has led to a higher eradication rate of H. pylori (82%) compared with metronidazole (56%) [1]. Furazolidone has been also effective in the clearance of H. pylori and resolution of acute gastric inflammation [3,37] and duodenal ulcer healing [15]. The results of this study suggest the recruitment of furazolidone as an effective, cheap and readily available antimicrobial [33] in quadruple therapy regimens especially in areas with high prevalence of metronidazole-resistant strains of H. pylori.
Acknowledgements
The authors wish to thank Mrs Janeshin for her assistance in the endoscopy room and Miss Alamshahi for helping to organize the manuscript.

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