Although the route of transmission of Helicobacter pylori remains unknown, drinking water has been considered a possible transmission vector. It has been shown previously that, in water, biofilms are a protective niche for several pathogens, protecting them from stressful conditions, such as low carbon concentration, shear stress, and less-than-optimal temperatures. In this work, the influence of these three parameters on the persistence and cultivability of H. pylori in drinking-water biofilms was studied. Autochthonous biofilm consortia were formed in a two-stage chemostat system and then inoculated with the pathogen. Total numbers of H. pylori cells were determined by microscopy using a specific H. pylori 16S rRNA peptide nucleic acid probe, whereas cultivable cells were assessed by standard plating onto selective H. pylori medium. Cultivable H. pylori could not be detected at any time point, but the ability of H. pylori cells to incorporate, undergo morphological transformations, persist, and even agglomerate in biofilms for at least 31 days without a noticeable decrease in the total cell number (on average, the concentration was between 1.54 ؋ 10 6 and 2.25 ؋ 10 6 cells cm ؊2 ) or in the intracellular rRNA content may indicate that the loss of cultivability was due to entry into a viable but noncultivable state. Unlike previous results obtained for pure-culture H. pylori biofilms, shear stress did not negatively influence the numbers of H. pylori cells attached, suggesting that the autochthonous aquatic bacteria have an important role in retaining this pathogen in the sessile state, possibly by providing suitable microaerophilic environments or linking biomolecules to which the pathogen adheres. Therefore, biofilms appear to provide not only a safe haven for H. pylori but also a concentration mechanism so that subsequent sloughing releases a concentrated bolus of cells that might be infectious and that could escape routine grab sample microbiological analyses and be a cause of concern for public health.Helicobacter pylori is one of the most prevalent pathogens in humans, especially in developing countries, where the incidence can be up to 90% of the population (16). Even though most individuals that are infected by this pathogen are asymptomatic, it is now well established that H. pylori infection can lead to the development of peptic and duodenal ulcer disease and gastric mucosa-associated lymphoid tissue lymphoma (8).The route of transmission of this pathogen is still unknown. Person-to-person transmission seems most likely as the only place where H. pylori has been systematically isolated is the human gastrointestinal tract (3). However, some authors have suggested that water, food, and animals can also be transmission vectors (3,7,10,19,28,37,38). The greatest obstacle to proving that water is a transmission route is the fact that H. pylori has never been cultured from drinking-water distribution systems (DWDS) using standard cultivation techniques (3, 18). Whether this is due to the fastidious nature o...