Optimizing the growth of stressed Helicobacter pylori

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and is responsible for causing gastric ulcers. H. pylori is known to become stressed and nonculturable after exposure to unfavorable conditions. In this study, we enhanced previously published resuscitation procedures, characterized conditions under which stressed H. pylori can be recovered, and formulated a selective and differential resuscitation medium. Results showed that a specialized broth supplemented with trace minerals and lysed human erythrocytes and serum is required for the recovery of nonculturable H. pylori. The type of stress was an important factor in the efficacy of resuscitation, with cells exposed to atmospheric oxygen more readily resuscitated than nutrient deprived cells. After resuscitation, culturable cells were recovered from previously nonculturable oxygen stressed cells (24 and 72 h of exposure) and nonculturable nutrient deprived cells (24 h of exposure). The length of time the cells were exposed to the stress was also an important factor in the recovery of stressed H. pylori. RNA levels were quantified and transcription of the cell division related gene, cdrA (HP0066), was assessed by qRT-PCR. The low levels of RNA detected in stressed cells, after resuscitation, support the idea that a small population of viable cells may be responsible for the colonies recovered on solid agar. The modification of the resuscitation broth into a selective and differential slant culture medium also allowed the recovery of stressed H. pylori. The methods presented here highlight the benefits and limitations of using human blood products for recovering nonculturable H. pylori.