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Mutations in the 23S rRNA gene of clarithromycin-resistant Helicobacter pylori from Japan
International Journal of Antimicrobial Agents 30 (2007) 250–254
Short communication
Mutations in the 23S rRNA gene of clarithromycin-resistant Helicobacter pylori from Japan Emiko Rimbara a , Norihisa Noguchi a,∗ , Hidenobu Kijima a , Tai Yamaguchi a , Takashi Kawai b , Masanori Sasatsu a a
b
Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan Endoscopy Center, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Received 26 February 2007; accepted 16 April 2007
Abstract The 23S rRNA gene in clinical isolates of Helicobacter pylori isolated between 1995 and 2004 from Japan was investigated and the relationship between mutations in this gene and clarithromycin susceptibility was studied. Among nine mutations that have previously been reported to confer clarithromycin resistance, an adenine → guanine transition at position 2142 (A2142G) or 2143 (A2143G) was detected in all clarithromycin-resistant strains (n = 67) but not in any clarithromycin-susceptible strains (n = 17). Mutations at positions 2182, 2223, 2244 and 2288 have previously been reported to confer clarithromycin resistance in H. pylori isolates from Bangladesh, China and Brazil. However, these mutations were not associated with clarithromycin resistance in H. pylori isolates from Japan in this study. Other mutations at positions 2115, 2144 and 2711, which have also been reported to confer clarithromycin resistance in H. pylori from Sweden and Italy, were not detected in the strains in this study. Our results suggest that susceptibility to clarithromycin is predicted by detection of mutations at positions 2142 and 2143 of the 23S rRNA gene in H. pylori isolates in Japan. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Helicobacter pylori; Clarithromycin; Antimicrobial resistance; 23S rRNA
1. Introduction Helicobacter pylori is a Gram-negative bacterium that infects the human stomach and was discovered in 1983. H. pylori causes peptic ulcers and chronic gastritis and is associated with gastric cancer [1]. Eradication therapy consisting of a proton pump inhibitor, amoxicillin and clarithromycin has been commonly used for H. pylori eradication in Japan [2]. However, eradication failure has been increasing recently [2], making it increasingly difficult to cure diseases caused by H. pylori infection. In Japan, clarithromycinresistant H. pylori have been increasing in recent times [3] and clarithromycin resistance is one of the major causes of eradication failure [4]. It has been shown that muta∗
Corresponding author. Tel.: +81 426 76 5615; fax: +81 426 76 5647. E-mail address: [email protected] (N. Noguchi).
tions in the peptidyltransferase-encoding region of 23S rRNA confers resistance to clarithromycin by decreasing the binding of clarithromycin to ribosomes in H. pylori [5]. Most findings show that the major point mutations are the adenine → guanine transition at either position 2142 (A2142G) or 2143 (A2143G), or the adenine → cytosine transition at position 2142 (A2142C) [6]. Furthermore, it has been reported that other mutations in the 23S rRNA gene in H. pylori are able to confer clarithromycin resistance in H. pylori [7–12]. Whilst the prevalence of A2142C/G and A2143G in clinical H. pylori isolates has been well investigated in Japan, there are few reports on mutations in the 23S rRNA gene other than at positions 2142 and 2143. As H. pylori are genetically diverse, the prevalence of mutations in 23S rRNA in certain areas other than Japan might not apply to Japan. In this study, mutations in the 23S rRNA gene in clinical isolates of H. pylori isolated between 1995 and 2004 in Japan were
0924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2007.04.009
E. Rimbara et al. / International Journal of Antimicrobial Agents 30 (2007) 250–254
investigated and the relationship between clarithromycin susceptibility and previously reported mutations in the 23S gene were studied. 2. Materials and methods
251
System (Promega, Madison, WI), and DNA sequencing was performed by auto DNA sequencer as reported previously [14]. DNA sequences of the 23S rRNA in H. pylori UA802, ATCC 700392 and ATCC 700824 were obtained from the DNA Data Bank of Japan (GenBank accession nos. U27270, AE000511 and AE001439, respectively).
2.1. Bacterial strains Eighty-one clinical H. pylori strains isolated between 1995 and 2004 at Tokyo Medical University Hospital in Japan as well as H. pylori strains UA802 [5], ATCC 700392 and ATCC 700824 were studied. H. pylori isolates were identified by oxidase production and the API Campy test (bioM´erieux, Marcy l’Etoile, France). Isolates were stored in a MicrobankTM (Pro-Lab Diagnostics, Ontario, Canada) in 20% glycerol/brain–heart infusion (BHI) broth at −80 ◦ C until tested. H. pylori strains were subcultured on BHI agar plates containing 5% horse blood at 35 ◦ C under microaerobic conditions until susceptibility tests were performed. 2.2. Susceptibility test The susceptibilities of H. pylori isolates to clarithromycin were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute [13]. H pylori isolates were considered resistant when the minimum inhibitory concentration (MIC) of clarithromycin was 1 g/mL. H. pylori ATCC 43504 was used as a quality control organism. 2.3. Polymerase chain reaction (PCR) and DNA sequence analysis For H. pylori isolated between 1995 and 2002 (n = 44), a 2150 bp fragment of the 23S rRNA gene (corresponding to positions 626–2774 of the 23S gene of H. pylori strain UA802 [5]) was amplified. For H. pylori isolated between 2003 and 2004 (n = 37), a 462 bp fragment of the 23S rRNA gene (corresponding to positions 1828–2290 of the 23S gene of H. pylori strain UA802 [5]) was amplified. Each fragment was purified with Wizard® SV Gel and PCR Clean-Up
3. Results 3.1. Clarithromycin susceptibilities of the strains studied Among the 84 strains studied, 67 clinical isolates were resistant to clarithromycin, whereas 14 clinical isolates as well as H. pylori UA802, ATCC 700824 and ATCC 700392 were susceptible to clarithromycin (Table 1). Among the 67 clarithromycin-resistant isolates, 19 isolates (28.4%) were highly resistant to clarithromycin (≥32 g/mL). 3.2. Correlation between clarithromycin susceptibility and mutations in the 23S rRNA gene According to previous reports [6–12], positions in the 23S rRNA gene that confer clarithromycin resistance in H. pylori are 2115, 2142, 2143, 2144, 2182, 2223, 2244, 2288 and 2711 (Fig. 1). Positions 2288 and 2711 of the 23S rRNA gene were analysed in 47 strains (including 44 isolates between 1995 and 2002, two ATCC strains and H. pylori UA802), whereas another seven positions were analysed in all 84 strains studied (Table 1). The mutations A2142G and A2143G were detected in 5 and 63 of the 67 clarithromycin-resistant strains, respectively. Furthermore, no clarithromycin-susceptible H. pylori strain had any mutation at positions 2142 and 2143 in the 23S rRNA gene in this study. All clarithromycin-resistant strains in this study had the mutation at either A2142G or A2143G, except one strain (TH3636). Strain TH3636 had both A2142G and A2143G in a single colony and was revealed to be highly resistant to clarithromycin (MIC = 256 g/mL). Among the five clarithromycin-resistant isolates with the mutation
Fig. 1. Positions in the 23S rRNA gene of Helicobacter pylori analysed in this study. The positions correspond to the 23S rRNA gene of H. pylori strain UA802.
T2711C
0a 0 0 0 82 (97.6) 16 (94.1) 66 (98.5) 19 (100.0)
C2244T G2223A T2182C
70 (83.3) 13 (76.5) 58 (86.6) 16 (84.2) 63 (75.0) 0 63 (94.0) 16 (84.2) MIC, minimum inhibitory concentration; CAM, clarithromycin. a Positions 2288 and 2711 in the 23S rRNA gene were analysed in 47 strains.
A2144T A2143G A2142G
5 (6.0) 0 5 (7.5) 4 (21.1) 0 0 0 0 84 17 67 19 <0.008 to >128 <0.008–0.063 2 to >128 32 to >128
A2115G
0 0 0 0
4. Discussion
Total CAM-susceptible strains CAM-resistant strains CAM highly resistant strains
No. (%) of strains with the following mutations in the 23S rRNA gene No. of strains MIC range (g/mL)
Table 1 Mutations in the 23S rRNA gene and clarithromycin susceptibilities in Helicobacter pylori
A2142G, the MIC of clarithromycin for one isolate was 4 g/mL, whilst those of the other four isolates were ≥32 g/mL. The mutation T2182C was detected in 70 (83.3%) of the 84 strains studied. There was no significant correlation between the mutation T2182C and susceptibility to clarithromycin. Among the clarithromycin-resistant isolates, there was also no significant correlation between the mutation T2182C and the level of clarithromycin resistance. Mutations G2223A and T2288C were detected in 82 (97.6%) of the 84 strains and in 45 (95.7%) of the 47 strains analysed in this study. The mutation C2244T was detected in two clarithromycin-resistant isolates; however it was also detected in clarithromycin-susceptible H. pylori UA802. The other three mutations analysed in this study, including A2115G, A2144T and T2711C, were not detected in any isolates.
3 (3.6) 1 (5.9) 2 (3.0) 0
45 (95.7)a 11 (100.0) 34 (94.4) 19 (100.0)
E. Rimbara et al. / International Journal of Antimicrobial Agents 30 (2007) 250–254
T2288C
252
In this study, we analysed nine positions of the 23S rRNA gene that have previously been reported to confer clarithromycin resistance in H. pylori. We investigated the correlation between these mutations and clarithromycin resistance in 81 clinical isolates in Japan. It has already been shown that mutations A2142C/G and A2143G in the 23S rRNA gene confer resistance to clarithromycin in H. pylori [5,6]. In this study, it was confirmed that clarithromycin-resistant H. pylori had mutations at either position 2142 or 2143 of the 23S rRNA gene. It was also previously reported that the mutation at position 2142 is able to confer higher clarithromycin resistance in H. pylori than that at position 2143 of the 23S rRNA gene [6]. Although most of the isolates with the A2142G mutation (four of five strains) showed high-level clarithromycin resistance (MIC ≥ 32 g/mL), one isolate with the mutation A2142G was not highly resistant to clarithromycin. The results suggest that the mutation A2142G was not necessary for high clarithromycin resistance. Whilst the cause of highlevel resistance is still controversial, it is possible that another mechanism might confer the high clarithromycin resistance in H. pylori. However, there was no difference with regard to the level of clarithromycin resistance in the prevalence of other mutations in the 23S rRNA gene in this study. To elucidate further the mechanisms of the high clarithromycin resistance in H. pylori, we also investigated the correlation between mutations in the ribosomal proteins (L4 and L22) and the level of clarithromycin resistance for 40 clinical isolates. Whilst a few mutations were detected, there was no mutation that was common and specific to isolates with a high level of resistance to clarithromycin (data not shown). Therefore, further investigation is required to account for high-level clarithromycin resistance in H. pylori. We showed that the TH3636 strain used in this study had both the mutation A2142G as well as A2143G. As
E. Rimbara et al. / International Journal of Antimicrobial Agents 30 (2007) 250–254
H. pylori has been shown to have two 23S rRNA genes (rrnA and rrnB) in its genome [15], we analysed each 23S rRNA gene and confirmed that only the rrnB gene had the A2143G mutation (data not shown), whereas we were unable to amplify the rrnA gene. It is therefore suggested that each mutation, A2142G and A2143G, was in the distinct 23S rRNA genes (rrnA and rrnB, respectively) in strain TH3636. It has also been shown that the mutation T2182C was detected with high frequency in isolates in Japan. Khan et al. [12] previously reported that the mutation T2182C is associated with clarithromycin resistance in clinical H. pylori isolates in Bangladesh. However, in this study the mutation T2182C was detected with high frequency not only in the clarithromycin-resistant isolates but also in the clarithromycin-susceptible isolates from Japan. Because H. pylori has genetic diversity, strains in Bangladesh might be distinguished from those in Japan. Therefore, it has been suggested that the mutation T2182C is not necessary for clarithromycin resistance in H. pylori in Japan. Fontana et al. [10] also reported that the mutation T2717C (corresponding to T2711C in this study), which is located in domain VI of 23S rRNA, confers a low level of clarithromycin resistance (MIC = 1 g/mL) in H. pylori isolates from Italy. However, the mutation T2711C was not detected in any strains in this study. As strains with low levels of clarithromycin resistance were not included in this study, it was suggested that the mutation T2711C was not involved in high levels of clarithromycin resistance. Further investigation of the relationship between the mutation T2711C and low-level clarithromycin resistance using isolates from Japan might be required. Mutations G2223A and T2288C, which were reported in clarithromycin-resistant H. pylori isolates from China [8], were detected in almost all (97.6% and 95.7%, respectively) clarithromycin-susceptible and -resistant isolates from Japan in this study. Therefore, it is suggested that adenine and cytosine at positions 2223 and 2288, respectively, were the wild-type in the isolates from Japan and are not related to clarithromycin resistance in H. pylori. On the other hand, the mutation C2244T, which was also previously reported to confer clarithromycin resistance in H. pylori isolates from Brazil [7] and China [8], was detected in two clarithromycin-resistant strains in this study. However, because clarithromycin-susceptible strain UA802 has the mutation C2244T, it is suggested that the mutation C2244T was not necessary for clarithromycin resistance in H. pylori. In conclusion, we confirmed that clarithromycin resistance could be explained by mutations at positions 2142 and 2143 of 23S rRNA in H. pylori isolated from Japan. Clarithromycin-resistant H. pylori has been strongly implicated in the failure of eradication of H. pylori in Japan. Therefore, administration of a susceptibility test before eradication therapy is recommended for successful treatment. However, the susceptibility test requires gastric biopsies
253
obtained in an endoscopy procedure, which is invasive for patients. It has already been shown that clarithromycinresistant H. pylori in faeces can be detected by analysis of the 23S rRNA gene amplified by primers specific to H. pylori [14]. The results of this study strongly suggest that analysis of positions 2142 and 2143 of the 23S rRNA gene from faeces could be applied to a clarithromycin susceptibility test instead of the susceptibility test by culture.
Acknowledgments We wish to thank Y. Suzuki, N. Hasegawa, N. Sekine and S. Soeda for their technical assistance. This work was supported by the High-Tech Research Centre Project for Private Universities provided by the Ministry of Education, Culture, Sports, Science and Technology and by the Matching Fund Subsidy for Private Schools of Japan.
References [1] Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, 7–14 June 1994. IARC Monogr Eval Carcinog Risks Hum 1994;61:1– 241. [2] Asaka M, Satoh K, Sugano K, et al. Guidelines in the management of Helicobacter pylori infection in Japan. Helicobacter 2001;6:177– 86. [3] Rimbara E, Noguchi N, Tanabe M, et al. Susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus in Tokyo, Japan, 1995–2001. Clin Microbiol Infect 2005;11:307–11. [4] Broutet N, Tchamgoue S, Pereira E, et al. Risk factors for failure of Helicobacter pylori therapy—results of an individual data analysis of 2751 patients. Aliment Pharmacol Ther 2003;17:99–109. [5] Taylor DE, Ge Z, Purych D, et al. Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations. Antimicrob Agents Chemother 1997;41:2621–8. [6] Versalovic J, Osato MS, Spakovsky K, et al. Point mutations in the 23S rRNA gene of Helicobacter pylori associated with different levels of clarithromycin resistance. J Antimicrob Chemother 1997;40:283– 6. [7] Ribeiro ML, Vitiello L, Miranda MC, et al. Mutations in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori isolates in Brazil. Ann Clin Microbiol Antimicrob 2003;2:11. [8] Hao Q, Li Y, Zhang ZJ, et al. New mutation points in 23S rRNA gene associated with Helicobacter pylori resistance to clarithromycin in northeast China. World J Gastroenterol 2004;10:1075–7. [9] Hulten K, Gibreel A, Skold O, et al. Macrolide resistance in Helicobacter pylori: mechanism and stability in strains from clarithromycintreated patients. Antimicrob Agents Chemother 1997;41:2550– 3. [10] Fontana C, Favaro M, Minelli S, et al. New site of modification of 23S rRNA associated with clarithromycin resistance of Helicobacter pylori clinical isolates. Antimicrob Agents Chemother 2002;46:3765– 9. [11] Toracchio S, Aceto GM, Mariani-Costantini R, et al. Identification of a novel mutation affecting domain V of the 23S rRNA gene in Helicobacter pylori. Helicobacter 2004;9:396–9.
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[12] Khan R, Nahar S, Sultana J, et al. T2182C mutation in 23S rRNA is associated with clarithromycin resistance in Helicobacter pylori isolates obtained in Bangladesh. Antimicrob Agents Chemother 2004;48:3567–9. [13] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. 12th ed. Twelfth Informational Supplement. M100-S12. Villanova, PA: CLSI; 2002.
[14] Rimbara E, Noguchi N, Yamaguchi T, et al. Development of a highly sensitive method for detection of clarithromycin-resistant Helicobacter pylori from human feces. Curr Microbiol 2005;51:1– 5. [15] Tomb JF, White O, Kerlavage AR, et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 1997;388:539– 47.
Short communication
Mutations in the 23S rRNA gene of clarithromycin-resistant Helicobacter pylori from Japan Emiko Rimbara a , Norihisa Noguchi a,∗ , Hidenobu Kijima a , Tai Yamaguchi a , Takashi Kawai b , Masanori Sasatsu a a
b
Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan Endoscopy Center, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Received 26 February 2007; accepted 16 April 2007
Abstract The 23S rRNA gene in clinical isolates of Helicobacter pylori isolated between 1995 and 2004 from Japan was investigated and the relationship between mutations in this gene and clarithromycin susceptibility was studied. Among nine mutations that have previously been reported to confer clarithromycin resistance, an adenine → guanine transition at position 2142 (A2142G) or 2143 (A2143G) was detected in all clarithromycin-resistant strains (n = 67) but not in any clarithromycin-susceptible strains (n = 17). Mutations at positions 2182, 2223, 2244 and 2288 have previously been reported to confer clarithromycin resistance in H. pylori isolates from Bangladesh, China and Brazil. However, these mutations were not associated with clarithromycin resistance in H. pylori isolates from Japan in this study. Other mutations at positions 2115, 2144 and 2711, which have also been reported to confer clarithromycin resistance in H. pylori from Sweden and Italy, were not detected in the strains in this study. Our results suggest that susceptibility to clarithromycin is predicted by detection of mutations at positions 2142 and 2143 of the 23S rRNA gene in H. pylori isolates in Japan. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Helicobacter pylori; Clarithromycin; Antimicrobial resistance; 23S rRNA
1. Introduction Helicobacter pylori is a Gram-negative bacterium that infects the human stomach and was discovered in 1983. H. pylori causes peptic ulcers and chronic gastritis and is associated with gastric cancer [1]. Eradication therapy consisting of a proton pump inhibitor, amoxicillin and clarithromycin has been commonly used for H. pylori eradication in Japan [2]. However, eradication failure has been increasing recently [2], making it increasingly difficult to cure diseases caused by H. pylori infection. In Japan, clarithromycinresistant H. pylori have been increasing in recent times [3] and clarithromycin resistance is one of the major causes of eradication failure [4]. It has been shown that muta∗
Corresponding author. Tel.: +81 426 76 5615; fax: +81 426 76 5647. E-mail address: [email protected] (N. Noguchi).
tions in the peptidyltransferase-encoding region of 23S rRNA confers resistance to clarithromycin by decreasing the binding of clarithromycin to ribosomes in H. pylori [5]. Most findings show that the major point mutations are the adenine → guanine transition at either position 2142 (A2142G) or 2143 (A2143G), or the adenine → cytosine transition at position 2142 (A2142C) [6]. Furthermore, it has been reported that other mutations in the 23S rRNA gene in H. pylori are able to confer clarithromycin resistance in H. pylori [7–12]. Whilst the prevalence of A2142C/G and A2143G in clinical H. pylori isolates has been well investigated in Japan, there are few reports on mutations in the 23S rRNA gene other than at positions 2142 and 2143. As H. pylori are genetically diverse, the prevalence of mutations in 23S rRNA in certain areas other than Japan might not apply to Japan. In this study, mutations in the 23S rRNA gene in clinical isolates of H. pylori isolated between 1995 and 2004 in Japan were
0924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2007.04.009
E. Rimbara et al. / International Journal of Antimicrobial Agents 30 (2007) 250–254
investigated and the relationship between clarithromycin susceptibility and previously reported mutations in the 23S gene were studied. 2. Materials and methods
251
System (Promega, Madison, WI), and DNA sequencing was performed by auto DNA sequencer as reported previously [14]. DNA sequences of the 23S rRNA in H. pylori UA802, ATCC 700392 and ATCC 700824 were obtained from the DNA Data Bank of Japan (GenBank accession nos. U27270, AE000511 and AE001439, respectively).
2.1. Bacterial strains Eighty-one clinical H. pylori strains isolated between 1995 and 2004 at Tokyo Medical University Hospital in Japan as well as H. pylori strains UA802 [5], ATCC 700392 and ATCC 700824 were studied. H. pylori isolates were identified by oxidase production and the API Campy test (bioM´erieux, Marcy l’Etoile, France). Isolates were stored in a MicrobankTM (Pro-Lab Diagnostics, Ontario, Canada) in 20% glycerol/brain–heart infusion (BHI) broth at −80 ◦ C until tested. H. pylori strains were subcultured on BHI agar plates containing 5% horse blood at 35 ◦ C under microaerobic conditions until susceptibility tests were performed. 2.2. Susceptibility test The susceptibilities of H. pylori isolates to clarithromycin were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute [13]. H pylori isolates were considered resistant when the minimum inhibitory concentration (MIC) of clarithromycin was 1 g/mL. H. pylori ATCC 43504 was used as a quality control organism. 2.3. Polymerase chain reaction (PCR) and DNA sequence analysis For H. pylori isolated between 1995 and 2002 (n = 44), a 2150 bp fragment of the 23S rRNA gene (corresponding to positions 626–2774 of the 23S gene of H. pylori strain UA802 [5]) was amplified. For H. pylori isolated between 2003 and 2004 (n = 37), a 462 bp fragment of the 23S rRNA gene (corresponding to positions 1828–2290 of the 23S gene of H. pylori strain UA802 [5]) was amplified. Each fragment was purified with Wizard® SV Gel and PCR Clean-Up
3. Results 3.1. Clarithromycin susceptibilities of the strains studied Among the 84 strains studied, 67 clinical isolates were resistant to clarithromycin, whereas 14 clinical isolates as well as H. pylori UA802, ATCC 700824 and ATCC 700392 were susceptible to clarithromycin (Table 1). Among the 67 clarithromycin-resistant isolates, 19 isolates (28.4%) were highly resistant to clarithromycin (≥32 g/mL). 3.2. Correlation between clarithromycin susceptibility and mutations in the 23S rRNA gene According to previous reports [6–12], positions in the 23S rRNA gene that confer clarithromycin resistance in H. pylori are 2115, 2142, 2143, 2144, 2182, 2223, 2244, 2288 and 2711 (Fig. 1). Positions 2288 and 2711 of the 23S rRNA gene were analysed in 47 strains (including 44 isolates between 1995 and 2002, two ATCC strains and H. pylori UA802), whereas another seven positions were analysed in all 84 strains studied (Table 1). The mutations A2142G and A2143G were detected in 5 and 63 of the 67 clarithromycin-resistant strains, respectively. Furthermore, no clarithromycin-susceptible H. pylori strain had any mutation at positions 2142 and 2143 in the 23S rRNA gene in this study. All clarithromycin-resistant strains in this study had the mutation at either A2142G or A2143G, except one strain (TH3636). Strain TH3636 had both A2142G and A2143G in a single colony and was revealed to be highly resistant to clarithromycin (MIC = 256 g/mL). Among the five clarithromycin-resistant isolates with the mutation
Fig. 1. Positions in the 23S rRNA gene of Helicobacter pylori analysed in this study. The positions correspond to the 23S rRNA gene of H. pylori strain UA802.
T2711C
0a 0 0 0 82 (97.6) 16 (94.1) 66 (98.5) 19 (100.0)
C2244T G2223A T2182C
70 (83.3) 13 (76.5) 58 (86.6) 16 (84.2) 63 (75.0) 0 63 (94.0) 16 (84.2) MIC, minimum inhibitory concentration; CAM, clarithromycin. a Positions 2288 and 2711 in the 23S rRNA gene were analysed in 47 strains.
A2144T A2143G A2142G
5 (6.0) 0 5 (7.5) 4 (21.1) 0 0 0 0 84 17 67 19 <0.008 to >128 <0.008–0.063 2 to >128 32 to >128
A2115G
0 0 0 0
4. Discussion
Total CAM-susceptible strains CAM-resistant strains CAM highly resistant strains
No. (%) of strains with the following mutations in the 23S rRNA gene No. of strains MIC range (g/mL)
Table 1 Mutations in the 23S rRNA gene and clarithromycin susceptibilities in Helicobacter pylori
A2142G, the MIC of clarithromycin for one isolate was 4 g/mL, whilst those of the other four isolates were ≥32 g/mL. The mutation T2182C was detected in 70 (83.3%) of the 84 strains studied. There was no significant correlation between the mutation T2182C and susceptibility to clarithromycin. Among the clarithromycin-resistant isolates, there was also no significant correlation between the mutation T2182C and the level of clarithromycin resistance. Mutations G2223A and T2288C were detected in 82 (97.6%) of the 84 strains and in 45 (95.7%) of the 47 strains analysed in this study. The mutation C2244T was detected in two clarithromycin-resistant isolates; however it was also detected in clarithromycin-susceptible H. pylori UA802. The other three mutations analysed in this study, including A2115G, A2144T and T2711C, were not detected in any isolates.
3 (3.6) 1 (5.9) 2 (3.0) 0
45 (95.7)a 11 (100.0) 34 (94.4) 19 (100.0)
E. Rimbara et al. / International Journal of Antimicrobial Agents 30 (2007) 250–254
T2288C
252
In this study, we analysed nine positions of the 23S rRNA gene that have previously been reported to confer clarithromycin resistance in H. pylori. We investigated the correlation between these mutations and clarithromycin resistance in 81 clinical isolates in Japan. It has already been shown that mutations A2142C/G and A2143G in the 23S rRNA gene confer resistance to clarithromycin in H. pylori [5,6]. In this study, it was confirmed that clarithromycin-resistant H. pylori had mutations at either position 2142 or 2143 of the 23S rRNA gene. It was also previously reported that the mutation at position 2142 is able to confer higher clarithromycin resistance in H. pylori than that at position 2143 of the 23S rRNA gene [6]. Although most of the isolates with the A2142G mutation (four of five strains) showed high-level clarithromycin resistance (MIC ≥ 32 g/mL), one isolate with the mutation A2142G was not highly resistant to clarithromycin. The results suggest that the mutation A2142G was not necessary for high clarithromycin resistance. Whilst the cause of highlevel resistance is still controversial, it is possible that another mechanism might confer the high clarithromycin resistance in H. pylori. However, there was no difference with regard to the level of clarithromycin resistance in the prevalence of other mutations in the 23S rRNA gene in this study. To elucidate further the mechanisms of the high clarithromycin resistance in H. pylori, we also investigated the correlation between mutations in the ribosomal proteins (L4 and L22) and the level of clarithromycin resistance for 40 clinical isolates. Whilst a few mutations were detected, there was no mutation that was common and specific to isolates with a high level of resistance to clarithromycin (data not shown). Therefore, further investigation is required to account for high-level clarithromycin resistance in H. pylori. We showed that the TH3636 strain used in this study had both the mutation A2142G as well as A2143G. As
E. Rimbara et al. / International Journal of Antimicrobial Agents 30 (2007) 250–254
H. pylori has been shown to have two 23S rRNA genes (rrnA and rrnB) in its genome [15], we analysed each 23S rRNA gene and confirmed that only the rrnB gene had the A2143G mutation (data not shown), whereas we were unable to amplify the rrnA gene. It is therefore suggested that each mutation, A2142G and A2143G, was in the distinct 23S rRNA genes (rrnA and rrnB, respectively) in strain TH3636. It has also been shown that the mutation T2182C was detected with high frequency in isolates in Japan. Khan et al. [12] previously reported that the mutation T2182C is associated with clarithromycin resistance in clinical H. pylori isolates in Bangladesh. However, in this study the mutation T2182C was detected with high frequency not only in the clarithromycin-resistant isolates but also in the clarithromycin-susceptible isolates from Japan. Because H. pylori has genetic diversity, strains in Bangladesh might be distinguished from those in Japan. Therefore, it has been suggested that the mutation T2182C is not necessary for clarithromycin resistance in H. pylori in Japan. Fontana et al. [10] also reported that the mutation T2717C (corresponding to T2711C in this study), which is located in domain VI of 23S rRNA, confers a low level of clarithromycin resistance (MIC = 1 g/mL) in H. pylori isolates from Italy. However, the mutation T2711C was not detected in any strains in this study. As strains with low levels of clarithromycin resistance were not included in this study, it was suggested that the mutation T2711C was not involved in high levels of clarithromycin resistance. Further investigation of the relationship between the mutation T2711C and low-level clarithromycin resistance using isolates from Japan might be required. Mutations G2223A and T2288C, which were reported in clarithromycin-resistant H. pylori isolates from China [8], were detected in almost all (97.6% and 95.7%, respectively) clarithromycin-susceptible and -resistant isolates from Japan in this study. Therefore, it is suggested that adenine and cytosine at positions 2223 and 2288, respectively, were the wild-type in the isolates from Japan and are not related to clarithromycin resistance in H. pylori. On the other hand, the mutation C2244T, which was also previously reported to confer clarithromycin resistance in H. pylori isolates from Brazil [7] and China [8], was detected in two clarithromycin-resistant strains in this study. However, because clarithromycin-susceptible strain UA802 has the mutation C2244T, it is suggested that the mutation C2244T was not necessary for clarithromycin resistance in H. pylori. In conclusion, we confirmed that clarithromycin resistance could be explained by mutations at positions 2142 and 2143 of 23S rRNA in H. pylori isolated from Japan. Clarithromycin-resistant H. pylori has been strongly implicated in the failure of eradication of H. pylori in Japan. Therefore, administration of a susceptibility test before eradication therapy is recommended for successful treatment. However, the susceptibility test requires gastric biopsies
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obtained in an endoscopy procedure, which is invasive for patients. It has already been shown that clarithromycinresistant H. pylori in faeces can be detected by analysis of the 23S rRNA gene amplified by primers specific to H. pylori [14]. The results of this study strongly suggest that analysis of positions 2142 and 2143 of the 23S rRNA gene from faeces could be applied to a clarithromycin susceptibility test instead of the susceptibility test by culture.
Acknowledgments We wish to thank Y. Suzuki, N. Hasegawa, N. Sekine and S. Soeda for their technical assistance. This work was supported by the High-Tech Research Centre Project for Private Universities provided by the Ministry of Education, Culture, Sports, Science and Technology and by the Matching Fund Subsidy for Private Schools of Japan.
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