Development of a rapid method to detect and to monitor bacteriophage amplification in contact with viable but non-culturable bacteria

Abstract

We propose in this study to develop a rapid, reliable, and non-culture method to detect and estimate bacteriophage (phage) titre as an alternative to the routine use of the double agar overlay assay (DLA). The present method is based on the analysis of nanoparticle (NPs) dispersion/aggregation dynamic in interaction with the phage. Titanium dioxide nanoparticles (TiO₂-NPs) were used as nanosensors to detect and monitor virions' titres in aqueous samples. Dispersion stability of TiO₂-NPs in aqueous suspension was investigated using a UV-Visible spectrophotometer. The comparison of NP spectral profiles with and without phage elucidated the impact of phage's titre on NP dispersion/aggregation behaviour in an aqueous solution. Indeed, the increase of nanoparticle dispersion stability is correlated with the increase of phage titre. Thus, based on this result, the phage was considered as a bio-dispersant agent. The determination of area under spectral profiles limiting the UV region [200-400 nm] was allowed to quantify, and compare the NPs bio-dispersion rate, in relation with added phage at different titres. In this study, this method was applied to monitor the phage amplification cycle for the detection of bacteria in viable but non-culturable (VBNC) state after water treatment by photocatalysis. The analysis of NP bio-dispersion rate shows an increase of TiO₂-NP dispersion stability correlated with an increase of free phage titration, mainly after the entry of target bacteria in VBNC state underestimated using a conventional method. Thus, this method could allow the establishment of new recommendations of wastewater treatment and assessment.

Keywords: NP dispersion rate; Nanoparticles; UV-vis spectrum; VBNC bacteria; phage.