A novel isolation protocol and probe‐based <scp>RT</scp>‐<scp>PCR</scp> for diagnosis of gastric infections with the zoonotic pathogen <i>Helicobacter suis</i>

Abstract

Abstract Background Helicobacter suis is a very fastidious microorganism associated with gastritis, gastric ulcers, and mucosa‐associated lymphoid tissue lymphoma in humans. In vitro isolation of this agent from human patients has so far been unsuccessful. Materials and methods A probe‐based real‐time PCR ( RT ‐ PCR ) for the rapid detection of H. suis in gastric biopsies was developed. Secondly, a mouse‐passage‐based protocol was optimized for isolation of low numbers of viable H. suis bacteria. Mice were inoculated with different numbers of viable H. suis (10 2 ‐10 8 ) and kept for 4 weeks to allow multiplication of this pathogen. Results The probe‐based real‐time PCR ( RT ‐ PCR ) exhibited a high degree of diagnostic specificity and analytical sensitivity, high linear correlations ( r 2 between 0.995 and 0.999), and high amplification efficiencies (&gt;90%) for H. suis . No cross‐reactivity was detected with human, porcine, non‐human primate, and murine DNA nor with DNA from other bacteria including Helicobacter spp. and Campylobacter spp. H. suis was successfully re‐isolated from the stomach of mice inoculated with at least 10 4 viable H. suis , using a biphasic medium ( pH 5), consisting of Brucella agar with Brucella broth on top, both supplemented with vitox supplement, Campylobacter‐selective supplement, amphotericin (5 μg/mL), HCl (0.05%), fetal bovine serum (20%), and linezolid (5 μg/mL). Linezolid was necessary to inhibit proliferation of contaminants, including lactobacilli. Conclusion The methods described above can be implemented for detection or isolation of H. suis from human gastric biopsies.