helicobacter pylori ( h. pylori ) is a gram negative , non - spore forming microaerophilic spiral resident of gastric mucosa in more than half of the world s population . the world health organization introduced this bacterium as a carcinogen class 1 agent in 1994 ( 1 ) . based on epidemiological studies , h. pylori is one of the most common pathogens that causes chronic inflammation of the gastrointestinal tract in humans and is an important risk factor in chronic gastritis , gastric ulcer , duodenal ulcer and gastric cancer ( 2 ) . h. pylori antibiotic resistance is the main factor affecting the effectiveness of current therapeutic regimens . prevalence of bacterial resistance varies in different geographic areas ; and it is correlated with the consumption of antibiotics in the general population ( 3 ) . triple therapy to eradicate h. pylori in which a bismuth preparation or proton pump inhibitor is combined with two drugs from clarithromycin , metronidazole , amoxicillin and tetracycline , is the most widely used regimen . however , h. pylori rapidly acquires resistance to many classes of antibiotics after repeated exposure . resistance is prevalent worldwide , especially in developing countries , and is considered the primary reason for failure to eradicate infection ( 4 ) . various researches have shown that resistance to metronidazole reduces the adequacy of treatment by 50% ( 5 ) . resistance to antibiotics is so high that in many countries it has led to removal of many first line antibiotic choices such as clarithromycin ( 6 ) . antimicrobial properties of nitazoxanide have been shown against helminthes , protozoa ( cryptosporidia , microsporidia , trichomonas , entamoeba , and giardia ) and many bacteria ( 8,9 ) . a treatment method called load therapy , which includes levofloxacin , omeprazole , alinia ( nitazoxanide ) and doxycycline , was used for seven days at doses of 100- 500- 40- 250 mg per day , respectively ; this method is the preferred treatment choice for h. pylori infections in the united states ( 10 ) . according to the high prevalence of h. pylori infection and its complications such as peptic ulcers , gastric adenocarcinoma and increasing resistance to metronidazole and the emergence of mdr strains that cause treatment failure , we aimed to examine the effectiveness of nitazoxanide against metronidazole resistant isolates . h. pylori strains were isolated from patients who referred to the endoscopy ward in laleh and kasra hospitals in tehran from november 2014 to july 2015 . following bacterial culture , the resistance pattern and minimal inhibitory concentrations ( mics ) toward metronidazole and nitazoxanide were determined . this study was conducted at kasra and laleh hospitals in tehran from november 2014 to july 2015 . h. pylori strains were isolated from 122 biopsy specimens of patients with gastrointestinal complaints who underwent endoscopy . exclusion criteria included treatment with proton pump inhibitor and use of antibiotics within the two weeks prior to sample collection . two biopsies per patient were obtained from antrum of stomach by a gastroenterologist , which were used for pathology and rapid urease test . the samples for culture were kept in transfer medium ( thioglycolate broth ) , which were subsequently processed for bacterial culture . biopsy specimens were cultured in brucella agar medium containing 10% horse blood , 10% fetal bovine serum and antibiotic supplement . the plates were incubated in microaerophilic atmosphere ( concentrations of 8 - 10% by volume co2 and 5 - 7% by volume oxygen ) and placed in a gas pak jar ( anaerocult c sachets , merck , germany ) ) for up to 10 days . colonies were identified by positive catalase , urease and oxidase tests as well as morphologic examinations . minimal inhibitory concentrations were determined by agar dilution method according to the clsi guidelines . a suspension with a turbidity equivalent to that of a mcfarland no.2 standard ( approximately 610cfu / ml ) an appropriate dilution of metronidazole was added at concentrations ranging from 0.5 to 64g / ml . following the preparation of microbial suspension , inoculation took place in plates containing mueller hinton agar by spotting 10l of bacterial suspension with different concentrations of metronidazole . within a short time after the absorption of the samples , plates were incubated under microaerophilic conditions at 37c for two days . the mic was determined as the lowest concentration of antimicrobial agent inhibiting the total growth of bacteria . resistance was considered when the metronidazole mic was greater than 4g / ml . after identifying metronidazole resistant the mic determination protocol towards nitazoxanide was similar to that metronidazole , except for the bacterial turbidity , which was set at mcfarland no.3 standard ( approximately 610cfu / ml ) . nitazoxanide is not yet available commercially in iran . since there is no determined breakpoint mic value for nitazoxanide against h. pylori , minimal inhibitory concentrations were determined by agar dilution method according to the clsi guidelines . a suspension with a turbidity equivalent to that of a mcfarland no.2 standard ( approximately 610cfu / ml ) an appropriate dilution of metronidazole was added at concentrations ranging from 0.5 to 64g / ml . following the preparation of microbial suspension , inoculation took place in plates containing mueller hinton agar by spotting 10l of bacterial suspension with different concentrations of metronidazole . within a short time after the absorption of the samples , plates were incubated under microaerophilic conditions at 37c for two days . the mic was determined as the lowest concentration of antimicrobial agent inhibiting the total growth of bacteria . resistance was considered when the metronidazole mic was greater than 4g / ml . after identifying metronidazole the mic determination protocol towards nitazoxanide was similar to that metronidazole , except for the bacterial turbidity , which was set at mcfarland no.3 standard ( approximately 610cfu / ml ) . nitazoxanide is not yet available commercially in iran . since there is no determined breakpoint mic value for nitazoxanide against h. pylori , from the 122 gastric biopsy specimens , 55 h. pylori isolates ( 45% ) were collected ; of which , 40 isolates ( 72.7% ) were shown to be resistant to metronidazole using agar dilution method . the time required for rapid urease test in the positive biopsy samples based on the severity of the infection varied from 20 minutes to 24 hours . the time required for rut ( rapid urease test ) in these colonies varied from 30 seconds to 10 minutes . the mic values for h. pylori isolates resistant to metronidazole are demonstrated in table 1 . the mic50 and mic90 values for metronidazole were 32 , 64 g / ml , respectively . the mic values for nitazoxanide in the h. pylori isolates resistant to metronidazole are presented in table 2 . the mic50 and mic90 values for nitazoxanide reported to be 8 and 8g / ml , respectively . comparison of the mic values of nitazoxanide and metronidazole revealed significant differences ( p<0.05 ) . the mic values of nitazoxanide for all the metronidazole resistant isolates are shown in figure 1 . 
   the mic values for nitazoxanide in the metronidazole resistant h. pylori isolates gastrointestinal disorders are very common in developing countries with poor health indicators ( 11 ) . the increasing resistance of microorganisms to antibiotics is commonly due to the lack of proper administration , inappropriate dosing , arbitrary use of drugs , and mutations in microorganisms ( 11 ) . the prevalence of h. pylori in iran , similar to other developing countries , is higher than in developed countries ( 12,13 ) . the prevalence of h. pylori infection varies in different areas of iran ( from 30.6% to 93.0% ) ( 14 - 16 ) . in this study , h. pylori resistance to metronidazole has been on the rise in iran : 73.4% in 2009 and 88.2% in 2011 ( 17 ) . metronidazole resistant isolates of h. pylori in this study was significantly high ( 72.7% ) . due to the increasing use of metronidazole for various parasitic and oral cavity infections , metronidazole is a common antibiotic used for h. pylori treatment in iran ; and drug resistant strains are becoming a major problem for therapeutic regiment ( 18 ) . this drug works by disrupting the enzyme pyruvate oxidation and energy metabolism , eventually causing death of microorganisms ( 19 ) . the american college of gastroenterology in its 2009 annual conference proposed a quadruple therapy for the treatment of ulcers caused by h. pylori infections . this four - drug regimen ( load ) includes levofloxacin , omeprazole , alinia ( nitazoxanide ) and doxycycline ( 10 ) . 
 
 in this study , increased levels of mics to values up to 32g / ml were confirmed for metronidazole . although most of h. pylori strains showed mic values higher than 32g / ml , lower mic values were found for nitazoxanide at the same concentrations . considering the very high levels of metronidazole resistance observed in this study , this medication might not be appropriate as the first - line therapy in iran . however , considering the relative effectiveness of nitazoxanide , this drug could be an acceptable and satisfactory alternative for patients in iran . this study was financially supported by a grant ( no.26419 - 30 - 04 - 94 ) from iran university of medical sciences ( tehran , iran ) , for which we are very grateful .
background : in this study , the efficacy of nitazoxanide in the treatment of helicobacter pylori   isolates , which were resistant to metronidazole , was examined . 
 methods : one hundred twenty two patients who underwent endoscopy examinations at kasra and laleh hospitals in tehran from november 2014 to july 2015 were enrolled . 
 helicobacter pylori   strains were isolated from the patients endoscopy biopsies by bacteriological culture . 
 those bacterial isolates resistant to metronidazole were examined for susceptibility to nitazoxanide . 
 serial agar dilution method was utilized to determine the minimum inhibitory concentrations for the antibiotics . 
 results : from 122 gastric biopsy specimens , 55 h. pylori isolates were recovered ( 45% ) ; of which , 40 ( 72.7% ) were resistant to metronidazole . comparing the mic values of nitazoxanide with metronidazole revealed significant differences ( p<0.05 ) . 
 the mic50 and mic90 values for nitazoxanide and metronidazole were 8 and 8g / ml , and 32 and 64g / ml , respectively . 
 conclusion : the high levels of metronidazole resistance suggest that this medication may not be beneficial for first - line therapy in iran . 
 however , considering the relative effectiveness of nitazoxanide , it may be considered a suitable alternative for patients in iran .