Department of Microbiology, Shanghai Second Medical University, Chongqing South Road 280, Shanghai, China 200025
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Abstract |
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Introduction |
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Materials and methods |
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A total of 153 consecutive clinical isolates of H. pylori, 81 from females and 72 from males, were isolated from gastric biopsy specimens taken between January 1998 and February 1999 in the Ninth People's Hospital of Shanghai. The specimens were inoculated directly on to brainheart infusion agar supplemented with 7% defibrinated sheep blood and cultures were incubated in microaerobic conditions for 48 h at 37°C. Isolates were identified, and stored at 80°C in brucella broth containing 30% glycerine until use. H. pylori NCTC 11637 (metronidazole MIC, 1 mg/L) was used as a control strain.
Antimicrobial agents
Metronidazole was purchased from Shanghai ChangZheng Pharmaceutical Factory, Shanghai, China. Pure powders of the other two antimicrobial agents were provided by Sichuan Pharmaceutical Co., Sichuan, China.
Determination of MICs
An agar dilution method was used to determine MICs. Bacteria were harvested from plates by suspending them in sterile 0.85% NaCl, yielding a viable count of about 3 x 1083 x 109 cfu/mL. Plates containing two-fold dilutions of each antibiotic were inoculated with H. pylori suspensions using a Steer's replicator to give an inoculum of approximately 106 cfu per spot. MICs were determined after 3 days' incubation. Resistance to metronidazole and amoxycillin was defined based on the European Helicobacter pylori Study Group breakpoints (MICs of >8 and >0.5 mg/L, respectively); tetracycline resistance was defined as an MIC of >16 mg/L.1
Determination of ß-lactamase production
ß-Lactamase production was determined by means of acidometry using ß-lactamase detection paper. A penicillinase-producing Staphylococcus aureus strain was used as a positive control.
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Results |
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Discussion |
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Tetracycline resistance in H. pylori was not reported until 1996, when Midolo and colleagues reported tetracycline-resistant strains from Australia.4 Piccolomini et al. reported that 6% of strains were tetracycline resistant in 1997 in Italy.1 We found a surprisingly high prevalence of tetracycline resistance, 58.8%; the tetracycline MIC for many strains was >32 mg/L, although the reasons for this are unknown. Tetracycline was a component of the bismuth-based triple-therapy regimen recommended in the 1990s for treating H. pylori infection, and has also been used extensively as therapy in other types of infection, such as non-gonococcal urethritis, in recent years. Whether the mechanism of tetracycline resistance is related to carriage of resistance plasmids, originating from other bacterial species, remains to be determined.
Amoxycillin resistance was not considered important until recently, when amoxycillin resistance in H. pylori isolates was identified in the USA, Canada and Italy. Resistance rates of 31%5 and 45%6 have been reported from Italy. Similarly, 41.2% of the isolates we tested were resistant to amoxycillin defined by the same breakpoints (MIC > 8 mg/L), or 71.9% as defined by European breakpoints (MIC > 0.5 mg/L).
In 1999, rates of resistance for other bacteria were also high in China: 81.8% of S. aureus7 and 60.2% of E. coli8 were resistant to amoxycillin. The prevalence of amoxycillin resistance was probably a result of indiscriminate use of this antimicrobial agent, because of the lack of clearly defined guidelines for the management of H. pylori-associated dyspepsia and other infections. Amoxycillin has also been one of the most commonly used antimicrobial agents in the community in China in recent years. Though the MICs for many resistant strains were high, no strain produced ß-lactamase. Dore et al. suggested that the mechanism of amoxycillin resistance may be related to the development of tolerance and that the antibiotic resistance phenotype may be lost upon freezing or storage.5 Amoxycillin MICs of all the strains we examined on multiple occasions, however, were stable before and after freezing for storage. We assume, therefore, that amoxycillin resistance is probably a result of alterations in penicillin-binding proteins.
Surprisingly, 39.2% of H. pylori isolates were resistant to all three antibiotics tested. The reason that so many multiresistant strains were identified may reflect extensive use of these three antibiotics in this area. This suggests that therapy regimens should be adjusted to include one of these three antibiotics combined with another agent for which rates of resistance are low, such as clarithromycin, which has good efficacy in vitro. To avoid increasing resistance, it is now probably essential to test antibiotic sensitivity of bacteria before treating patients, rather than giving empirical treatment.
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Acknowledgments |
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Notes |
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References |
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2 . Liu, W. Z., Shi, Y., Wu, S. M., Zang, J. Z., Xu, W. W., Xiao, S. D. et al. (1998). Study on short term triple therapy combining omeprazole, clarithromycin and furazolidone or metronidazole to eradicate infection of Helicobacter pylori. Shanghai Medicine Journal 21, 4023.
3 . Zhang, Z. H. (1997). Study on biological characterization of Helicobacter pylori. In Study on Helicobacter pylori in China, (Liu, Z. G., Liu, S. L. & Ma, J. W. S., Eds), pp. 7889. Science and Technology Publishing House, Beijing.
4 . Midolo, P. D., Korman, M. G., Turnidge, J. D. & Lambert, J. R. (1996). Helicobacter pylori resistance to tetracycline. Lancet 347, 11945.[ISI][Medline]
5 . Dore, M. P., Piana, A., Carta, M., Atzei, A., Are, B. M., Mura, I. et al. (1998). Amoxycillin resistance is one reason for failure of amoxycillinomeprazole treatment of Helicobacter pylori infection. Alimentary Pharmacological Therapy 12, 6359.
6 . Dore, M. P., Sepulveda, A. R., Mura, I., Realdi, G., Osato, M. S. & Graham, D. Y. (1997). Explanation for variability of omeprazole amoxicillin therapy? Tolerance of H. pylori to amoxicillin. Gastroenterology 112, A105 (Abstract).
7 . Liu, Y., Pan, X. H. & Sheng, X. (1999). Analysis of resistance in Staphylococcus aureus. Shanghai Journal of Medical Laboratory Sciences 14, 208.
8 . Wang, Y. Z., Zhang, X. & Liu, A. R. (1999). Resistance to sixteen antibiotics resistance in E. coli. Shanghai Journal of Medical Laboratory Sciences 14, 106.
Received 8 October 1999; returned 25 November 1999; revised 4 January 2000; accepted 23 February 2000