Effect of Chlorine on Incorporation of Helicobacter pylori into Drinking Water Biofilms

The use of a specific peptide nucleic acid (PNA) probe demonstrated that Helicobacter pylori persisted inside biofilms exposed to low concentrations of chlorine (0.2 and 1.2 mg liter⁻¹) for at least 26 days, although no culturable cells were recovered. Coupled with data obtained using viability stains in pure culture, this result suggests that H. pylori can survive chlorination but remain undetectable by culture methods, which can be effectively replaced by PNA hybridization.Helicobacter pylori is a Gram-negative microorganism that colonizes the human stomach and can cause gastric ulcers that can degenerate into gastric carcinoma (5). The route of transmission for this pathogen is not well known, and even though culturable H. pylori has never been isolated from drinking water distribution systems (DWDS), molecular techniques such as PCR have detected the presence of H. pylori DNA in potable water (6, 15, 17), indicating that this environment could act as a reservoir for this bacterium.Chlorine is the most commonly used disinfectant worldwide to ensure the safe distribution of water to the consumer (19). Although studies conducted by Johnson et al. (14) and Baker et al. (4) have shown that H. pylori is inactivated by chlorine, their conclusions were based on the lack of recovery using standard culture plating methods which fail to consider cells that have entered a viable but nonculturable (VBNC) state. Recently, Moreno et al. (16) applied molecular techniques to demonstrate that H. pylori can survive in low concentrations of chlorine in a VBNC state. However, all these studies were performed with pure cultures using suspended cells, and until now, there have been no studies reporting on the effect of chlorination on H. pylori when associated with heterotrophic biofilms where, as is well known, microorganisms become more resistant to the biocide effect of chlorine (10).In a recent study, we demonstrated that H. pylori can be incorporated into drinking water biofilms and remain viable in the lower layers of these structures (11). It is therefore important to understand the ability of this pathogen to be incorporated and survive in heterotrophic biofilms formed in chlorinated waters. If this pathogen can remain viable under these conditions, it might therefore represent a risk to public health when released into the bulk fluid.The aim of this work was to study the effect of low concentrations of chlorine on H. pylori cells both when associated with heterotrophic biofilms and when suspended in pure culture, to assess whether the biofilm can provide protection against disinfection.