The T4SS-positive H. pylori strains NCTC11637 [1 ], P12 [46 (link)], and G27 [47 (link)] were grown from stocks stored at -80 °C (BHI medium containing 20% glycerol). The bacteria were grown on GC agar plates containing 10% horse-serum, 10 μg/mL vancomycin, and 4 μg/mL amphotericin. The microaerophilic growth conditions for the bacteria were provided in a 2.5-L anaerobic jar (Oxoid, Wesel, Germany) with a CampyGen pack [48 (link)]. The bacteria were grown for 48 h and subcultured on a fresh plate for 24 h before being harvested for infection experiments [49 (link)]. The isogenic mutant NCTC11637ΔcagA was grown on GC agar plates as above, but the plates further contained 8 μg/mL kanamycin.
Campygen pack
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United Kingdom
The CampyGen pack is a laboratory equipment product used to generate a microaerobic atmosphere suitable for the growth of Campylobacter species. The product provides a controlled and reliable method for creating the specific gas environment required for the cultivation of this bacterial genus.
Automatically generated - may contain errors
4 protocols using campygen pack
1
Culturing H. pylori Strains for Infection
2
Maintaining Campylobacter Cultures for Research
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A list of strains used in this study is provided in Table 1 . All Campylobacter stocks were maintained at −80 °C as 1 ml aliquots in 20 % (v/v) glycerol in Muller Hinton (Oxoid) broth. The Campylobacter strains were routinely sub-cultured on mCCDA (modified Campylobacter blood free selective agar base, Oxoid) grown for 48 h at 37 °C in a microaerobic atmosphere (10 % CO2, 5 % O2, 85 % N2 v/v) in gas jars containing a CampyGen® pack (Oxoid) or in an anaerobic workstation (Don Whitley scientific, UK).
3
Generating Isogenic H. pylori Mutants from Wild-type Strains
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Seven H. pylori wild-type isolates from different Asian countries are cagPAI- and CagA-positive (Table 1 ). Isogenic ΔcagA and ΔcagL knockout mutants were generated according to standard procedures [52 (link), 53 (link)]. All Helicobacters were raised on GC agar plates supplemented with nystatin (1 μg/mL), trimethoprim (5 μg/mL), vancomycin (10 μg/mL) and horse serum [54 (link), 55 (link)]. The antibiotics were purchased from Sigma-Aldrich (St. Louis, MO/USA). The agar plates were cultivated for 2 days at 37 °C in anaerobic jars containing CampyGen packs (Oxoid, Wesel/Germany) generating an atmosphere of 85 % N2, 10 % CO2 and 5 % O2 [56 (link)].
Characteristics of H. pylori strains and encoded CagA proteins used in this study
| H. pylori strain | Origin | Pathology | CagA EPIYA-type | Protein sequence | Reference |
|---|---|---|---|---|---|
| Ind69 | Indonesia | Gastric ulcer | ABD | LC007101 | [97 ] |
| FD453 | Malaysia | Functional dyspepsia | ABD | This study | [36 (link)] |
| Mand38 | Myanmar | Gastritis (Antral predominant mild gastritis) | ABD | LC007102 | This study |
| CH7 | China | Pre-cancer surveillance study | ABD | This study | This study |
| TN2-GF4 | Japan | Gastric ulcer | ABD | LC007103 | [98 (link)] |
| 2002-14 | Mexico | Dyspepsia | ABD | JN390453 | [99 (link)] |
| Shi470 | Peruvian Amazon | Not evaluated directly (Non-atrophic gastritis assumed) | ABD | YP_001910294.1 | [100 (link)] |
4
Isolation and Preservation of Anaerobic Bacteria
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Stool samples from the calves and dog were subcultured on Tryptone soy agar plates containing 5% sheep’s blood (Cleveland Scientific, Bath, OH, United States) and supplemented with antibiotics (TSA-CVA: 20 μg/mL cefoperazone, 10 μg/mL vancomycin and 2 μg/mL amphotericin B) upon arrival. All human clinical isolates were subcultured onto Bolton agar (Bolton Broth, Thermo Fisher Scientific, Pittsburgh, PA, United States) containing 1.5% Bacteriological Agar (Neogen, Lansing, MI, United States) upon arrival in the laboratory. All plates were placed in anaerobic jars containing CampyGen packs (Oxoid, Basingstoke, United Kingdom) and incubated for 48 h at 37°C. Isolates were selected and streaked again for growth in Tryptone Soy agar (TSA) and purification; bacterial growth was collected and stored at –80°C in TSB + 15% glycerol until further use.
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