The Evolution of Helicobacter pylori Antibiotics Resistance Over 10Years in Beijing,ChinaWen Gao,*,1Hong Cheng,*,1Fulian Hu,*Jiang Li,*Lihui Wang,*Guibin Yang,†Le Xu‡and Xiaoli Zheng‡*Department of Gastroenterology,Peking University First Hospital,Beijing,China,†Department of Gastroenterology,Aerospace Clinical College of Peking University,Beijing,China,‡Department of Gastroenterology,Beijing Hospital,Beijing,ChinaHelicobacter pylori is a Gram-negativeflagellated spiral bacteria.Infection with H.pylori is mainly acquired in childhood.H.pylori infection is recognized as a causal factor in the pathogenesis of chronic gastritis,peptic ulcer,gastric cancer,and gastric MALT lymphoma.H.pylori eradication treatment is indicated in patients with peptic ulcer disease,MALToma,atrophic gastritis, post-gastric cancer resection,or patients who arefirst-degree relatives of patients with gastric cancer[1]. Among numerous eradication regimens,a proton pump inhibitor and combination of two antibiotics(amoxicil-lin,clarithromycin,and metronidazole)are considered to be the most effective andfirst-line therapy regimens recommended by Maastricht III Consensus and in China[1,2].However,antibiotic resistance of H.pylori, especially to clarithromycin and metronidazole, strongly undermined the efficacy of eradication treat-ment.The reported frequencies of resistance to anti-biotics varied widely between geographic regions.A European study involving17countries and1274H.pylori isolates showed a mean resistance rate(deter-mined by E-test)to amoxicillin0.8%;to clarithromycin 9.9%;and to metronidazole33.1%[3].Another recent study from French examined530H.pylori strains iso-lates from2004to2007,among those,26%(138⁄530) strains were resistant to clarithromycin,61% (324⁄530)to metronidazole,and13.2%(70⁄530)to ciprofloxacin,whereas no resistance against amoxicillin and tetracycline was observed[4].As culture-based antimicrobial susceptibility data are not always avail-able and the H.pylori eradication regimen especially antibiotics involved should be chosen based on local resistant epidemiologic data and an empirical basis,it is important to understand the regional antibiotics resis-tance status and trend of this bacterium.The aim of this study was to assess the prevalence of antibiotics (amoxicillin,clarithromycin,metronidazole,tetracy-cline,levofloxacin,and moxifloxacin)resistance of H.pylori strains isolated from Beijing in recent 10years.KeywordsHelicobacter pylori,antibiotic resistance, amoxicillin,clarithromycin,metronidazole, tetracycline,levofloxacin,moxifloxacin.Reprint requests to:Fulian Hu,Professor, Department of Gastroenterology,Peking University First Hospital,Beijing100034, China.E-mail:hufulian@1These authors contributed equally to the study.Supported by Beijing Medicine Research and Development Fund(No.2005-1008).AbstractObjectives:To evaluate Helicobacter pylori antibiotics resistance evolution from2000to2009to amoxicillin,clarithromycin,metronidazole,tetra-cycline,levofloxacin and moxifloxacin in Beijing,China.Methods:A total of374H.pylori strains isolated from374subjects who had undergone upper gastrointestinal endoscopy from2000to2009were collected and examined by E-test method for antibiotics susceptibility. Results:The average antibiotics resistance rates were0.3%(amoxicillin), 37.2%(clarithromycin),63.9%(metronidazole),1.2%(tetracycline),50.3% (levofloxacin)and61.9%(moxifloxacin).Overall resistance to clarithro-mycin,metronidazole,andfluoroquinolone increased annually(from14.8 to65.4%,38.9to78.8%,and27.1to63.5%,in2000or2006–2007to 2009,respectively).The secondary resistance rates were much higher than primary rates to these antibiotics,which also increased annually in recent 10years.Conclusions:The trend of clarithromycin,metronidazole,andfluoroquino-lone resistance of H.pylori increased over time and the resistance to amoxi-cillin and tetracycline was infrequent and stable in Beijing.Clarithromycin, metronidazole,andfluoroquinolone should be used with caution for H.pylori eradication treatment.Helicobacter ISSN1523-5378Materials and MethodsPatients and Isolation of H.pylori Strains Demographic and endoscopic data were recorded in each case.All patients were asked about previous H.pylori eradication therapy and antibiotics use for other infections.Patients who did not remember their previous antibiotics use or lacked prescription records were regarded as previously untreated group.Three biopsy specimens were taken from gastric antrum from each patient,one for rapid urease test, one for histopathologically examination.One of the gastric mucosal biopsy specimen obtained from gastric antrum was grinded,planted,and primarily cultured on 8%defibrinated sheep blood with3.9%agar medium (Columbia Agar Base;Oxoid LTD,Basingstoke,Hamp-shire,UK)with0.5%trimethoprim,0.3%vancomycin, and0.2%amphotericin at37°C under microaerophilic conditions(5%O2,10%CO2,85%N2).Plates were incubated for at least3days,up to7days prior to passage.The bacterial strains obtained from primary culture were identified as H.pylori strains by colony morphology,Gram’s stain,urease reaction,oxidase reaction,and catalase reaction.All strains were stored at)80°C in brain heart infusion broth(BHI;Difco Laboratory,Detroit,MI,USA)supplemented with30% glycerol.Before the antibiotics E-test,H.pylori strains were cultured for1–2passages on8%sheep blood with 3.9%agar medium at37°C under microaerophilic conditions.A total of374H.pylori strains isolated from374 patients who had undergone upper gastrointestinal endoscopy in GI department of Peking University First Hospital,Aerospace Clinical College of Peking University and Beijing Hospital from2000to2009were collected and involved in antimicrobial susceptibility test.Determination of MIC for E-testAlthough agar or broth dilution methods are estab-lished for standard susceptibility test for H.pylori,they are difficult to perform routinely and E-test has been widely performed and yielded reliable results equivalent to agar dilution method[5].In this study,all374 isolated H.pylori strains were cultured and performed E-test to identify the minimal inhibitory concentration (MIC)value to metronidazole,amoxicillin,clarithro-mycin,tetracycline,levofloxacin,and moxifloxacin (E-test strips from AB Biodisk,Solna,Sweden).The H.pylori strains were considered amoxicillin, clarithromycin,metronidazole,tetracycline,levofloxa-cin and moxifloxacin resistant with MICs‡1,‡1,‡8,‡1,‡1,‡1mg⁄L,respectively[6].ResultsThe374patients consisted of213(57.0%)men and 161(43.0%)women,median age49years(range13–83years).Of the374subjects,290(77.5%)patients never received H.pylori eradication treatment before, while84(22.5%)patients failed in their previous anti-H.pylori treatment once or more times.Endoscopic diagnosis showed that162patients had chronic gastri-tis,160had duodenal ulcer,36had gastric ulcer,8had both gastric and duodenal ulcer,7had gastric cancer, and1had gastric MALToma(Table1).Annual prevalence rates of overall,primary and secondary antibiotic resistance rates of H.pylori strains to amoxicillin,clarithromycin,and metronidazole were showed in Table2,resistant rates to tetracycline, levofloxacin and moxifloxacin were shown in Table3. The resistance status to tetracycline,levofloxacin and moxifloxacin was not examined until the year of2006–2007and2008.The trend of resistance prevalence toTable1General data of the374subjectsYear Total Gender Treatment DiagnosisN Male Female Untreated Treated G DU GU DU+GU Ga MALT200054351947715343110 200171442763835278010 2002–2003271892257128000 2004–20052719824312105000 2006–200780423871936377631 2008633330392434225110 2009522230242833180010 Total374213(57%)161(43%)290(77.5%)84(22.5%)162(43.3%)160(42.8%)36(9.6%)8(2.1%)7(1.9%)1(0.3%)M,male;F,female;G,gastritis;DU,duodenal ulcer;GU,gastric ulcer;DU+GU,duodenal ulcer+gastric ulcer;Ca,gastric cancer;MALT,gastric mucosa-associated lymphoid tissue lymphoma.Gao et al.Evolution of H.pylori Antibiotics Resistanceclarithromycin,metronidazole and levofloxacin was shown in Figs 1–3.H.pylori resistance to amoxicillin was found in only one case in all 374strains (MIC =1mg ⁄L,0.27%),which was isolated from an untreated patient in theyear of 2000.This H.pylori strain was actually triply resistant to amoxicillin,clarithromycin and metronidazole.The overall resistance rate to clarithromycin rose from 14.8%in 2000to 65.4%in 2009(Fig.1),while the primary resistance rate to clarithromycin was 12.8%in the year of 2000,rose to its peak of 38.5%in the year of 2006–2007and 2008,then decreased to 25%in 2009(Fig.2).Compared with primary resis-tance status,the secondary resistance rate dramatically increased from 28.6%(2⁄7)in 2000to 100%(28⁄28)in 2009(Fig.3).Metronidazole resistance rate was relatively high with an average value of 63.9%(239⁄374),ranged from 38.9%in 2000to 78.8%in 2009,with a highest rate of 83.6%in 2007(Table 2,Fig.1).When it came to primary and secondary resistance rate,the latter was even higher.The peak primary resistance rate was 83.1%in 2006–2007,and then decreased a little to 66.7%in 2008and 2009(Fig.2).The secondary metro-nidazole resistance rate arrived 100%in the year of 2004–2005and 2008,with an average rate of 89.3%(Table 2,Fig.3).Table 3Annual prevalence of antibiotic resistance of Helicobacter pylori strains to tetracycline,levofloxacin,and moxifloxacin from 2006to 2009Primary Secondary Overall TLeMxT LeMxTLeMx2006–20070⁄41(0)10⁄40(25.0)N ⁄C1⁄8(12.5)3⁄8(37.5)N ⁄C1⁄49(2.0)13⁄48(27.1)N ⁄C20080⁄39(0)18⁄39(46.2)11⁄27(40.7)0⁄24(0)18⁄24(75.0)16⁄18(88.9)0⁄63(0)36⁄63(57.1)27⁄45(60.0)20091⁄24(4.2)10⁄24(41.7)10⁄24(41.7)0⁄28(0)23⁄28(82.1)23⁄28(82.1)1⁄52(1.9)33⁄52(63.5)33⁄52(63.5)Overall1⁄104(1.0)38⁄103(36.9)21⁄51(41.2)1⁄60(1.7)44⁄60(73.3)39⁄46(84.8)2⁄164(1.2)82⁄163(50.3)60⁄97(61.9)Values given in parentheses are percentages.T,tetracycline;Le,levofloxacin;Mx,moxifloxacin.Table 2Annual prevalence of antibiotic resistance of Helicobacter pylori strains to amoxicillin,clarithromycin,and metronidazole from 2000to 2009Primary Secondary Overall ACMzA CMzACMz20001⁄47(2.1)6⁄47(12.8)16⁄47(12.8)0⁄7(0)2⁄7(28.6)5⁄7(71.4)1⁄54(1.9)8⁄54(14.8)21⁄54(38.9)20010⁄63(0)8⁄63(12.7)20⁄63(12.7)0⁄8(0)4⁄8(50.0)6⁄8(75.0)0⁄71(0)12⁄71(16.9)26⁄71(36.6)2002–20030⁄22(0)2⁄22(9.1)12⁄22(9.1)0⁄5(0)4⁄5(80.0)4⁄5(80.0)0⁄27(0)6⁄27(22.2)16⁄27(59.3)2004–20050⁄24(0)5⁄24(20.8)17⁄24(20.8)0⁄3(0)3⁄3(100.0)3⁄3(100.0)0⁄27(0)8⁄27(29.6)20⁄27(74.1)2006–20070⁄71(0)27⁄71(38.0)57⁄71(38.0)0⁄9(0)8⁄9(88.9)8⁄9(88.9)0⁄80(0)35⁄80(43.8)65⁄80(81.3)20080⁄39(0)15⁄39(38.5)26⁄39(38.5)0⁄24(0)21⁄24(87.5)24⁄24(100.0)0⁄63(0)36⁄63(57.1)50⁄63(79.4)20090⁄24(0)6⁄24(25.0)16⁄24(25.0)0⁄24(0)28⁄28(100.0)25⁄28(89.3)0⁄52(0)34⁄52(65.4)41⁄52(78.8)Overall1⁄290(0.3)69⁄290(23.8)164⁄290(56.6)0⁄84(0)70⁄84(83.3)75⁄84(89.3)1⁄374(0.3)139⁄374(37.2)239⁄374(63.9)Values given in parentheses are percentages.A,amoxicillin;C,clarithromycin;Mz,metronidazole.Evolution of H.pylori Antibiotics Resistance Gao et al.The resistance to tetracycline was infrequent.It hap-pened in one of forty-nine(2.0%)in2006–2007,zero of sixty-three in2008,and one offifty-two(1.9%)in 2009(Table3).It was reported that there was cross-resistance between levofloxacin and moxifloxacin.Although not all H.pylori strains that received levofloxacin suscepti-bility test(n=163)were screened by moxifloxacin (n=97),all97H.pylori strains whose resistance status to the two antibiotics were examined simultaneously were resistant to both of them,with an average rate of50.3and61.9%,respectively(Table3).The second-ary resistance rate was much higher than primary rate.The primary resistance rate to levofloxacin and moxifloxacin was36.9and41.2%,respectively (Table3),while the secondary rate was73.3and 84.8%,respectively(Table3).The primary levofloxa-cin resistance rate rose from25%in2006–2007to 41.7%in2009(Fig.2),while the secondary resistance rate rose from37.5%in2006–2007to82.1%in2009 (Table3,Fig.3).DiscussionThe European multicenter study confirmed that the E-test showed excellent intra-and interlaboratory cor-relation with agar dilution for amoxicillin and clarithro-mycin[7,8].It was performed in the present study for at least twice for each isolated H.pylori strain to get sta-ble and reliable results.Reports of amoxicillin resistance are infrequent.The loss of the penicillin-binding protein was found associ-ated with resistance to it[9].Resistance to amoxicillin has been estimated to be<1%in most studies[10,11], but also at a high rate(8.8%)in Japan[12].In the present study,there was one H.pylori strain resistant to it,with a prevalence rate of0.3%(1⁄374).Hence,the amoxicillin resistance’s role in clinical practice may even be marginalized[13],which means this antibiotic could be chosen to be prescribed in most suitable cases in clinical practice.Although very high resistance rates to amoxicillin(71.9%)have been reported in some studies[14],however,these results must be interpreted with caution until the strains have been explored in depth[10].The prevalence of H.pylori resistance to metronida-zole varies from20to40%in Europe and the USA to 50–80%in developing countries[10].A previous study performed in China showed a resistance rate of77.8% (119⁄153)to metronidazole.In the present study,the overall metronidazole resistance rate was63.9% (239⁄374),which was higher than that in Europe and similar to that in China.The primary and secondary resistance rate was56.6%(164⁄290)and89.3% (75⁄84),respectively.As the Maastricht III Consensus Report stated that the PPI-clarithromycin-metronidazole regimen is preferable in populations with<40%metro-nidazole resistance[1],a regimen including metronida-zole is not suitable and should not be chosen at least as first-line treatment therapy in Beijing,China. Resistance to macrolides(clarithromycin is most widely used)is produced by a mutation in the2143 and2144position in the V domain of the rRNA23S [15].Resistance to clarithromycin is considered to be caused by the previous consumption of macrolides.The resistance of this antibiotic seriously affected the eradi-cation rate of H.pylori infection as PPI-clarithromycin-amoxicillin regimen was recommended asfirst-line therapy in most countries[1].Gao et al.Evolution of H.pylori Antibiotics ResistanceThe clarithromycin resistance status varied a lot in different regions of the world.Before the year2000,it was estimated to be<4%in Canada[16],which has already reached10–15%in the USA based on data from clinical trials[10,17].A survey from Japan observed an average resistance rate of16.4% (577⁄3521)from1996to2008and it has increased gradually to approximately30%from1996through 2004,remained unchanged since2004[11].A survey from Taiwan reported a10.6%resistance rate to this antibiotic,the resistance rate elevated significantly aftera failed clarithromycin-based triple therapy(78.7vs10.6%,p<.001)[18].Compared with the resistance status in developed countries and regions in Asia,the resistance rate in Beijing was pretty high,especially in strains isolated from previous treatment failure patients. In our present study,the overall resistance to clarithro-mycin increased from14.8%in2000to65.4%in2009, while the increase in primary resistance rate to clari-thromycin was12.8%in the year of2000,rose to its peak of38.5%in the year of2006–2007and2008, decreased to25%in2009.Clarithromycin was intro-duced to clinical practice in China in1995and then was largely used for the treatment of respiratory diseases,which may be a factor in the emergence of macrolide-resistant strains[19].Compared with primary resistance status,the second-ary resistance rate to clarithromycin dramatically increased from28.6%(2⁄7)in2000to100%(28⁄28) in2009,with an average rate of83.3%(70⁄84).From the year of2000to2009,the trend of secondary resis-tance rate was keeping increasing,even the actual number of H.pylori strains involved was little.It was thought that the clarithromycin resistance is easily acquired,and H.pylori strains often became resistant to it after previous treatment with this antibiotic[20].The Maastricht III Consensus Report stated that the thresh-old of clarithromycin resistance at which antibiotic should not be used or a clarithromycin susceptibility test should be performed is15–20%[1].According to the consensus and the resistance characteristics of the antibiotic,the regimen that comprised clarithromycin should be chosen cautiously.To patients who had failed in previous clarithromycin containing treatment,this antibiotic should not be chosen or readministered, except with the support from antimicrobial susceptibil-ity test.However,in some regions of China,it was common that clarithromycin was readministered to cure H.pylori infection again and again even after mul-tiple times of failure in eradication treatment without susceptibility test,which might be an important reason of refractory H.pylori infection and the severe resistance status of this antibiotic.The tetracycline resistance mechanism has been described as a change in three contiguous nucleotides in the16S rRNA gene(AGA926-928RTTC)[21].Resis-tance to tetracycline is very low,or even absent,in most countries[4,10].There were also high resistance rate reports in Korea(5.3%)[22]and Bulgaria(primary resistance 4.4%and secondary resistance13.3%), which might be because of the high consumption of tetracycline in previous years[23].Tetracycline is rarely prescribed in China recently, especially in cities such as Beijing.In the present study, there were two H.pylori strains resistant to tetracycline, with a resistance prevalence rate of1.2%(2⁄164).It would be an option after failure infirst-or second-line therapy treatment.Fluoroquinolones(levofloxacin)-based triple therapy achieved good eradication rates(90–94%)in Italy[24] and in Germany(86.7%)[25].High eradication rate (82.4%)was also reported in China recently[26].As other bacteria,resistance of H.pylori tofluoroquinol-ones is because of point mutations in the quinolone resistance determining regions of gyrA[27].Resistance tofluoroquinolones also mirrors the use of these kinds of drugs.Thefluoroquinolone resistance rate was relatively low in European and American coun-tries.It was reported that the resistance rate was 3.9%(ciprofloxacin)in eastern European countries in 1998[28],11.5%(levofloxacin)in Hong Kong[29]. In the present study,the levofloxacin and moxifloxa-cin resistance tests were performed on H.pylori strains isolated in2006–2009,with an average rate of50.3 and61.9%,respectively.The average primary resistance rate to levofloxacin was36.9%,while the average secondary resistance rate increased to73.3%, suggesting the easy-to-acquired resistance characteris-tics.In our hospital,the prescription offluoroquino-lone topped other antibiotics in recent years,which might be an important cause of its high resistance rates.The relatively highfluoroquinolone resistance rate observed in our study contrasted with the pretty high eradication rate of more than80%of levofloxacin-or moxifloxacin-based triple regimen[26,30],indicating that the susceptibility or resistance status in vitro did not always predict treatment success or failure,at least asfluoroquinolone was mentioned.However,in our study,the number of strains per year for secondary antibiotic resistance status analysis was low,so only a trend can be considered,and this trend was toward an increased prevalence over time. However,with a so high rate of resistance,it should be necessary to test the antimicrobial susceptibility of the H.pylori strains,using either the culture or theEvolution of H.pylori Antibiotics Resistance Gao et al.available molecular methods,particularly in case of secondary resistance(but not only perhaps). ConclusionThe high prevalence of clarithromycin,metronidazole, andfluoroquinolone resistance of H.pylori strains particularly in cases of secondary resistance highlighted that the management of the treatment of H.pylori infection is becoming a problem.Even knowing the susceptibility of H.pylori,eradication rates do not achieve100%,as the results observed in vivo by following in vitro susceptibility to anti-H.pylori antibi-otics are often disappointing[31].More than20%of patients will fail to eradicate H.pylori infection even with the current most effective treatment regimens. Antibiotic resistance to clarithromycin has been identi-fied as one of the major factors affecting the eradication rate of H.pylori infection,as PPI-amoxicillin-clarithro-mycin regimen is recommended asfirst-line treatment in most countries.The resistance rate to clarithromycin seemed to be relatively high and increasing in China annually.Alternative antibiotics should be considered as a choice offirst-line or rescue therapy.Amoxicillin and tetracycline might be a good option as their resis-tance rates were very low.However,levofloxacin or moxifloxacin-basedfirst-line or rescue therapy may constitute an encouraging strategy,as in vitro antimicrobial susceptibility does not necessarily lead to eradication in vivo.References1Malfertheiner P,Megraud F,O’Morain C,et al.Current con-cepts in the management of Helicobacter pylori infection:theMaastricht III Consensus Report.Gut2007;56:772–81.2Hu FL,Hu PJ,Liu WZ,et al.Third Chinese National Consensus Report on the management of Helicobater 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