Helicobacter pylori Growth and Urease Detection in the Chemically Defined Medium Ham's F-12 Nutrient Mixture

Abstract

Obstacles continue to hinder in vitro studies of the gastric human pathogen Helicobacter pylori, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate that H. pylori grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only ϳ10 3 CFU/ml. All H. pylori strains (n ‫؍‬ 21), including fresh clinical isolates, grew in serum-free F-12. H. pylori grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of H. pylori in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), ␤-cyclodextrin (200 g/ml), or cholesterol (50 g/ml). H. pylori also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar, H. pylori only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 g/ml), ␤-cyclodextrin (200 g/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or ␤-cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar for H. pylori. Several lines of evidence suggested that BSA itself is not responsible for H. pylori growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of H. pylori using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding of H. pylori metabolism, virulence factors, and transmission, and result in improved recovery and identification of H. pylori from clinical specimens.

A supplemented soft agar chemotaxis assay demonstrates the Helicobacter pylori chemotactic response to zinc and nickel

“…Brucella broth supported some motility, but a large number of cells were observed to swim only when FBS was present. FBS is known to promote robust H. pylori growth (Testerman et al, 2001), but our data show that it also promotes motility. For this reason, we used FBScontaining medium (BB10) for all further analyses.…”

“…H. pylori requires zinc for growth in vitro (Testerman et al, 2001(Testerman et al, , 2006. We do not know whether zinc is limiting in the stomach, but studies have found that H. pylori infection lowers mucosal zinc concentrations (Sempértegui et al, 2007), possibly due to the presence of neutrophils, which produce the zinc-chelating agent calprotectin (Kehl-Fie & Skaar, 2010).…”

“…pylori does not require additional nickel for growth (Testerman et al, 2001(Testerman et al, , 2006, although it requires nickel as a cofactor for two critical enzymes: urease, which mediates pH homeostasis, and hydrogenase, which is used for hydrogen metabolism . The basal level of nickel in the medium is apparently sufficient for these enzymes, but it is not yet known whether in vivo nickel is limiting or toxic.…”

Reduced infectivity of waterborne viable but nonculturable Helicobacter pylori strain SS1 in mice

“…Briefly, pellets were re‐suspended in inoculating fluid IF‐0a GN/GP (1.2×), and then supplemented to a final concentration of 0.05% bovine serum albumin (BSA) and 1.25 mmol/L NaHCO 3 . ( H. pylori has been shown to use more carbon sources and grow successfully in media containing BSA,28, 29 so it was included to ensure better visualization of metabolic activity in the VBNC state.) Dye mix D (Biolog) was then added to achieve a final concentration of 0.01%; 100 μL of this solution was pipetted into each well of the PM1 plate, which was then incubated in microaerobic conditions for 48 hours.…”

Fate of Helicobacter pylori artificially inoculated in lettuce and carrot samples

“…This could be attributed partially to the high level of genetic diversity presented by H. pylori strains, which may cause fail of growth in currently available defined media (19).…”