Detection of Helicobacter pylori in biofilms by real-time PCR.
Helicobacter pylori is a cause of peptic ulcer disease and a causative agent of gastric cancer. Currently, a possible waterborne route of transmission or a possible survival in drinking water biofilms is discussed. H. pylori, like many other bacterial strains, has the ability to enter the viable but nonculturable state (vbnc) in case of unfavorable conditions. Therefore it is necessary to develop new analysis tools for vbnc bacteria. We established a fast and reliable method to detect H. pylori in drinking water biofilms by quantitative real-time PCR which makes it redundant to use difficult cultivation methods for nonculturable bacteria. With this method it was possible to identify water biofilms as a niche for H. pylori. The real-time PCR analysis targets the ureA subunit of the Helicobacter pylori urea gene which showed high specificity and sensitivity. The quantitative real-time PCR was used to detect H. pylori in biofilms of different age, unspiked and spiked with predetermined levels of cells. The drinking water biofilms were generated in a silicone-tube model. The DNA-sequences for probe and primers showed no cross-homologies to other related bacteria and it was possible to detect less than 10 genomic units of H. pylori. This novel method is a useful tool for a fast screening of drinking water biofilms for H. pylori. The results suggest that drinking water biofilms may act as a reservoir for H. pylori which raises new concerns about the role of biofilms as vectors for pathogens like Helicobacter pylori.
Linke
S
S
Institute for Hygiene and Public Health, University of Bonn, Sigmund-Freud-Strasse 25, Klinikgelände 35, 53105 Bonn, Germany. Stefan.Linke@ukb.uni-bonn.de
Lenz
J
J
Gemein
S
S
Exner
M
M
Gebel
J
J
D001483:Base Sequence; D018441:Biofilms; D004269:DNA, Bacterial / Q000032:analysis; D004784:Environmental Monitoring / Q000379:methods*; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D014871:Water Microbiology; D014873:Water Pollutants; D014881:Water Supply
D004269:DNA, Bacterial; D014873:Water Pollutants