Use of RNA-based genotypic approaches for quantification of viable but non-culturable Salmonella sp. in biosolids

Recent research efforts demonstrated an increase in fecal coliform counts in anaerobically digested biosolids after dewatering. Variety of bacteria enters viable but nonculturable (VNC) state as a survival response when exposed to environmental stress. Increase in coliform concentration after digestion and dewatering processes have been attributed to cells going into a viable but non-culturable state implying that traditional coliform enumeration methods are not sufficient to determine number of viable cells. Therefore, this research has been undertaken to develop a method for rapid and accurate quantification of viable but non-culturable pathogens in biosolids via monitoring and quantifying stress-related genes in Salmonella sp. The proposed method has the potential to allow accurate detection of pathogens in biosolids even when the cells are non-culturable due to environmental stress. The research proposed identification of stress related genes in Salmonella when cells are exposed to heat for different durations by using available Salmonella microarrays. In the context of this study the identified stress genes can be quantified through reverse transcription, complementary DNA (cDNA) synthesis, and amplification of cDNA via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Then quantity of mRNA can be correlated to cell viability and cells ability to grow, i.e., their culturability. Development of a novel approach to understand the pathogen behaviour in biosolids is key to ensure low public health risks from biosolids. Nevertheless, the initial results suggest that intact RNA isolation from biosolids is still challenging task.