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International Journal of Molecular and Clinical Microbiology 2 (2012) 148-152
Resistance Rates to Various Antimicrobial Agents of Helicobacter
pylori Isolates in Eastern Turkey
Gokben Ozbey1*, Ibrahim Halil Bahcecioglu2, Mehmet Nuri Acik3

1Vocational School of Health Services, Firat University, 23119, Elazig, Turkey
2Department of Gastroenterology, Faculty of Medicine, Firat University, 23119, Elazig, Turkey
3Vocational School of Health Services, Bingol University, 12000, Bingol, Turkey The aim of this study was to determine the resistance rates of Helicobacter pylori (H. pylori) to various antimicrobial agents. The agar disk diffusion method (Kirby Bauer) was used to determine the sensitivity of H. pylori isolates to various antimicrobials. Of the 61 H. pylori isolates tested, no isolates was resistant to amoxycillin and tetracycline. The resistance rates were 42.6% for metronidazole, 21.3% for clarithromycin, and 3.3% for levofloxacin. Compared to clarithromycin and metronidazole, levofloxacin showed the lowest resistance. This is the first report on the resistance rates of H. pylori to antibiotics in Elazig Province, East of Turkey. This study suggests that the large scale studies is needed to help us to understand better the effect of resistance on the H. pylori eradication. 1. Introduction
Megraud, 1997). In recent years, resistance to amoxycillin, clarithromycin and metronidazole Helicobacter pylori (H. pylori) is associated have been on the increase in a number of with a variety of disorders including chronic gastritis, peptic ulcer, gastric adenocarcinoma, inhibition of H. pylori growth in vitro lymphoma (Amieva and El-2PDU  &RUUHD (Bauernfeind, 1997), and levofloxacin-based and Houghton, 2007). Therefore, eradication of triple therapy is effective as first-line anti- H. pylori colonization is important to prevent H. pylori therapy (Nista et al  Rispo et al., peptic ulceration and gastric carcinogenesis resistance in H. pylori has been found to be antibiotics used for the treatment of H. pylori linked to mutations in the quinolone resistance- infections are amoxycillin, tetracycline, determining regions (QRDRs) of gyrA (Gerrits metronidazole, and clarithromycin (Unge, 1998). The prevalence of H. pylori infection has compliance, and resistance to antibiotics are become increasingly widespread in Turkey. causes of treatment failure (%URXWHW HW DO  Therefore, the eradication of this bacterium has *Corresponding author Dr. Gokben Ozbey Tel: +90 424 2370079 Fax: +90 424 2415544 E-mail: [email protected] G. Ozbey et al.,/ International Journal of Molecular and Clinical Microbiology 2 (2012) 148-152 become a major public health concern worldwide. solution and adjusted to the density equal to 3.0 However, the resistance rate of H. pylori to McFarland standard (1x109 cfu/ml) (Xia et al, antibiotics in Elazig Province, East of Turkey has 1994). The suspension was spread on Mueller- not yet been reported. The aim of this study was to Hinton blood agar plates (Oxoid). The antibiotic investigate the resistance rates of H. pylori to clarithromycin (15 μg), tetracycline (30 μg), amoxycillin (10 μg), levofloxacin (5 μg) were 2. Material and Methods
aseptically placed onto the agar. Inhibition zone diameters were measured in milimeters after 3 days 2.1. Patients and H. pylori isolates of incubation at 37°C under microaerophilic Sixty one H. pylori isolates used in this study conditions and determined as resistant (R) or were isolated from the antrum of Turkish patients susceptible (S) (Boyanova et al, 2000). A zone size (29 males and 32 females, age range 20 to 80 years, <16 mm was evaluated as resistant for average 47) with gastrointestinal complaints PHWURQLGD]ROH %R\DQRYD HW DO  0LVKUD HW DO admitted to Gastroenterology Department of Firat 2006), ”25 mm for amoxicillin resistance (Lang and University Hospital, during 2009 and 2010. Patients Garcia, 2004) and ”30 mm for clarithromycin and collected endoscopically in two groups as gastritis WHWUDF\FOLQH UHVLVWDQFH %R\DQRYD HW DO  in 51 cases and peptic ulcer in 10 cases, Kulsuntiwong et al., 2008). Reference H. pylori consecutively enrolled and selected at random. strains (clinical strains) were used as positive This study was approved by the Medical Ethics control (kindly provided by Dr. Vildan CANER). Committee of Firat University and informed consent was provided from all patients prior to 3. Results
3.1. Isolation and identification results of H. pylori A total of 61 H. pylori isolates was isolated 2.2. Isolation and identification of H. pylori from 51 patients with gastritis and 10 patients immediately streaked onto Columbia agar base (Oxoid, Basingstoke, UK) containing 7% laked 3.2. Antimicrobial susceptibility testing results horse blood (SR0048C, Oxoid, Basingstoke, UK) The results of the susceptibility testing for and H. pylori supplement SR0147E (trimethoprim 5 H.pylori isolates are shown in Table 1. Sixty one mg/l, vancomycin 10 mg/I, amphotericin B 5 mg/I isolates tested against the different antibiotics. The and cefsulodin 5 mg/I) (Oxoid). Plates were highest resistance rate was for metronidazole 26 incubated at 37ºC for 4 to 7 days in microaerophilic (42.6%), followed by 13 (21.3%) to clarithromycin, conditions obtained by a gas generating kit and 2 (3.3%) to levofloxacin. No resistance to amycillin and tetracycline in our study was detected. (Chomvarin et al., 2006). Identification of H. pylori was evaluated by observation of the colony Table 1. Resistance rates to antimicrobial agents of H. pylori
morphology, gram-staining and biochemical methods (oxidase, and catalase activities) (Goodwin Antibiotics
Number of
Resistant isolates %
Reference H. pylori strains (clinical strains) (provided by Dr. Vildan CANER, Pamukkale University, Faculty of Medicine, Department of Medical Biology, Denizli-Turkey) were used as a 4. Discussion
2.3. Antimicrobial susceptibility testing Antimicrobial susceptibility testing was H. pylori is increasingly resistant to antibiotics, performed by using disk diffusion method. H. pylori especially metronidazole and clarithromycin colonies were suspended in 1.0 ml sterile saline 0HJUDXG  'H )UDQFHVFR HW DO  G. Ozbey et al.,/ International Journal of Molecular and Clinical Microbiology 2 (2012) 148-152 Determining the antibiotic resistance among Silva et al., 200). The prevalence of H. pylori H. pylori isolates would improve the eradication of resistance to tetracycline is low (<3%) in all countries, except in Africa (43.9%) (De Francesco Numerous studies have performed to investigate et al., 2010). Tetracycline resistance in Turkey is the prevalence of H. pylori resistance to antibiotics approximately 0-4% (KantarcHNHQ HW DO  $JHO (Megraud and Lehours, 2007). Resistance to HW DO  %DNLU 2]EH\ HW DO  metronidazole has been observed worldwide Amoxycillin resistance has been associated with (Eltahawy, 2002) and showed commonly in several a mutation in the pbp-1A gene and altered uptake of countries, ranging from 10 to 90% with the lowest beta-lactams after long exposure of H. pylori to resistance rates in Europe and Australia, the highest amoxicillin (DeLoney and Schiller, 2000). Japanese rates in Africa whereas in other developing authors have reported an increase in H. pylori countries, the rate of resistance to metronidazole resistance rates to amoxicillin from 2000 to 2003 ranges from 80 to 90% (Quintana-Guzman et al., (Watanabe et al., 2005). The results of the present 1998). Therefore, metronidazole should be preferred study are similar to a previous study (Yetgin, 2006) to amoxicillin in first-line therapy in Europe in Turkey where no resistance to amoxycillin and (Malfertheiner et al., 2007) but not in Asian patients (De Francesco et al., 2010). The resistant rate (42.6%) to metronidazole in this study is consistent The prevalence of levofloxacin resistance varies with a previous report (41.9%) in Turkey (Bakir ranging from 14.3% in Japan to 16.8% in Belgium, Ozbey et al., 2009) but lower than previous reports 17% in Brazil, 18% in Hong Kong, 21.5% in Korea, (49.2% and 53%, respectively) performed by 22.1% in Germany, %19.1 in Italy and 25.5% in Kantarceken et al. (2000) and Agel et al. (2000). Turkey %RJDHUWV HW DO  Coelho HW DO  This may be explained by the wide use of *ORFNHU HW DO  .LP HW DO  :DWDQDEH HW metronidazole in treatment of gynecologic infection DO  :RQJ HW DO  Zullo et al., 2007). The and intestinal parasitic infections, which occur in resistance towards levofloxacin is rapidly increasing worldwide and a cross-resistance between Resistance rates to clarithromycin has been clarithromycin and levofloxacin resistance have reported as 10-15% in USA, 10% in France, 16% in been also reported (Zullo et al., 2007). This could be a cause for concern in using levofloxacin in those Meyer et al., 200 )UDVHU HW DO  6WUHHW HW DO areas where primary clarithromycin resistance is  %URXWHW HW DO  6LPVHN HW DO  Resistance rates to clarithromycin (21.3%) was This is the first report on the resistance rates of found in this study. The major cause for H. pylori to antibiotics in Elazig Province, East of clarithromycin resistance is a previous use of Turkey. In our study, metronidazole resistance was macrolides, and therefore, an increased prevalence found to be high. We found that levofloxacin is showed in most countries (Megraud and Lehours, showed the lowest resistance compared to clarithromycin and metronidazole. Levofloxacin Tetracycline is an antibiotic which is commonly may be used in eradicating H. pylori. However, cost used in first- and second-line regimens for the of levofloxacin should be taken into account when treatment of H. pylori (Megraud and Marshall, dealing with widespread infection. Further research  Gisbert and Pajares, 2001) and widely and an alternative therapy method is also needed to available and cheap, had advantage that resistance improve the H. pylori eradication rate. against it was rare in H. pylori (Ribeiro et al., 2004). However, in the past few years, the incidence of Acknowledgments
tetracycline resistance has increased, especially in The material used in this study obtained from the countries where tetracycline can be obtained project funded by the Firat University Scientific without prescription (5HDOGL HW DO  Wu et al., Research Projects Unit (FUBAP 1609). We thank to 2000). This increase is a serious concern because it management of Elazig Veterinary Control and negatively affects the efficacy of tetracycline- Research Institute for their invaluable help during containing regimens (*LVEHUW DQG 3DMDUHV  our study. The authors gratefully acknowledge Dr. Vildan CANER, Department of Medical Biology, G. Ozbey et al.,/ International Journal of Molecular and Clinical Microbiology 2 (2012) 148-152 Faculty of Medicine, Pamukkale University, use and levofloxacin resistance in Helicobacter pylori Denizli, Turkey for supplying some clinical strains Chomvarin, C., Kulsantiwong, P., Chantarasuk, Y., of H. pylori and Dr. Yakut AKYON YILMAZ, Chantrakooptungool, S., Kanjanahareutai, S., 2006. Department of Medical Microbiology, Faculty of Comparison of media and antibiotic supplements for Medicine, Hacettepe University, Ankara, Turkey for isolation of H. pylori from gastric biopsies. Southeast. her technicial support and help and Dr. Gireesh Asian J. Trop. Med. Public Health. 37, 1163-1169. Rajashekara (Ohio Agricultural Research and Coelho, L.G., Moretzsohn, L.D., Vieira, W.L., Gallo, M.A., Passos, M.C., Cindr, J.M., Cerqueira, M.C., Vitiello, L., Ribeiro, M.L., Mendonca, S., Pedrazzoli-Júnior, J., Castro, Research Program, The Ohio State University, L.P., 2005. New once-daily, highly effective rescue triple Wooster, OH, USA) for his valuable advice. therapy after multiple H. pylori treatment failures: a pilot study. Aliment. Pharmacol. Ther. 21, 783-787. References
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