A Novel Isolation Protocol and Probe‐based RT‐PCR for Diagnosis of Gastric Infections with the Zoonotic Pathogen Helicobacter Suis

BackgroundHelicobacter 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 methodsA 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 4weeks to allow multiplication of this pathogen.ResultsThe 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 (>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 (5g/mL), HCl (0.05%), fetal bovine serum (20%), and linezolid (5g/mL). Linezolid was necessary to inhibit proliferation of contaminants, including lactobacilli.ConclusionThe methods described above can be implemented for detection or isolation of H.suis from human gastric biopsies.