The Korean Journal of Helicobacter and Upper Gastrointestinal Research Vol. 12, No. 2, 82-87, June 2012 http://dx.doi.org/10.7704/kjhugr.2012.12.2.82 광주전남지역에서 Helicobacter pylori 제균항생제내성률분석 Analysis of Antimicrobial Resistance of Helicobacter pylori in Gwangju, Chonnam Provinces 전남대학교의과대학내과학교실 Min Hyung Kim, Hyun Soo Kim, Sun Young Park, Chang Hwan Park, Sung Kyu Choi, Jong Sun Rew Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea Background/Aims: A triple therapy, comprised of a proton pump inhibitor, clarithromycin, and amoxicillin or metronidazole, is the most commonly used treatment for Helicobacter pylori (H. pylori) eradication. But, H. pylori have become resistant to antimicrobial agents, reducing eradication rates. Thus, we investigated the distribution of minimal inhibitory concentration (MIC) for these drugs and analyzed the factors that affect it. Materials and Methods: From July 2008 through December 2009, we performed tissue culture with gastric mucosa from 51 patients who were infected with H. pylori and measured MIC by Epsilometer test (E-test). Results: The mean age of the group was 56.8±13.8 years, and the male to female ratio was 2:1. Resistance rates of H. pylori to amoxillin, clarithromycin, metronidazole, ciprofloxacin, levofloxacin, tetracycline were 2%, 15.7%, 21.6%, 25.5%, 25.5%, and 0%, respectively. Statistically, there were no significant relationship between resistance rate and other factors including age, sex, alcohol or smoking. Follow up test revealed eradication failure in 4 of 22 patients but, MIC for amoxillin and clarithromycin of these four patients were considered to belong to the susceptible group. Three patients belonging to the clarithromycin-resistant group (MIC>256μg/mL), were eradicated. In addition, one of the three patients showed resistant to not only clarithromycin (MIC 12μg/mL), but also amoxiciliin (MIC 0.6μg/mL). Conclusions: The microbial susceptability test of each antibiotics does not accurately reflecte successful eradication, but further investigation is needed to identify the factors that influence the eradication of H. pylori. (Korean J Helicobacter Up Gastrointest Res 2012;12:82-87) Key Words: Helicobacter pylori; Micribial sensitivity test 김민형, 김현수, 박선영, 박창환, 최성규, 유종선접수일 :2012년 4월 1일승인일 :2012년 5월 25일연락처 : 김현수광주시동구제봉로 42 우편번호 : 501-757 전남대학교의과대학내과학교실 Tel: 062-220-6201 Fax: 062-225-8578 E-mail: dshskim@jnu.ac.kr 서론 Helicobacter pylori (H. pylori) 감염은만성위염, 소화성궤양의원인인자이며, 위선암과점막연관림프조직형위림프종 (gastric mucosa associated lymphoid tissue lymphoma) 의발생과밀접한연관이있다. 또한 H. pylori의치료는궤양의 재발억제뿐아니라위암예방을위해서도필요하다. 1 현재가장많이사용하고있는 H. pylori 제균요법은프로톤펌프억제제 (proton pump inhibitor, PPI) 를중심으로두가지항생제 (amoxicillin, clarithromycin) 를병용하는삼제병합요법이널리시행되고있다. 2 삼제요법제균은도입당시에는효과적이었으나항생제내성이증가하면서제균율이감소하여메타분석에서제균율이 70 95% 로다양하게보 82
김민형외 : 광주전남지역에서 Helicobacter pylori 제균항생제내성률분석 83 고되고있다. 3-5 국내에서도 1991년부터 1998년까지는제균율이 90% 이상으로보고되었으나 6 1998년에서 2003년까지의여러연구에서는 79.2 91.4% 정도를보였고, 7-10 2003년이후의제균율은 77.6 87.2% 정도로점차감소하는경향이다. 11,12 H. pylori 제균에는환자의연령, 성별, 약제의순응도, 흡연등여러가지요인이영향을줄수있으나, 13,14 제균율감소의가장큰원인은 metronidazole, clarithromycin, amoxicillin 등여러항생제에대한 H. pylori 내성률의증가로생각된다. 15-17 본연구에서는광주, 전남지역에서분리된 H. pylori 균주의항생제내성률을분석하고, 실제 H. pylori 제균에사용하는여러항생제의최소억제농도 (minimal inhibitory concentration, MIC) 에영향을주는인자및이들이제균치료결과에미치는영향을확인하고자하였다. 대상및방법 1. 대상환자 2008년 7월부터 2009년 12월까지전남대학교병원에서상부위장관내시경을시행하여 H. pylori 양성소화성궤양으로확인되었거나, 위선종등의점막하박리술후 H. pylori 양성으로확인된 51명을대상으로하여 H. pylori 배양및제균요법에사용되는항생제들에대한 MIC를분석하였으며, MIC에영향을주는인자를분석하기위하여의무기록지를후향적으로조사하였다. 2. 방법 1) H. pylori 배양 : 소화성궤양환자나, 위선종으로점막하박리술을시행받은환자에서상부위장관내시경으로위생검조직을채취하였으며, 조직을멸균분쇄하여 3개의 chocolate agar에넣어배양하였다. 이후 chocolate agar에키운 H. pylori 균주를멸균된면봉을사용하여긁어서 freezing media (brucella broth와 5% fetal bovine serum, 40% glycerol 로구성 ) 가들어있는 15 ml conical tube에덩어리없이잘풀어 cryo tube에분주하고알코올램프로입구를화염멸균한다음표기 (labelling) 하여 70 o C에보관하였다. 2) H. pylori 항생제감수성검사 : MIC 검사는 H. pylori 제균요법에사용되고있는 amoxicillin, clarithromycin, metronidazole, ciprofloxacin, levofloxacin, tetracycline을대상으로하였고 MIC를검사하기위하여 Epsilometer test (E-test) 를사용하였다. 90 mm 1 plate (chocolate agar) 에 1 strip을사용하여균을도포한후 3 5일정도충분히성장시키고나서 MIC를판독하였으며, 두명의관찰자를두고실험을진행하였다. H. pylori에대한항생제내성기준은오늘날 자주이용되고있는 MIC값을사용하였으며, 18 본연구에서는 amoxicillin의경우 MIC가 0.5μg/mL 이상일때, clarithromycin의경우는 MIC>1μg/mL, metronidzole은 MIC>8 μg/ml을기준으로하였고, 이외에 ciprofloxacin과 levofloxacin은 MIC>1μg/mL, tetracycline은 MIC>4μg/mL를기준으로하여평가하였다 (Table 1). 3) H. pylori 제균치료 : 상부위장관내시경검사를통하여신속요소분해효소검사 (CLO test R ) 를시행하여양성으로나온환자를 H. pylori 양성으로판정하였으며, 이환자들을대상으로 PPI (pantoprazole 40 mg 혹은 lansoprazole 30 mg 중한가지 ) 와 amoxicillin 1 g, clarithromycin 500 mg을하루 2회씩 1주일동안제균치료하였다. 치료완료후에위내시경검사를통한조직생검이나신속요소분해효소검사, 13 C 요소호기검사 (urea breath test) 를시행하였으며위세가지검사중한가지라도양성으로나온경우는제균치료실패로판정하였다. 3. 자료분석 연속변수는평균 ± 표준편차로표시하였고, 통계처리는 SPSS for Windows version 17.0 (SPSS Inc., Chicago, IL, USA) 을이용하였으며모든자료는 P value가 0.05 미만일때유의하다고판정하였다. Table 1. Cut-off Values of Antibiotic Resistance in Helicobacter pylori Isolates Antibiotics 결 1. 대상환자의특성 분석에포함된전체대상환자는 51명이었고, 이중남자는 34명 (66.7%), 여자는 17명 (33.3%) 이었다. 연령범위는 25 83 세로서전체평균연령은 56.8±13.8세였으며, 성별평균연령은남자 54.2±14.6세, 여자 62.0±10.5세였다. 치료전시행한상부위장관내시경에서나타난병변의종류는위궤양 9명 (17.6%), 십이지장궤양 3명 (5.9%), 위선종및조기위암에 과 MIC value (μg/ml) Amoxicillin 1.0, 0.5 or 0.25 Clarithromycin >1.0 Metronidazole >8 or 4 Tetracycline >4 or 2 Azithromycin >2.0 or 1.0 Ciprofloxacin >1.0 Levofloxacin >1.0 Moxifloxacin >1.0 MIC, minimal inhibitory concentration.
84 Korean J Helicobacter Up Gastrointest Res: 제 12 권제 2 호 2012 Table 2. Clinical Characteristics of the Patients Patients (n=51) Sex Male 34 (66.7) Female 17 (33.3) Age (yr) 56.8±13.8 Male 54.2±14.6 Female 62.0±10.5 Endoscopic diagnosis Gastric ulcer 9 (17.6) Duodenal ulcer 3 (5.9) EMR or ESD 35 (68.6) MALT lymphoma 2 (3.9) Gastritis 2 (3.9) Values are presented as n (%) or mean±sd. EMR, endoscopic mucosal resection; ESD, endoscopic submucosal dissection. Fig. 2. Minimal inhibitory concentration (MIC) distribution of clarithromycin for Helicobacter pylori (H. pylori). Eight cases were resistant (MIC>1.0μg/mL) to clarithromycin. Fig. 1. Minimal inhibitory concentration (MIC) distribution of amoxicillin for Helicobacter pylori (H. pylori). One case (2%) was resistant (MIC>0.5μg/mL) to amoxicillin. Fig. 3. Minimal inhibitory concentration (MIC) distribution of tetracycline for Helicobacter pylori (H. pylori). 0% were resistant (MIC>4μg/mL) to tetracycline. 대하여점막절제술이나점막하박리술을시행한경우가 35 명 (68.6%), mucosa-associated lymphoid tissue (MALT) 림프종과위축성위염이각각 2명 (3.9%) 이었다 (Table 2). 2. 삼제요법후제균율 H. pylori 균주를분리하여검사를시행했던총 51명의환자중실제 H. pylori 제균치료를받은환자는 31명이었고, 이중제균치료를완료한후제균성공여부를확인한환자는 22명이었다. 또한이중제균실패로판정받은환자는 4 명으로 18% 에해당되었다. 3. H. pylori 균주의항생제에대한 MIC 및내성률 Amoxicillin에대한 MIC 분포를관찰한결과 0.5μg/mL 이상을내성기준으로보았을때 2% 에서내성이있었고, clarithromycin에대한 MIC 분포는분리균주의 84% 정도가 0.0625μg/mL 이하였지만, 높은 MIC를보이는균주가있어 15.7% 가내성균에속했다. Tetracyline의경우모든균주에서 0.125μg/mL 이하의 MIC를보여내성균은없었다. Metronidazole에대한 MIC는매우다양한값에분포하고있었다. Metronidazole의내성 cut-off value는 8μg/mL로하였으며, 이기준에따른내성률은 21.6% 로나타났다. Quinolone 항생제는 ciprofloxacin과 levofloxacin를대상으로하였
김민형외 : 광주전남지역에서 Helicobacter pylori 제균항생제내성률분석 85 Table 3. Prevalence of Antibiotic Resistance among Helicobacter pylori isolates a Resistant strains/ total strains Resistance rate (%) Amoxicillin 1/51 1.9 Clarithromycin 8/51 15.7 Tetracycline 0/51 0 Metronidazole 11/51 21.6 Ciprofloxacin 13/51 25.5 Levofloxacin 13/51 25.5 Fig. 4. Minimal inhibitory concentration (MIC) distribution of metronidazole for Helicobacter pylori (H. pylori). Eleven cases (21.6%) were resistant (MIC>8μg/mL) to metronidazole. Fig. 5. Minimal inhibitory concentration (MIC) distribution of ciprofloxacin and levofloxacin for Helicobacter pylori (H. pylori). Thirteen cases (25.5%) were resistant (MIC>1.0 μg/ml) to ciprofloxacin and levofloxacin. 으며, 두항생제사이에 MIC는거의차이가없었고내성률은모두 25.5% 였다 (Fig. 1 5, Table 3). 한편우리나라에서 H. pylori 제균치료의일차약제로사용되고있는 amoxicillin과 clarithromycin 모두에대해내성을나타낸균주는 1.9% 였다 (Table 4). 4. 항생제내성이 H. pylori 제균에미치는영향 제균에실패한 4명모두항생제감수성검사에서는 amoxicillin과 clarithromycin 모두에내성기준이하의 MIC를보이는균주였다. Clarithromycin에내성기준이상의 MIC를보여내성균주로판단되었던 8균주 (15.7%) 중실제제균 a Resistant cut-off values was defined as 0.5μg/mL for amoxicillin, >1.0μg/mL for clarithromycin, 4μg/mL for tetracycline, 8μg/mL for metronidazole, and 1.0μg/mL for ciprofloxacin and levofloxacin. Table 4. The multi-drug Resistance in Helicobacter pylori Strains Resistant antibiotics a (n) Type of multi-drug resistant Strains, n (%) 0 27 (52.9) 1 CH 3 (5.9) MT 5 (9.8) Q 10 (19.6) 2 CH+MT 2 (3.9) MT+Q 1 (1.9) 3 AM+CH+MT 1 (1.9) CH+MT+Q 2 (3.9) a Resistant cut-off values was defined as 0.5μg/mL for amoxicillin (AM), >1.0μg/mL for clarithromycin (CH), 8μg/mL for metronidazole (MT), and 1.0μg/mL for ciprofloxacin and levofloxacin (Q). Table 5. The Effects of Resistance of Amoxicillin and Clarithromycin on the Eradication Rate of H. pylori after PPI Triple Therapy Amoxicillin Clarithromycin Patients (n) Eradication rate of H. pylori, n (%) Success Failure Susceptible Susceptible 19 15/19 (78.9) 4/19 (21.0) Susceptible Resistant 2 2 (100) 0 (0) Resistant Susceptible 0 - - Resistant Resistant 1 1 (100) 0 (0) H. pylori, Helicobacter pylori; PPI, proton pump inhibitor. 치료시행후제균여부를확인한것은 3명이었으나, 이들은실제치료후모두제균성공으로확인되었으며, 이들중한균주는 clarithromycin 뿐만아니라 amoxicillin에도내성기준이상의 MIC를보였으나성공적으로제균치료가
86 Korean J Helicobacter Up Gastrointest Res: 제 12 권제 2 호 2012 이루어졌다 (Table 5). 고찰 H. pylori 감염은균주의다양성과숙주감수성에따라소화성궤양, 만성활동성위염, 위 MALT 림프종및위암등다양한상부위장관질환을유발하는것으로알려져있어그제균치료가널리시행되고있으나, 최근항생제내성이증가하면서그제균율이점차감소하고있다. 국내에서 amoxicillin에대한 H. pylori의내성률은다양하게보고되었는데, 2000년이전의한연구에서는 agar dilution법으로측정했을때 amoxicillin에대한 H. pylori의내성균은전혀없다고보고한바있으나, 16 2003년에는 18.5% 로높아진내성률을보고하였다. 15 본연구에서는 MIC 0.5 μg/ml 이하를내성기준으로보았을때 2% 에서내성을보였다. 현재우리나라에서 amoxicillin이 H. pylori 제균치료의일차약제로사용되고있으므로향후보다많은연구가필요하리라생각된다. Clarithromycin의경우 1999년에 E-test에의한국내내성률이 7.1%, 10.2% 로보고된바있고, 19,20 2004년에 agar dilution법에의한경우는 13.8% 로보고되었다. 15 본연구에서는 15.7% 로이전까지의연구들보다는약간높은내성률을보여계속적인내성률증가를시사하는것으로생각되며, 추후지속적인항생제내성률관찰이필요하겠다. Metronidazole에대한 H. pylori의국내내성률보고를보면 35 66% 정도로다양하기는해도모두비교적높은내성률을보이고있으나 15,19-21 본연구에서는 21.6% 로이전의연구들보다는낮은내성률을보였다. Tetracyline의경우에도 agar dilution법에의한국내내성률이 12.3% 로보고되었지만 15 본연구에서는내성균주를보이지않았다. 최근까지보고된항생제내성에대한연구들을보면국내에서삼제요법으로주로사용되고있는 metronidazole과 clarithromycin, amoxicillin에대한내성이전반적으로증가하는추세임을관찰할수있어, 11 H. pylori에대한감수성검사를시행하여제균율이높은약제의조합사용을권하기도한다. 10 그러나본연구에서실제제균치료에실패한환자들의균주를확인해보았을때모두항생제감수성검사에서는 amoxicillin과 clarithromycin 에내성기준이하의 MIC를보였으며, clarithromycin에내성을보일것으로생각되었던 8균주중제균치료후제균여부를확인했던 3명의환자모두에서성공적으로제균치료가되었다. 뿐만아니라이 3균주중하나는 clarithromycin는물론 amoxicillin에도내성기준이상의 MIC를보였으나제균성공을보인것이었다. 항생제내성률의증가와 H. pylori 제균율감소의연관성에대한연구들을보면내성률에따라제균율이차이가있다는보고도있으나, 22 일본 23,24 과국내 25 의일부연구에서는항생제내성률증가에따른제균율의변화가없다는보고도있어이에대한결론을도출하기에는아직논란의여지가있다. 이번연구는연구에포함된환자의수가적다는점과더불어, 의무기록지열람을통한후향적방법으로이루어졌기때문에과거제균치료여부나항생제사용의병력, 약물순응도에대한정확한조사가어려워이들요인과 H. pylori 제균여부에대한검사가통일되지않았으므로제균율에미치는영향을배제할수없다는한계점이있겠다. H. pylori 제균치료를시행함에있어서, 치료전균주에대한항생제의 MIC를조사, 평가하는것만으로제균의성공여부를정확히예측하기는힘들것으로생각되며, 보다많은환자를대상으로한전향적연구를통해제균여부에영향을미칠수있는다양한요인들에대한종합적분석이필요할것으로보인다. 참고문헌 1. Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med 2002;347:1175-1186. 2. Kim JG. Treatment of Helicobacter pylori infection. Korean J Gastroenterol 2005;46:172-180. 3. Huang JQ, Hunt RH. Treatment after failure: the problem of "non-responders". Gut 1999;45(Suppl 1):I40-I44. 4. Laheij RJ, Rossum LG, Jansen JB, Straatman H, Verbeek AL. Evaluation of treatment regimens to cure Helicobacter pylori infection--a meta-analysis. Aliment Pharmacol Ther 1999;13:857-864. 5. Perri F, Qasim A, Marras L, O'Morain C. Treatment of Helicobacter pylori infection. Helicobacter 2003;8(Suppl 1):53-60. 6. Kim BW, Choi MG, Moon SB, et al. Pooled analysis of antibiotic therapy for Helicobacter pylori eradication in Korea. Korean J Gastroenterol 1999;34:42-49. 7. Lee DS, Kim SW, Byun BH, et al. The therapeutic effect of triple therapy in Helicobacter pylori infection. Korean J Gastroenterol 1998;32:32-37. 8. Kim JH, Lee KT, Lee SM, et al. Efficacy of ten days of clarithromycin, amoxicillin and omeprazole in eradicating Helicobacter pylori infection. Korean J Med 1999;56:581-589. 9. Song HJ, Yang YS, Lee IS, et al. Efficacy and tolerability of pantoprazole-based triple therapy in eradication of Helicobacter pylori in patients with peptic ulcer and/or gastritis. Korean J Gastroenterol 2000;36:185-191. 10. Chung WC, Cho YS, Jeong JJ, et al. Eradication rate of Helicobacter pylori according to the diseases and therapeutic regimens, and reinfection rate after successful Eradication in a tertiary clinic. Korean J Gastroenterol 2003;41:1-8.
김민형외 : 광주전남지역에서 Helicobacter pylori 제균항생제내성률분석 87 11. Choi YS, Cheon JH, Lee JY, et al. The trend of eradication rates of first-line triple therapy for Helicobacter pylori infection: single center experience for recent eight years. Korean J Gastroenterol 2006;48:156-161. 12. Na HS, Hong SJ, Yoon HJ, et al. Eradication rate of first-line and second-line therapy for Helicobacter pylori infection, and reinfection rate after successful eradication. Korean J Gastroenterol 2007;50:170-175. 13. Cutler AF, Schubert TT. Patient factors affecting Helicobacter pylori eradication with triple therapy. Am J Gastroenterol 1993;88: 505-509. 14. Graham DY, Lew GM, Malaty HM, et al. Factors influencing the eradication of Helicobacter pylori with triple therapy. Gastroenterology 1992;102:493-496. 15. Kim JM, Kim JS, Jung HC, Kim N, Song IS. Antibiotic resistance of Helicobacter pylori isolated from Korean patients in 2003. Korean J Gastroenterol 2004;44:126-135. 16. Kim JJ, Reddy R, Lee M, et al. Analysis of metronidazole, clarithromycin and tetracycline resistance of Helicobacter pylori isolates from Korea. J Antimicrob Chemother 2001;47:459-461. 17. Eun CS, Han DS, Park JY, et al. Changing pattern of antimicrobial resistance of Helicobacter pylori in Korean patients with peptic ulcer diseases. J Gastroenterol 2003;38:436-441. 18. Kim JM. Antibiotic resistance of Helicobacter pylori isolated from Korean patients. Korean J Gastroenterol 2006;47:337-349. 19. Yoo HM, Lee YC, Park HJ, Kim WH, Lee KW, Park IS. Clinical characteristicsof patients with failed eradication of Helicobacter pylori and antibiotic resistance. Korean J Gastroenterol 1999;33: 311-320. 20. Kim N, Lim CN, Lim SH, et al. Establishment of an Helicobacter pylori-eradication regimen in consideration of drug resistance, recrudescence and reinfection rate of H. pylori. Korean J Med 1999;56:279-291. 21. Han DS, Cho YJ, Son JH, Park KN, Kang JO. Antimicrobial resistances of Helicobacter pylori in Korean patients with duodenal ulcer. Korean J Gastroenterol 1997;29:727-733. 22. Chi CH, Lin CY, Sheu BS, Yang HB, Huang AH, Wu JJ. Quadruple therapy containing amoxicillin and tetracycline is an effective regimen to rescue failed triple therapy by overcoming the antimicrobial resistance of Helicobacter pylori. Aliment Pharmacol Ther 2003;18:347-353. 23. Murakami K, Sato R, Okimoto T, et al. Efficacy of triple therapy comprising rabeprazole, amoxicillin and metronidazole for second-line Helicobacter pylori eradication in Japan, and the influence of metronidazole resistance. Aliment Pharmacol Ther 2003; 17:119-123. 24. Kamada T, Haruma K, Hata J, et al. The long-term effect of Helicobacter pylori eradication therapy on symptoms in dyspeptic patients with fundic atrophic gastritis. Aliment Pharmacol Ther 2003;18:245-252. 25. Lee JH, Cheon JH, Park MJ, et al. The trend of eradication rates of second-line quadruple therapy containing metronidazole for Helicobacter pylori infection: an analysis of recent eight years. Korean J Gastroenterol 2005;46:94-98.