**4.** *H. pylori* **susceptibility to antibiotics**

During the last decade, an increased number of *H. pylori* have become resistant to antibiotics, especially to clarithromycin and levofloxacin [29]. The resistance rates to metronidazole have always been more than 15% worldwide, but the increasing resistance rates to clarithromycin and levofloxacin in some areas have become higher than 10–15%. Thus, these antibiotics are not recommended for first-line therapy of *H. pylori* without prior susceptibility testing [21]. It is common to treat *H. pylori* infections without prior susceptibility testing, and different studies show a much lower resistance rate to clarithromycin in *H. pylori* from untreated patients than in *H. pylori* from previously treated patients [30–32]. It is therefore of the greatest importance to make susceptibility testing after the first treatment failure.

The susceptibility testing of *H. pylori* can be done by various methods. The most common are dilution methods, disk diffusion, and E-test.

The dilution method is regarded to be the golden standard for susceptibility testing. A twofold dilution row of the test antibiotic is made. A standard number of bacteria (McFarland 3) are added to each tube with antibiotics. The bacterial growth is inhibited by high concentrations of antibiotics. The first tube with bacterial growth is called the minimal inhibitory concentration (MIC). *H. pylori* should be grown for 48–72 hours under microaerobic conditions. It may be difficult to find a suitable media in which *H. pylori* grows fast enough, and the slightest contamination will grow faster than *H. pylori* and thereby spoil the susceptibility testing.

The disk diffusion test requires a small tablet of an antibiotic. The tablet is placed on the agar plate and is incubated for 3 days. After 3 days, there will be a zone around the tablet with no

**Figure 3.** Reading guide for E tests. (A) Colonies of a metronidazole-resistant subpopulation in the ellipse minimum inhibitory concentration (MIC) >32; (B) trailing of microcolonies at the end point MIC 0.5 μg/ml. Warburton-Timms and McNulty [85].

growth of *H. pylori*. This is the inhibition zone, and the diameter of the zone can be translated to an MIC value, which shows whether or not the bacteria are resistant to the antibiotic. To make the susceptibility testing of *H. pylori*, a McFarland 3.0 dilution of *H. pylori* and Mueller-Hinton agar plates with 10% blood or chocolate ager plates should be used and incubated in microaerobic conditions at 37° C.

The E-test is a stripe with a concentration gradient of an antibiotic. The stripe is placed on the agar plate and is incubated for 3 days. After 3 days, there will be a droplet shape around the stripe with no growth of *H. pylori* (**Figure 3**). That concentration where *H. pylori* grows close to stripe is the MIC value [33].
