Harboring of Helicobacter pylori by Amoebae in Water Systems

While person-to-person transmission is most common, a need exists to understand the fate of Helicobacter pylori in the most likely environmental vehicle: drinking water. Exposure to this pathogen through drinking water may be related to its ability to parasitize and replicate in amoebae. Helicobacter pylori expressing the green fluorescent protein (gfp-HP) were sub-cultured on Tryptic Soy Agar with 5% sheep's blood at 37oC under microaerophilic conditions for 48 h, which resulted in >90% viable cells as determined by the BacLight Live/DeadTM assay. Direct interference microscopy was used to confirm the replicative form of the bacterium (helical as opposed to the dormant coccoid shape). The ratio of gfp-HP to amoebae was 100:1, and the co-culture was exposed to free chlorine or gentamicin and washed to kill/remove extra-amoebic bacteria and to simulated drinking water conditions. The co-culture was followed over time using four methods to determine whether intra-amoebic bacteria are viable: 1) direct epifluorescent microscopy of the gfp-HP within the amoeba through use of Z-stacking image analyses to confirm their presence within the amoeba cell membrane; 2) quantitative spectrophotometry of the gfp emission spectra; 3) quantitative polymerase chain reaction (qPCR) assay using Helicobacter pylori-specific probes; and 4) aliquots of co-culture amoeba lysate subjected to conditions suitable for growing H. pylori. Results indicate that the concentration of H. pylori cells increased under the co-culture conditions. Replication and viability in the presence of commonly used disinfection methods is also being further assessed.