In contrast to what has been previously published and recommended (1
), we found that culture, which is a prerequisite for antimicrobial susceptibility testing (23
), is also an accurate way to diagnose H. pylori
infection. Despite the fact that there were 47 centers involved in this study, the recovery rate was higher than 90% and even slightly better than the sensitivity of the UBT. The most probable explanation for this high yield was the use of a rigorous and standardized protocol, identical in the five laboratories involved in performing the bacterial culture. The critical points were the grinding of biopsies, the use of several kinds of agar media including two which were freshly prepared, and the follow-up readings of the plates over a 12-day period. Moreover, a transport system which maintained the biopsies at a low temperature, avoided dessication and contact with air (26a
), and assured delivery within 24 h was used.
We have consistently used Wilkins-Chalgren agar enriched with human blood for culture for the past 10 years following an initial comparative study of media (25
). It has provided the best results in clinical practice compared to other media, but these findings have never been formally published (22
). Human serum is considered the best growth supplement (38
). Cefsulodin is a good selective agent for gram-negative bacilli including Pseudomonas aeruginosa
) and has been proposed in a commercially available selective supplement (6
). The improved growth capacity of fresh media has been stressed by others (12
), as well as the use of selective and nonselective media (33
A reference method for studying the susceptibility of H. pylori
to clarithromycin was recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (29
) after this study was performed. The NCCLS method also uses agar dilution but differs on the following points: Wilkins-Chalgren agar was used instead of Mueller-Hinton agar; 10% instead of 5% blood was used; there was a larger bacterial inoculum (109
instead of 107
CFU/ml), which was prepared in brucella broth from a 48-h plate instead of in saline from a 72-h plate; and a shorter incubation time (48 instead of 72 h) was used. The higher inoculum was used in order to detect possible resistant mutant colonies and to be able to read the plates within 48 h. The agar dilution method, which is considered to be the reference method for performing susceptibility tests for most other bacteria, was also used to test metronidazole and amoxicillin.
This is the first time that a frequency distribution of the logarithms of the MICs for the three antibiotics has been obtained on such a large random sample of strains cultured from duodenal ulcer disease patients who are possibly representative of those in northern Europe.
The MIC distribution for amoxicillin against H. pylori
was normal, with the highest MIC at 0.5 μg/ml. It is necessary to seek a resistance mechanism, such as a modification in penicillin binding proteins, before categorizing these strains as resistant. We found no resistant strains, in contrast to the findings of van Zwet et al. (35
), or tolerant strains, as recently identified by Dore et al. (7
The MIC distribution for clarithromycin against H. pylori
was normal for 97% of the strains. A point mutation on the 23S rRNA gene (30
) was detected in all resistant strains except one by PCR-RFLP, leading one to think that an A2142C mutation could be present. The proportion of resistant strains was very low in this study. This may reflect the limited use of macrolides in countries from the northern part of Europe, especially Scandinavia. However, the resistance rate was also surprisingly 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% (24
). Another explanation may be that strains isolated from patients with duodenal ulcer disease are more likely to be susceptible to macrolides. Further studies should be performed to address this question.
The MIC distribution of metronidazole against H. pylori
showed a normal distribution for part of the strains. A tendency for a second mode was observed for strains with MICs higher than 8 μg/ml. The resistance mechanism of H. pylori
to metronidazole is not well known. It may concern the enzymes involved in the reduction of the nitro group, but alternate pathways may exist, and therefore the observed MICs would be the result of a complex phenomenon (32
). The breakpoint of 8 μg/ml has been proposed for metronidazole resistance based on studies using bismuth-based triple therapies (11
), and this figure was applied in this study. This breakpoint was valid for the MC group but not for the OMC group. The stepwise test indicated that the threshold of 32 μg/ml would be more appropriate if the latter regimen is used.
In contrast to clarithromycin resistance, resistance to metronidazole (>8 μg/ml) was found in 27% of the strains, with a marked variation between the different countries. Surprisingly, the frequency of resistance was only 16% in France; in previous studies the frequency was double this value. In Norway it was 42%. The reason for this high resistance rate in Norway is unclear.
The Etest has proven to be an accurate method to test the susceptibilities of fastidious organisms, including H. pylori
, to antibiotics. However, there is some concern regarding the value of Etest results for testing the effect of metronidazole on this bacterium. While we found an excellent agreement between the agar dilution method and the Etest for clarithromycin, there was an unacceptable discrepancy rate of 22% for metronidazole. The reason is unclear, but in this study, the plates were not preincubated in an anaerobic atmosphere (4
). On the other hand, for the agar dilution test, the plates were prepared extemporaneously and therefore they were more likely to have a constant redox potential than the plates used for the Etest. In previously performed studies, the correlation was indeed far from perfect: the percentage of agreement within plus or minus 1 dilution was 89 (31
) and 83% (8
), but only 56% in the study of Cederbrant (5
). Contrary to these results, Hirschl et al. found the Etest to be accurate and precise (14
The clinical relevance of H. pylori
resistance to antibiotics is an important aspect to consider (26
). Unfortunately, it was not possible to draw any conclusions for clarithromycin because of the low number of resistant strains encountered. With regard to metronidazole, there was an important impact of the resistance to this drug when it was used only with clarithromycin but the addition of omeprazole had a beneficial effect. The reason is not clear since metronidazole activity is not supposed to be pH dependent and, even more importantly, the diffusion of the drug in the gastric lumen decreases when the pH increases (9
). Therefore, it is most likely that the beneficial effect involves clarithromycin, whose activity is pH dependent, and/or a synergism between the two compounds. In another study, where amoxicillin was given instead of clarithromycin with metronidazole and lansoprazole, there was a lower cure rate (2
A consequence of the high eradication rate is a low occurrence of secondary resistance. Indeed, in the group receiving OAC, no secondary resistance was noted. In the group treated with OMC, four strains resistant to clarithromycin and four strains resistant to metronidazole were observed, and even more were observed in the group treated with MC, with five strains resistant to clarithromycin and eight resistant to metronidazole.
The occurrence of resistance to metronidazole in the group receiving AC was unexpected. In fact, the determination of these MICs was repeated and most of them were found to be in the susceptible range. The finding of resistance was probably due to technical problems related to the method of metronidazole testing. Even with agar dilution, the reproducibility is not 100% satisfactory. The recent discovery that mutations of the nitroreductase gene can result in resistance to metronidazole gives us some hope that in the near future molecular tests will be developed (10
In conclusion, culture of H. pylori proved to be an achievable goal even in a large randomized clinical trial and, as expected, susceptibility testing is important in explaining the results observed. Since the prevalence of primary resistance varies between different areas, it is of paramount importance to include culture and susceptibility testing in future trials. Even though the Etest has proved to be a very satisfactory test for clarithromycin, its use for metronidazole requires further standardization.