The detection, characterization, and cultivation of nonculturable Helicobacter pylori

Abstract

Transmission of a bacterial pathogen from host to host is a complex process that may involve survival of the pathogen outside the host for considerable lengths of time. The bacterium Helicobacter pylori causes severe gastritis and gastric ulcers, and infection can increase the risk of stomach cancer. The main mode of transmission is believed to be the oral-oral route, however other routes of transmission such as drinking water have been implicated but have not been proven due to difficulty in culturing this organism. In this dissertation, the environmental transmission of H. pylori was investigated using several approaches. A primary objective of this study was to determine if H. pylori could be detected in an environmental reservoir readily consumed by humans, such as drinking water. H. pylori was detected by PCR but not culture in drinking water and biofilms that were obtained from groundwater and municipal systems. H. pylori contamination was sporadic and not associated with measured environmental factors, such as pH or temperature. Growth curve analysis of laboratory grown H. pylori showed that the cells exhibited a switch from a spiral to coccoid morphology as they aged or were exposed to stressful culture conditions. However, results showed that cell morphology was not indicative of culturability, with spiral forms dominant in early nonculturable samples. Microarray analysis of the transition to a nonculturable state showed that cells under oxygen stress quickly modified their transcriptional activity while the cells exposed to nutrient deprivation had nearly undetectable changes in transcriptional activities. Resuscitation of the stressed cells showed that type of stress and length of exposure affected regrowth of H. pylori. The oxygen stressed cells increased virulence factor transcription while nutrient deprived cells decreased transcription of the same factors. This observation led to the conclusion that oxygen stressed and nutrient deprived cells are metabolically active but react differently to in vitro culture conditions with starved cells likely undergoing nutrient shock. Collectively these data suggest that H. pylori can persist and are metabolically active under stressful conditions posed by the environmental mode of transmission.