Effects of ozone and chlorine disinfection on VBNC Helicobacter pylori by molecular techniques and FESEM images

ABSTRACT

Helicobacter pylori is a pathogen bacteria associated with chronic gastritis, peptic ulceration, and gastric carcinoma. H. pylori has a spiral morphology, which under certain conditions of stress becomes a coccoid form. This type of morphology has been linked to a viable but non-culturable (VBNC) state, which is thought to allow its persistence in the environment. Membrane damage in VBNC H. pylori in water as a mechanism for inactivation using ozone (O₃) and chlorine disinfection has not been reported in the literature. In this paper, disinfection assays with ozone and chlorine were conducted to evaluate their effects on VBNC H. pylori cells. The use of fluorescent dyes such as propidium monoazide (PMA) coupled with quantitative real-time polymerase chain reactions produced results necessary to assess the viability of the microorganism and demonstrate the effect of each disinfectant on the bacterial count. Applying ozone showed a 5-log bacterial reduction using a disinfectant concentration and exposure time (CT) of 4 mg min/L. Chlorine disinfection for the same 5-log reduction required a higher CT value. Field emission scanning electron microscope images of ozone-treated VBNC H. pylori also showed severe cell damage. The use of PMA revealed that chlorine produced physical damage in the membrane in addition to the known inhibiting effect on cell enzymatic processes. These findings are important for the detection and control of VBNC H. pylori cells in drinking water systems.

KEYWORDS:

• Chlorine
• FESEM
• ozone
• qPCR-PMA
• VBNC Helicobacter pylori

Acknowledgements

The authors appreciate the participation of Mr Ignacio Monje Ramirez for his assistance in testing ozone treatment. Also to Ms Berenit Mendoza Garfias for the support provided in the critical point drying of samples analyzed by FESEM.

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCiD

María Teresa Orta de Velásquez http://orcid.org/0000-0001-9936-1997

Isaura Yáñez Noguez http://orcid.org/0000-0003-1129-1790

Beatriz Casasola Rodríguez http://orcid.org/0000-0003-4671-5477

Priscila Ivette Román Román http://orcid.org/0000-0002-4653-3000

Additional information

Funding

This research was supported of the Consejo Nacional de Ciencia y Tecnologia (CONACYT). Projects PDCPN2014-01 number [247605].