Tracking persistence of Helicobacter pylori in potable water biofilms using novel 16S rRNA peptide nucleic acid probes

A two stage chemostat system was used to study the pathogenic bacteria H. pylori association to drinking water biofilms. After allowing one week old biofilms to be grown on stainless steel coupons the system was inoculated with the pathogen that was detected using the recently established technique of 16S rRNA peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH). Results show that H. pylori can successfully incorporate within biofilms and its presence was detected for up to five days, either in the basal layer or inside typical biofilm structures such as stacks or fronds. The PNA FISH protocol appears to be a promising new technique for the in situ visualization of microorganisms in biofilms, especially because the hydrophobic nature of the PNA molecule allows a better diffusion through the constituents of the biofilm matrix allowing an improved discrimination of microorganisms inside these naturally occurring structures. A setback in the application of this methodology was the presence of autofluorescent microorganisms. This problem can be minimized by comparing the morphologic characteristics of these suspected false positives with typical H. pylori morphology. If questions subsist, the visualization of the biofilm under different filter blocks can also improve the degree of certainty in the identification, since the reporter probe has usually a very distinctive pattern of fluorescence when compared to the autofluorescent microorganisms