Lyophilized H. pylori stocks (ATCC 43504, 43579, 49503, 51652, and 51653, Rockville, MD) were each reconstituted with 0.5 ml Brucella broth (Difco, Detroit, MI) with 0.1% cyclodextrin (BBCD) and transferred to 25 ml of BBCD in a 25 cm2 tissue culture flask. Similarly, 1.0 ml of a frozen 15% glycerol stock containing Sydney Strain I (University of Delaware) was thawed and added to 25 ml BBCD in a 25-cm2 tissue culture flask. Flasks were incubated at 37°C on a rocking platform (28 cycles per minute) under microaerophilic conditions using the BBL Campy Pack Plus System (Becton Dickinson, Cockeysville, MD). After three days, 5 ml of each culture was transferred to 100 ml BBCD in a 150 ml filter unit receiver flask (Nalgene, Rochester, NY) and incubated at 37°C with rocking as described above in the GasPak CO2 Jar System (Becton Dickinson, Cockeysville, MD). After four days, 6 drops of broth culture were used to inoculate 4 plates of Trypticase Soy Agar with 5% sheep blood (Becton Dickinson, Cockeysville, MD) for each strain. All plates were incubated inverted in a GasPak CO2 Jar System. Each remaining broth culture was harvested by centrifugation at 10,000 g for 10 minutes at 4°C. Pellets were resuspended in phosphate buffered saline (PBS) with 5 mM MgCl2 and washed a total of five times. After four days, the plate cultures were harvested by scraping the colonies from the agar, suspending them in PBS with 5 mM MgCl2, and washing as described above. The protein concentration of each suspension was determined using a Bradford microassay (BioRad, Richmond, CA).
Wet preparations and Gram-stained (Becton Dickinson, Cockeysville, MD) smears were prepared for all cultures at times of transfer and harvest. These were examined using a Zeiss Axioplan Photomicroscope with 63× and 100× objectives under brightfield, phase contrast, and differential interference contrast settings.
Harvested bacteria from broth and plate cultures were diluted to a concentration of 0.5 mg/ml in sample buffer containing 0.1 M dithiothreitol as the reducing agent. The samples were electrophoresed on 11% SDS polyacrylamide mini gels using a Hoeffer Mighty Small II electrophoresis unit (Pharmacia Biotech, Piscataway, NJ). Separated antigens were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) using a Nova blot semi-dry transfer system (Pharmacia Biotech, Piscataway, NJ). Membranes were blocked overnight at 4°C with 0.5% bovine serum albumin in PBS, dried, and stored desiccated until use.
Membranes were incubated with polyclonal rabbit anti H. pylori [diluted 1:1000 in blotting buffer (PBS with 1% nonfat dry milk)] or serum from a patient with H. pylori (diluted 1:500 in blotting buffer) for 1 hour at room temperature. Membranes were washed three times with PBS and then incubated with a 1:1000 dilution of biotinylated goat anti rabbit IgG (H + L) or a 1:1000 dilution of biotinylated goat anti human IgG (H + L) (Kirkegaard and Perry Labs, Gaithersburg, MD). Membranes were washed with PBS as described above and incubated with a 1:1000 dilution of peroxidase conjugated streptavidin (Kirkegaard and Perry Labs, Gaithersburg, MD) for 1 hour at room temperature. Membranes were washed again as described and incubated with 4-chloro-1-naphthol substrate (7.8 mM 4-chloro-1-naphthol diluted 1:2 with 1:1500 30% H2O2 in citrate phosphate buffer, pH 4.0). Membranes were washed with distilled water, air dried, and evaluated for banding patterns.
DNA was isolated from harvested cultures of H. pylori from either plate or broth medium. Briefly, 7.5 volumes of 6 M guanidine HCl/0.1 M sodium acetate, pH 5.5 was added to 100 μl aliquots of harvested bacteria. Preparations were mixed by inversion and placed on an orbital shaker (Lab-Line, Melrose Park, IL) at 150 rpm for 60 minutes at room temperature. Samples were centrifuged at 18,000 × g for 30 minutes at 4°C. Each supernatant was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1), followed by addition of 0.1 volume 3 M sodium acetate and 2.5 volumes 95% ethyl alcohol to precipitate the DNA. Samples were placed at -20°C for a minimum of 60 minutes, followed by centrifugation as described above. Pellets were washed with 70% ethyl alcohol, dried, and resuspended in sterile Tris-EDTA (TE) buffer.
PCR and urease gene fingerprinting analysis
A 2.4 kilobase (kb) product encompassing the H. pylori urease A and urease B (ureA and ureB) structural genes was amplified as described by Foxall  using primers HpR1 (5' AGGAGAATGAGATGA 3') and HpR2 (5' ACTTTATTGGCTGGT 3'). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Each reaction contained 100 nanograms (ng) H. pylori genomic DNA in a 50 μl reaction volume (2.0 ng/μl) unless otherwise noted. Amplification was carried out in a Perkin Elmer 9600 thermocycler (Foster City, CA). Reaction parameters were as follows: denaturation for 5 minutes at 95°C, followed by 34 cycles of 94°C for 1 minute, 53°C for 1 minute, and 72°C for 3 minutes. This was followed by 1 cycle of 94°C for 1 minute, 53°C for 1 minute, and 72°C for 5 minutes. Following amplification, 20 μl samples of each reaction were mixed with 5 μl gel loading solution (Sigma Biochemical, St. Louis, MO) and analyzed on 0.7% Seakem GTG agarose gels (FMC Bioproducts, Rockland, ME) to verify the amplification of the 2.4 kb product. Amplified products were ethanol precipitated and resuspended in sterile Tris-EDTA buffer. Each DNA sample was incubated with 20 U of restriction enzyme Hae III (Promega, Madison, WI) in the appropriate buffer overnight at 37°C. Individual restriction patterns for each strain were analyzed by electrophoresis of each digested sample on 4.0% Nusieve 3:1 agarose (FMC Bioproducts, Rockland, ME) gels.