A novel isolation protocol and probe-based RT-PCR for diagnosis of gastric infections with the zoonotic pathogen Helicobacter suis

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 (10²-10⁸) 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 (r² 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 10⁴ 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.

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