Viability Determination of Helicobacter pylori Using Propidium Monoazide Quantitative PCR Gemma Agustı´, Francesc Codony, Mariana Fittipaldi, Ba ´ rbara Adrados and Jordi Morato ´ Laboratori de Microbiologia Sanita ` ria i Mediambiental (MSM Lab) – Aquasost, UNESCO Chair in Sustainability, Universitat Polite ` cnica de Catalunya, 08222 Terrassa, Barcelona, Spain Helicobacter pylori is a Gram-negative microaerobic curved rod bacterium. This bacterium causes gastric diseases and peptic ulcers and plays an important role in gastric cancer and lymphoma. For this reason, H. pylori has been classified by the World Health Organization as a class I carcinogen [1]. It has been estimated that more than half of the human population is currently infected with H. pylori, although most people never manifest symptoms of the infection [2]. H. pylori exists in a bacillary form (rod or spiral) in the natural habitat within the human host, but detri- mental environmental circumstances have been observed to cause a switch from bacillary to coccoid forms. Formation of nonculturable coccoid forms is a common feature among bacillary Gram-negative bacte- ria subjected to stress conditions, such as starvation, temperatures changes, and antibiotic treatment [3]. Some authors contend that coccoid form is a degen- erative form and others suggest that coccal shapes are viable but nonculturable forms. These two hypotheses are supported by diverse studies. Whether the H. pylori coccoid forms is degenerative, or viable but noncultur- able, remains open to question [3]. Several reports have argued that coccal shapes may constitute a resistance form under adverse environmen- tal conditions. These reports have demonstrated, in coccoid bacterial forms, the ability to synthesize DNA [4], the presence of intact cellular structures by electron microscopy [5,6], high enzymatic activity [7–9], and a high capacity to cause gastric inflammation in BALB ⁄ c A mice [10]. However, studies also exist that support the opposite hypothesis. Inhibition of protein synthesis, DNA degradation, and low RNA concentrations have been observed in coccoid forms [11]. Nonviability of the coccoid form was shown in vivo by the inability to Keywords Helicobacter, coccoid forms, cellular viability, qPCR, propidium monoazide. Reprint requests to: Francesc Codony, Laboratori de Microbiologia Sanita `ria i Mediambiental, (MSM Lab) – Aquasost, UNESCO Chair in Sustainability, Universitat Polite ` cnica de Catalunya, Violinista Vellsola ` 37, 08222 Terrassa, Barcelona, Spain. E-mail: codony@oo.upc.edu Abstract Background: While Helicobacter pylori exists in a bacillary form in both the natural habitat and the human host, detrimental environmental circum- stances have been observed to lead to the conversion of H. pylori from the bacillary to the coccoid form. However, the viability or nonviability of coccoid forms remains to be established in H. pylori. The aim of this study was to determine whether the quantitative PCR combined with propidium monoazide could be an alternative and good technique to determine H. pylori viability in environmental samples and, to contribute to under- standing of the role of the H. pylori forms. Materials and Methods: Viability, morphological distribution, and the number of live H. pylori cells were determined using a propidium mono- azide-based quantitative PCR method, at various time points. Results: Under adverse environmental conditions was observed the conver- sion of H. pylori from the bacillary to the coccoid form, and the decrease in amplification signal, in samples that were treated with propidium mono- azide, over the time. Conclusions: Incorporation of propidium monoazide indicates that there is an increase in H. pylori cells with the damaged membrane over the study, leading to the manifestation of cellular degeneration and death. Conse- quently, quantitative PCR combined with propidium monoazide contributes to our understanding of the role of H. pylori cells, under adverse environ- mental conditions. Helicobacter ISSN 1523-5378 ª 2010 Blackwell Publishing Ltd, Helicobacter 15: 473–476 473