Growth and survival of Helicobacter pylori in defined medium and susceptibility to Brij 78

Abstract

The gastrointestinal pathogen Helicobacter pylori requires supplementation with either fetal calf serum (FCS), bovine serum albumin (BSA), or (2,6-dimethyl)-beta-cyclodextrin (CD) for growth in a complex or defined medium. Because the availability of medium in which all components were chemically defined would facilitate metabolic studies of H. pylori, growth of the type strain, ATCC 43504, was compared in a defined medium with different growth additives. The dependency of H. pylori growth on FCS or BSA in a defined medium could partially be replaced by dependency on CD and cholesterol when the last two components were both added to the defined medium. Growth and cell yield were not affected by the addition of glucose, but the culture viability (numbers of CFU per milliliter was extended. Because therapeutic antifoams are used to relieve gastrointestinal symptoms we studied whether the unique susceptibility of H. pylori to the emulsifier polyoxyethylene-20-stearylether (Brij 78) was growth dependent or medium specific. The bactericidal activity exerted in buffer at pH 5 was independent of the preculture medium, and a 5-h exposure of the bacteria to 1.28 to 2.56 microg of Brij 78 per ml reduced the numbers of viable bacteria by >5 log10. The MICs (0.16 to 0.32 microg/ml) were lower than the corresponding minimal bactericidal concentrations in different growth media and were affected by FCS or BSA. In conclusion, CD plus cholesterol promotes the growth of H. pylori in a serum-free defined medium in which glucose enhances cell viability. The antibacterial activity exerted by Brij 78 is neither growth dependent nor medium specific.

FIG. 1

Growth of H. pylori ATCC 43504 in BB (A) and defined medium (B) supplemented with different additives: no additive (•), 10% FCS (▪), and 0.1% CD (▾). Growth was determined by estimation of viable counts (numbers of CFU per milliliter.

FIG. 2

Growth patterns of H. pylori ATCC 43504 in a defined medium with 0.2% glucose supplemented with different additives. ⧫, defined medium; ▪, defined medium plus 2.0 μg of cholesterol per ml; ▴, defined medium plus 0.1% CD; ▾, defined medium plus 2.0 μg of cholesterol per ml plus 0.1% CD; •, defined medium plus 0.5% BSA. Growth was determined by reading the optical density at 560 nm (OD₅₆₀).

FIG. 3

Effect of glucose on growth of H. pylori ATCC 43504 in a defined medium plus BSA. •, no glucose; ▪, 0.2% glucose; ▴, 1% glucose.

FIG. 4

Growth and viability of H. pylori ATCC 43504 in a defined medium plus BSA in the presence of different carbon sources: 0.2% glucose (⧫), 0.2% pyruvate (▪), 0.2% succinate (▴), 0.2% citrate (▾), or no additional carbon source (•).

FIG. 5

Viability after prolonged incubation of H. pylori ATCC 43504 in a defined medium plus BSA without glucose (░⃞) and with 0.2% glucose (□).