Abstract
Helicobacter pylori which infects more than 50% of the global population, has
been detected in water, but its transmission pathway remains unknown. Freeliving
amoebae (FLA) are ubiquitous pathogens commonly found in water. It
has been previously shown that FLA can act as hosts for some pathogens, protecting
them, and thus acting as Trojan Horses. Inside FLA, amoeba resisting
bacteria are more resistant to adverse conditions.
The aim of this work was to investigate the presence of H. pylori cells inside
amoebae in drinking and waste water samples from Eastern Spain.
Methods: Nineteen drinking and 31 waste water samples were filtered
through 3 lm nitrocellulose filters. Membranes were placed in Non-Nutrient-
Agar and the culture was maintained until amoebae were observed. Afterwards,
extra-amoebic bacteria were killed using sodium hypochlorite. Samples were
treated with propidium monoazide to eliminate exogenous DNA and dead cells.
DNA from protozoa was extracted and specific H. pylori qPCR was performed.
Results: DNA from H. pylori cells inside FLA were identified in 11 out of 50
water samples; 7 in waste water and 4 in drinking water. PMA treatment allowed
the detection of DNA from H. pylori cells only from the inside of live amoebae.
Conclusions: The combination of sodium hypochlorite treatment prior to
analysis with PMA-qPCR is a rapid and specific method to detect H. pylori cells
inside FLA. Thus, our results demonstrate that FLA may be a H. pylori reservoir
in water, showing that they could protect the bacterium from disinfection treatments
and harsh environmental conditions.
