Abstract
Metronidazole and tetracycline E tests were compared to an agar dilution method for the antimicrobial susceptibility testing of Helicobacter pylori. Sixteen strains were tested by using tetrazolium egg yolk (TEY) agar. The characteristic E test inhibition ellipse was clearer on TEY agar than on standard blood agar and gave results comparable to those of the agar dilution test. The use of TEY medium is preferable to that of blood agar medium in E test MIC determinations for H. pylori.
The gastric pathogen Helicobacter pylori is the major etiological agent of chronic gastritis and peptic ulcers and a major risk factor for gastric cancer (2, 7). Eradication of H. pylori prevents peptic ulcer recurrence and may also decrease the prevalence of gastric cancer in high-risk populations (2–4). Though metronidazole is often used to treat H. pylori in the United States, resistance to metronidazole is common in developing countries (8), leading to very frequent failure of metronidazole-based anti-H. pylori therapy (6). Increasing resistance to several other antimicrobial agents, including clarithromycin, has focused new attention on the need for reliable methods for determining drug susceptibility (1, 9). The current standard method uses agar dilution to determine the MIC of an antimicrobial agent. However, this technique is time consuming and tedious.
The Epsilometer test (E test; AB Biodisk, Solna, Sweden) is simple to use and yields MICs that agree well with those obtained by the “gold standard” agar dilution methods and that are relatively unaffected by variations in inoculum size. The E test is becoming increasingly popular for the determination of MICs for H. pylori, usually by using one of the many blood agar-based media, although the medium, inoculum size, and growth conditions have not been standardized. Our previous results showed that tetrazolium egg yolk (TEY) agar is extremely convenient for determining the MICs of metronidazole and other antimicrobial agents by standard agar dilution methods (9). Egg yolk emulsion, which forms the base of egg yolk agar, is available commercially in the United States (Oxoid USA Inc., Columbia, Md.). TEY agar contains an oxidation-reduction indicator (tetrazolium red) which changes from yellow to red when bacterial growth occurs: even the very small red H. pylori colonies are easily visible, and confluent growth is very obvious against the yellow background of the agar (9). In contrast, on blood agar, the growth or elliptical zone of H. pylori is hard to see, making the endpoint or elliptical zone difficult to establish with certainty.
The purpose of this study is to test the validity of using TEY agar for E testing. We have compared E test MIC determinations with those obtained in parallel using our miniwell MIC technique. The E test strips for metronidazole and tetracycline were used as recommended by the manufacturer. MICs were determined in the miniplate format in TEY agar as described previously (9).
Sixteen different H. pylori isolates were revived from cryoconserved strains that were grown on blood agar (brucella agar with 5% sheep blood, amphotericin [6 μg/ml], and Skirrow’s antibiotics [10 μg of vancomycin, 5 μg of trimethoprim, 2.5 IU of polymyxin B per ml]). Colonies were then picked and grown on a standard blood agar plate without antimicrobial agents. The strains were adjusted to a density of 0.5 to 1 McFarland standard (1.5 × 108 to 3 × 108 CFU), 100 μl was spread on 90-mm-diameter TEY agar petri plates, and 7 μl was inoculated into the antibiotic-containing TEY agar in each well of standard 24-well tissue culture plates (9). E test strips were placed on the plates as soon as the inoculum was absorbed into the agar. The plates were incubated at 37°C under microaerophilic conditions (5% O2, 10% CO2, and 85% N) (9). Three days later the MIC was read in both the E strip and the miniwell plates. The MIC was defined as the lowest concentration of each antibiotic resulting in complete inhibition, i.e., no colonies (5). The two tests were considered to be in agreement when their MICs were within 2 dilutions of each other and remained within the same category of resistant or susceptible (10).
The E test was also quite reproducible. The coefficient of agreement when the test was done in duplicate was 86% for metronidazole (n = 15 strains) and 93% for tetracycline (n = 14 strains).
The E test was also extremely easy to read, since there was a clearly defined elliptical inhibitory zone against the yellow background. There was 100% agreement between the E test and the miniwell format for the MICs of both metronidazole and tetracycline by the 2-dilution definition. In 8 of 16 and 13 of 16 strains tested for metronidazole and tetracycline susceptibility, respectively, there was a difference of 1 dilution between MICs determined by the two methods. Occasionally a H. pylori strain that was resistant to metronidazole produced an unclear zone in the E test due to the presence of very small colonies in the inhibitory zone. The correlation between the results of the two methods assessed for metronidazole and tetracycline is shown in the Table 1.
TABLE 1.
Comparison of MICs for H. pylori isolates obtained by agar dilution in miniwell format and by E test in petri dishes
| Isolate no. | Metronidazole MIC (μg/ml)
|
Tetracycline MIC (μg/ml)
|
||
|---|---|---|---|---|
| E test | Miniwell | E test | Miniwell | |
| 1 | 32 | 64 | 0.5 | 1 |
| 2 | 6 | 8 | 0.125 | 0.5 |
| 3 | 0.75 | 2 | 0.25 | 1 |
| 4 | 32 | 32 | 0.25 | 1 |
| 5 | 32 | 16 | 0.016 | 0.06 |
| 6 | 0.094 | 0.25 | 0.064 | 0.06 |
| 7 | 0.064 | 0.5 | 0.125 | 0.125 |
| 8 | 0.25 | 1 | 0.064 | 0.125 |
| 9 | 0.25 | 1 | 0.125 | 0.5 |
| 10 | 32 | 16 | 0.25 | 0.5 |
| 11 | 32 | 64 | 0.19 | 0.25 |
| 12 | 0.5 | 0.5 | 0.19 | 0.06 |
| 13 | 3 | 2 | 0.047 | 0.125 |
| 14 | 32 | 64 | 0.016 | 0.125 |
| 15 | 1.5 | 1 | 0.5 | 0.25 |
| 16 | 2 | 2 | 0.25 | 0.25 |
In general, MIC results in the miniwell format were slightly higher than those obtained with the E test for the two drugs. Our results validate the use of the E test on TEY agar to determine the MIC of metronidazole or tetracycline appropriate for testing strains of H. pylori. The test was reproducible and accurate. The TEY agar produced a clear and easily readable inhibitory zone, and there was good agreement with the MICs given by the agar dilution technique. E test zones were generally clear, and MIC endpoints were easily read. Some resistant strains tested against metronidazole had an elliptical zone of inhibition that was not smooth at the E strip intersection. The highest drug concentration recorded on the E strip for metronidazole was 32 μg/ml, but in Peru we have found H. pylori strains that are resistant to up to 128 μg of metronidazole/ml. It is doubtful that this difference is of clinical significance. A Belgian study (1) also compared the E test and the agar dilution technique for 12 different antibiotics, including metronidazole and tetracycline. Results rarely differed by more than 2 dilution steps, and 44% of their strains were resistant to metronidazole, thus confirming the report of 50% in our previous study (9).
In this study we found three H. pylori strains for which the tetracycline MIC was 1 μg/ml in the miniwell determination. Borderline tetracycline-resistant strains such as these will bear watching to determine if tetracycline-resistant strains develop in the future. In many midlevel developing countries like Peru, where cost and simplicity are important and where metronidazole and perhaps clarithromycin resistance are common, the E test performed on TEY agar provides a cheap and simple method for the determination of antimicrobial resistance in H. pylori strains.
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