DVC-FISH and PMA-qPCR techniques to assess the survival of Helicobacter pylori inside Acanthamoeba castellanii.
Free-living amoebae (FLA) are ubiquitous microorganisms commonly found in water. They can act as Trojan Horses for some amoeba-resistant bacteria (ARB). Helicobacter pylori is a pathogenic bacteria, suggested to be transmitted through water, which could belong to the ARB group. In this work, a co-culture assay of H. pylori and Acanthamoeba castellanii, one of the most common FLA, was carried out to identify the presence and survival of viable and potentially infective forms of the bacteria internalized by the amoeba. Molecular techniques including FISH, DVC-FISH, qPCR and PMA-qPCR were used to detect the presence of internalized and viable H. pylori. After 24 h in co-culture and disinfection treatment to kill extra-amoebic bacteria, viable H. pylori cells were observed inside A. castellanii. When PMA-qPCR was applied to the co-culture samples, only DNA from internalized H. pylori cells was detected, whereas qPCR amplified total DNA from the sample. By the combined DVC-FISH method, the viability of H. pylori cells in A. castellanii was observed. Both specific techniques provided evidence, for the first time, that the pathogen is able to survive chlorination treatment in occurrence with A. castellanii and could be very useful methods for performing further studies in environmental samples.
Research Institute of Water and Environmental Ingeneering (IIAMA), Polytechnic University of Valencia, 46022 Valencia, Spain.
Laura
L
Moreno-Mesonero
Research Institute of Water and Environmental Ingeneering (IIAMA), Polytechnic University of Valencia, 46022 Valencia, Spain. Electronic address: ymoren@upv.es.
Yolanda
Y
Moreno
Research Institute of Water and Environmental Ingeneering (IIAMA), Polytechnic University of Valencia, 46022 Valencia, Spain.
José Luis
JL
Alonso
Biotechnology Department, Polytechnic University of Valencia, 46022 Valencia, Spain.
M Antonia
MA
Ferrús
D048248:Acanthamoeba castellanii / Q000382:microbiology*; D054879:Halogenation; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D054884:Host-Pathogen Interactions; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D050296:Microbial Viability; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D014871:Water Microbiology
Acanthamoeba castellanii; Co-culture; DVC-FISH; H. pylori; PMA-qPCR