OPTIMISATION OF GROWTH OF HELICOBACTER PYLORI IN LIQUID CULTURE

Abstract

Helicobacter pylori is a microaerophilic, glucose non-fermenting bacterium found closely attached to gastric epithelial cells beneath a protective layer of mucus in the human stomach. The bacterium has been associated with active chronic gastritis and peptic ulcer disease. The ability to obtain a better growth yield of this bacterium would benefit medical investigations into H. pylori by consistently yielding larger quantities of cells for study. The objective of this project is to study the conditions for growth of H. pylori with respect to both medium and gas conditions in shake flask and fermenter cultures, and identify the environmental and process conditions that yield higher viable cell densities. The liquid medium recommended at the start of this project for culture of H. pylori consisted of brain-heart infusion broth (a mixture of brain-heart solids, meat peptone and casein hydrolysate with glucose, sodium chloride and di-sodium hydrogen phosphate) supplemented with 4 g/L yeast extract and 10% horse serum. The maximum viable count and OD of the cultures was not changed when the yeast extract and horse serum concentrations were reduced by half, and when brain-heart solids and meat peptone were removed from the medium. Our studies showed that carbon dioxide (CO2) and oxygen (O2) affected growth of H. pylori in shake flask cultures. H. pylori was able to grow in flasks containing air only, provided that the flasks were sealed so CO2 produced by the bacteria could accumulate. The maximum viable count was the same in flasks containing 5% or 10% O2, and less in flasks containing 21 % O2. We also attempted to identify the cause for the cessation of growth. A specific, inhibitory metabolite was not identified, although the metabolites ammonia, acetate and carbon dioxide were considered. Perfusion experiments, where the spent medium was removed by a cross-flow filter and replaced with fresh medium, yielded higher cell densities than in shake flasks and batch fermentations. The maximum cell density was 2x109 cfu/mL when the cultures were perfused, compared with cell densities of 9x108 cfu/mL achieved in shake flask cultures and batch fermentations. The higher cell density in perfused cultures seems to indicate that the cause of cessation of growth is either nutrient-limitation or product-inhibition.