Antibiotic Susceptibility of Helicobacter pylori Clinical Isolates: Comparative Evaluation of Disk-Diffusion and E-Test Methods

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1 CURRENT ROBIOLOGY Vol. 53 (2006), pp DOI: /s Current Microbiology An International Journal ª Springer Science+Business Media, Inc Antibiotic Susceptibility of Helicobacter pylori Clinical Isolates: Comparative Evaluation of Disk-Diffusion and E-Test Methods K. K. Mishra, S. Srivastava, A. Garg, A. Ayyagari Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, , Lucknow, India Received: 21 March 2006 / Accepted: 13 June 2006 Abstract. Antimicrobial susceptibility of 25 Helicobacter pylori strains isolated from patients with acid peptic diseases were tested for in vitro sensitivity to commonly used antibiotics using disk-diffusion and E-test, methods. All strains tested were susceptible to tetracycline by E-test, with the minimum inhibitory concentration () values being <0.125 lg/ml for all strains except for 6 (<0.023 lg/ml). However 1 strain was resistant by disk-diffusion method. One strain was resistant to clarithromycin both by disk diffusion and E-test ( <48 lg/ml), and 1 strain was resistant only by disk diffusion. Only one strain was resistant to amoxicillin by disk diffusion and E-test ( >256 lg/ml). For ciprofloxacin, three strains were resistant by disk diffusion and two by E-test ( <32 lg/ml). Sixteen strains were resistant to metronidazole by disk diffusion and E-test ( 8 lg/ml), and 1 was resistant only by E-test ( <48 lg/ml). Overall, 64% of the strains were resistant to metronidazole. The for metronidazole was also tested by agar-dilution method, and metronidazole resistant strains had an, >8 lg/ml. The disk-diffusion method showed excellent correlation with E-test results; there was 100% agreement for amoxicillin a other antibiotics showed 90% to 95% accuracy. Disk diffusion is cheaper than E-test (approximately 2.6 cents vs. US$2.60), is easy to perform, and is a reliable method for testing H. pylori susceptibility to antimicrobial agents in the clinical microbiology laboratory. Correspondence to: A. Ayyagari; a_ayyagari@yahoo.com Helicobacter pylori eradication treatment is indicated in all patients with active or recurrent peptic ulceration [1]. Eradication treatment usually consists of a proton pump inhibitor or an H 2 -receptor antagonist (e.g., ranitidine) in combination with two antibiotics [2]. The rationale behind the combination of two antibiotics is the increased success of the eradication therapy and decreased rates at which secondary antibiotic resistances develop. The prevalence of clarithromycin, metronidazole, and amoxicillin resistances varies between countries and is highest for metronidazole [3 8]. Resistance to tetracycline and ciprofloxacin has been reported but appears uncommon [9 13]. Treatment for H. pylori infection is often started on an empirical basis, and if the infecting strain is resistant, its successful eradication is compromised. Therefore, it is important to know about the antibiotic resistance pattern of local H. pylori strains. Currently there are no standard testing recommendations for this fastidious organism, and interpretative criteria for susceptibility or resistance have yet to be standardized. There are several problems with antimicrobial susceptibility testing of H. pylori [14 16]. Standardization of a test procedure that is simple and fast and can be applied in routine laboratories to allow a faster identification of resistant strains and thus selection of more effective therapies is now the most pressing need [17 20]. Agar or broth dilution methods have been used, but they are difficult to perform routinely [18]. Disk-diffusion testing because it is simple, easy to perform, economical is often used [21]. The E-test has proven to be an accurate method to be test susceptibilities of fastidious organisms, including H. pylori, to antibiotics. The E-test has a more stable pattern of

2 330 CURRENT ROBIOLOGY Vol. 53 (2006) antibiotic release and has been found to tolerate a prolonged incubation [22]. This is the main reason why the E-test, and not the disk diffusion method, has been recommended for H. pylori. However, some concern, exists regarding the value of E-test results for determining the effect of different antibiotics on this bacterium. Moreover the E-test is economically impractical (approximately US$2.60/strip) for clinical laboratory use when testing individual isolates. The purpose of this study was to assess the reliability of results obtained by using the disk-diffusion method for determining the qualitative susceptibility of H. pylori. To accomplish this, we compared the results obtained by the disk-diffusion method with those obtained by the E-test and agar dilution method. Materials and methods Bacterial strains. Twenty-five H. pylori strains (gastritis n = 12, peptic ulcer, n = 8, carcinoma stomach n = 3, and portal hypertension [PHT] n = 2) were isolated from the gastric biopsy specimens of 25 patients attending the endoscopy units in the Gastroenterology Department, Sanjay Gandhi Postgraduate Institute of Medical Sciences, (Lucknow, India) for upper gastrointestinal complaints. None of the patients had received any antimicrobial therapy for at least 4 weeks before the study. H. pylori CCUG (provided by B. Kaijser, University of Goteborg, Sweden) was used as control strain. Culture of H. pylori from gastric biopsy specimens. For bacterial culture, gastric biopsy samples were homogenized and cultured on Brucella chocolate agar (Difco) containing antibiotic supplement (vancomycin 6 mg/l, polymyxin-b 2500 IU/L, and amphotericin-b 2 mg/l) as described earlier with 7% sheep blood [23 25]. The plates were incubated at 37 C in microaerophilic condition and were observed after 72 hours. Organisms were identified as H. pylori based on colony morphology modified Gram staining, and positive oxidase, catalase, and rapid urease tests [24, 25]. Histologic sections of formalin-fixed biopsy specimens were stained with haematoxylin and eosin as well as Giemsa to verify the presence of H. pylori. Antibiotic susceptibility test. Antimicrobial sensitivity of H. pylori isolates and the reference strain (CCUG 17874) were detected by conventional agar disk-diffusion procedure [l8, 23, 26]. The bacterial suspension [28] (McFarland tube no. 3) of H. pylori was plated on Brucella chocolate agar plates. The disks (6-mm diameter, Hi Media, India) of different antibiotics (amoxicillin [10] ], tetracycline [30 ], clarithromycin [15 ], ciprofloxacin [5 ] and metronidazole [10 ]) were placed on the plates and incubated at 37 C in a microaerophilic chamber for 72 hours and examined for the diameter of the inhibition zone, which was measured in millimeters, with the measuring caliper and noted. Based on the National Committee for Clinical laboratory Studies (NCCLS) guidelines for the fastidious organism H. influenza [27] zone size 18 mm was considered resistant to amoxicillin (an analog of ampicillin); 30 mm was considered resitant to ciprofloxacin clarithromycin, and tetracycline. For metronidazole 16-mm zone size was considered to be resistant. For the purpose of analysis the nonsusceptible categories of intermediate and resistant were combined into a single resistant category. The minimum inhibitory concentration for different antibiotics was determined by E-test (Epsilometer test; AB Biodisk Solna, Sweden) and agar-dilution methods. Determination of by E-test. Plates containing Brucella chocolate agar with 7% sheep blood were used for test. Antimicrobial agents (amoxicillin, metronidazole, tetracycline, and clarithromycin) were tested at concentrations ranging from to 256 lg/ml; however, ciprofloxacin was tested at concentration ranges, to 32 lg/ml. The 140-mm diameter agar plates were inoculated by confluent swabbing of the surface with the adjusted inoculum suspensions (McFarland tube no. 3). Five E-test strips were aseptically placed onto the dried surfaces of each inoculated agar plate. The plates were incubated at 37 C under microaerophilic conditions and 100% relative humidity. The s were read after 72 hours of incubation on the basis of the intersection of the elliptical zone of growth inhibition with the scale on the E-test strip per manufacturerõs instructions [46]. interpretive criteria. There is no standardized breakpoint for H. pylori except for clarithromycin. The cut-off point for different antibiotics was considered on the basis of NCCLS guidelines and previously published studies [27, 43, 49]. The isolates were considered resistant when the value was greater than the susceptible breakpoint of 8 lg/ml for metronidazole [28], >2 lg/ml amoxicillin and >2 lg/ml for clarithromycin [27]. Since the resistant breakpoints for tetracycline and ciprofloxacin are not established for H. pylori, we defined resistance as s of >2 lg/ml for tetracycline and >2 lg/ml for ciprofloxacin [9, 44 45, 48]. Determination of by agar dilution methods. In addition, to metronidazole was also tested by agar dilution method [28]. Briefly, the Brucella chocolate agar 90-mm plates were prepared with different concentration of metronidazole [2, 4, 8, l6, and 32 lg/ml]. The plates were inoculated with 1 to 2 ll suspension of each H. pylori strains (McFarland No. 3) [28]. A control plate without metronidazole was inoculated in each series. H. pylori CCUG was used as control. All plates were incubated at 37 C for 72 hours under microaerophilic conditions and 100% relative humidity. After incubation, the plates were examined visually, and the lowest concentration of antibiotic showing complete inhibition of bacterial growth was recorded as the for that antibiotic. Results Antibiotic susceptibility test by disk diffusion. Antimicrobial resistance of 25 H. pylori isolates to different antibiotics as determined by disk-diffusion is listed in Table 1. The maximum resistance of H. pylori isolates was for metronidazole with 16 isolates (64%) whereas 9 isolates (36%) were sensitive by diskdiffusion method. The frequency of resistance to other antibiotics such as amoxicillin, tetracycline, clarithromycin, and ciprofloxacin was 1 (4%), 1 (4%), 2 (8%), and 3 (12%) isolates, respectively. Of the 25 isolates tested, 9 of 12 isolates from gastritis, 3 of 8 from duodenal ulcer, 1 of 2 primary hyperthyroidism from and all isolates from stomach carcinoma patients showed resistance to metronidazole, Multiple resistance was observed in 3 of 25 (12%) isolates. One was resistant to

3 K. K. Mishra et al.: Antimicrobial susceptibility testing 331 Table 1. Comparison of antimicrobial susceptibility test results by disk-diffusion method and E-test for 25 isolates of H. pylori E-test Disk-diffusion method n (%) of isolates n (%) of isolates Antimicrobial agents Range S R S R Amoxicillin (96) 1 (4) 24 (96) 1(4) Tetracycline (100) 0 (0) 24 (96) 1 (4) Clarithromycin (96) 1 (4) 23 (92) 2 (8) Ciprofloxacin (92) 2 (8) 22 (88) 4 (12) Metronidazole < (32) 17 (68) 9 (36) 16 (64) S, sensitive; R, resistant. Table 2. Resistance profile of 25 H. pylori isolates by disk diffusion and E-test methods Lab no. Diag Amoxicillin Metronidazole Tetracyclin Clarithromycin Ciprofloxacin Gastritis R 256 R >256 R <0.032 R <0.75 R > DU S <0.016 S <0.016 S <0.125 S <0.016 S < DU S >0.047 S <0.016 S <0.125 S <0.023 S < DU S <0.016 S <0.016 S <0.125 S <0.016 S < PHT S <0.016 S >256 S <0.125 S <0.016 S < Ca S <0.016 R >256 S <0.032 S <0.016 R < Ca S <0.032 R >256 S <0.125 S <0.016 S < DU S <0.016 R <48 S <0.125 S <0.50 S > PHT S <0.016 R 256 S <0.032 R <48 R > Gastritis S <0.016 R >256 S <0.032 S <0.016 S < Gastritis S <0.016 R 256 S <0.032 S <0.016 S < Gastritis S <0.016 R 256 S <0.125 S <0.016 S < DU S <0.016 R >256 S <0.032 S <0.016 S < Gastritis S <0.016 R <94 S <0.125 S <0.016 S < Gastritis S <0.016 S <1 S <0.125 S <0.016 S < Gastritis S <0.016 S <1 s <0.125 S <0.016 S < Gastritis S <0.016 R >256 S <0.125 S <0.016 S < Gastritis S <0.016 R >256 S <0.125 S <0.016 S < DU S <0.016 R >256 S <0.125 S <0.016 S < Gastritis S <0.016 R >256 S <0.125 S <0.016 S < Gastritis S <0.016 R >256 S <0.125 S <0.016 S < Ca S <0.016 R >256 S <0.125 S <0.016 S < Gastritis S <0.016 S <1 S <0.125 S <0.016 S < DU S S <1 S <0.125 S <0.016 S < DU S <0.016 S <1 S <0.125 S <0.016 S <0.125 DU, duodenal ulcer; Ca, carcinoma stomach; PHT, portal hypertension; S - sensitive; R - resistant. metronidazole and ciprofloxacin, a second was resistant to metronidazole, ciprofloxacin and clarithromycin, and the third was resistant to all the five drugs tested (Table 2). for H. pylori by E-test. The range of for amoxicillin was to 256, for tetracycline was to 0.125, for clarithromycin was to 48, for ciprofloxacin was to 32, and for metronidazole was to 256. Because more than 90% of the strains were sensitive at lower concentration, the 50 s and 90 s for amoxicillin, tetracycline, clarithromycin, and ciprofloxacin were <0.016, <0.125, <0.023, and <0.125 respectively. However, for metronidazole 50 and 90 were <192 and <256 lg/ml, respectively. The details of the for each isolate are listed in Table 2. All strains tested were susceptible to tetracycline by E-test; the values were lg/ml for all strains except 6 (<0.023 lg/ml). However, when tested by disk diffusion, 1 strain was found to be resistant. The

4 332 CURRENT ROBIOLOGY Vol. 53 (2006) results of E-tests and disk-diffusion methods are listed in Table 1. One strain was resistant to clarithromycin by the disk-diffusion and E-test ( 48 lg/ml), and 1 strain was resistant only by disk diffusion. Only 1 strain was resistant to amoxicillin by disk diffusion and E-test ( >256 lg/ml). For ciprofloxacin, 3 strains were resistant by disk diffusion and two by E-test, ( <32 lg/ml). The range of metronidazole value was broad (0.016 to 256). Sixteen strains were resistant to metronidazole by disk diffusion and E-test ( > 8 lg/ ml), and 1 was resistant only by E-test ( <48 lg/ ml). Overall, 64% of the strains were resistant to metronidazole. Resistances to amoxicillin, tetracycline,clarithromycin, and ciprofloxacin was found to be relatively low compared with metronidazole (Table 1). The E-test and disk- diffusion methods showed good reproducibility. H. pylori by agar dilution. In addition, the of metronidazole was also tested by agar-dilution method. All of the metronidazole-resistant strains had s >8 lg/ml by agar-dilution method. None of the metronidazole-resistant strains showed any zone of inhibition around (8 lg/ml) the strip by E-test. The of metronidazole range from to 256 lg/ml by E-test and 2 to 32 lg/ml by agar-dilution method, and a susceptibility classification scheme was defined with a unique breakpoint of 8 lg/ml, which separates the strains into two categories ( <8 lg/ml = susceptible strains, and 8 lg/ml = resistant strains). Discussion Susceptibility testing of H. pylori is increasingly important because of the primary or acquired resistance to various drugs, which can decrease the efficacy of the treatment of gastric infection. Treatment failure is characterized by reappearance of symptoms and by H. pylori positive tests even after therapy. We used Brucella chocolate agar with 7% sheep blood for culture of H. pylori and antimicrobial susceptibility testing. This medium was found to support good growth of H. pylori [23 25] and E-test manufacturer also recommend brucella agar with 5% to 10% sheep blood [46]. In the present study, we found that the majority of H. pylori isolates were sensitive to antibiotics (amoxicillin, tetracycline, clarithromycin and ciprofloxacin) tested in vitro except metronidazole, and resistances to amoxicillin (4%), tetracycline (4%), clarithromycin (8%), ciprofloxacin (12%) were relatively lower than metronidazole (64%) (Table 1). The results of the present study are consistent with the previous multicentric study [44] and report published from Kolkata, India [47]. These observations suggest the use of these antibiotics in patients for H. pylori eradication. Reports regarding metronidazole-resistant H. pylori from Western countries have quoted figures ranging from 15% to 27% [29, 30]. The low proportions of resistant strains may be due to the fact that in The United States and some Eurppean countries, metronidazole is neither marketed nor used very commonly. In the United Kingdom 25% to 26.6% H. pylori strains were resistant to metronidazole [29, 30]. On the contrary, resistance to metronidazole as high as 84% has been reported from Africa [31] and as high as 85% from Kolkata, India [47]. The rates of metronidazole resistance found in our study are lower than in Africa but definitely higher than in developed countries. Sixteen of our strains were found to be metronidazole resistant by disk-diffusion method and 17 by E-test ( in 12 strains >256 lg/ml, in 3 strains <192 lg/ml, 1 and in strain each <48 and <94 lg/ml). In India, metronidazole is cheaper, readily available, and used extensively for self-medication. The resistance mechanism of H. pylori to metronidazole is not well known [34]. It may concern the enzymes involved in the reduction of the nitro group, but alternate pathways may exist; therefore, the observed s would be the result of a complex phenomenon [32]. Another probable reason might be plasmid-mediated resistance. For metronidazole susceptibility testing, the agar-dilution method correlated very well with the disk-diffusion method and E-test. In our study, 2 strains were found to be clarithromycin resistance by disk diffusion and 1 by E-test ( <8 lg/ml). The prevalence of clarithromycin resistance [33] (10%) tends to be lower in countries in which clarithromycin only recently has become available and in which other macrolides are not widely used. The resistance rate was also reported to be low in France, which is contrary to results obtained in several other studies involving hundreds of strains, where it was in the range of 10% [33]. Genetic studies have revealed that clarithromycin resistance is often associated with point mutation of the 23S rrna gene [35]. For ciprofloxacin, three strains were resistant by disk diffusion and two by E-test ( 32 lg/ml) (Table 1). Although ciprofloxacin is not a drug of choice in the therapy of H. pylori infection, its combination with amoxicillin could be considered [36]. Compared with the low prevalence ( 1%) of ciprofloxacin resistance in H. pylori in most studies [37], the present study showed that 12% and 8% of strains were resistance to ciprofloxacin by disk diffusion and E-test, respectively.

5 K. K. Mishra et al.: Antimicrobial susceptibility testing 333 There is a trend toward an increased rate of resistance in developed countries. This may be caused by an increased use of these compounds to treat H. pylori infection [36]. All strains were uniformly susceptible to tetracycline by E-test, except one, which was resistant by disk diffusion (Table 1). One strain found to be amoxicillin resistant by disk diffusion method was also resistant by E-test ( >256 lg/ml) (Table 1). By evaluation of diagnostic test, the accuracy between E-test and the disk-diffusion method for amoxicillin was excellent (100%). In some of the previous studies, none of the strains was found to be resistant to amoxicillin or tetracycline [38 41]. The previous data suggest that the rate of resistance to amoxicillin and tetracycline agents is still low and does not constitute a significant problem among patients with H. pylori infections. According to a previous report, the disk-diffusion method has not been recommended for bacterial species, which require a long incubation time because of the pattern of antibiotic release from the disks [42]. E-test, which has more stable pattern of antibiotic release has been found to tolerate a prolonged incubation [22]. However, we have found that prolonged incubation puts reliability at risk, even when the E-test is used (data not shown). Therefore, whichever method is used, a short incubation time is needed for resistance testing. In this study, we showed that the disk-diffusion method is equivalent to the E-test for in vitro antibiotic resistance determination for H. pylori. Thus, this study suggests that disk diffusion might be a good alternative for determining the antibiotic sensitivity of H. pylori strains. To conclude, the disk-diffusion method is costeffective (approx. 2.6 cents vs. US$2.6), is easy to perform, and does not require any special materials or techniques compared with the E-test susceptibility method. We found 100% agreement between the diskdiffusion method and the E-test for amoxicillin, where as other antibiotic showed 90% to 95% accuracy. In conclusion, the disk diffusion method is useful for in vitro susceptibility testing of H. pylori strains, which is required before attempting a second eradication after initial failure. ACKNOWLEDGMENTS K.K.M. is thankful to S.R. Naik, Department of Gastroenterolgy, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS) (Lucknow, India) for extending clinical support to carry out the study. Literature Cited 1. National Institute of Health Consensus conference (1994) Helicobacter pylori in peptic ulcer diseases. Development Panel on Helicobacter pylori in Peptic Ulcer Diseases. 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