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

Abstract

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.

Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria

“…RNA-based genotypic approaches targeting stress related genes have been made to identify VBNC cells of Salmonella spp. in biosolids qPCR (76). For the detection of V. cholerae in the VBNC state, 16S rRNA and mRNA for tuf, rpoS , and relA have been targeted (77).…”

Defining Established and Emerging Microbial Risks in the Aquatic Environment: Current Knowledge, Implications, and Outlooks

“…Since the introduction of the concept or sublethally injured or viable but nonculturable (VBNC) cells by Byrd and Colwell in the 1980's [1], there is increasing evidence for the existence of such a state in microbes, particularly in the aquatic environment that elicits a myriad of interrelated sub-lethal microbial stresses such as nutrient starvation and osmotic stress [2, 3] (Table 1). This is a cause for concern because of evidence that microbial pathogens in such a state may still retain their capacity to cause infections after ingestion by fish, animals, or by humans, despite their inability to grow under conditions employed in laboratory-based procedures for determining their presence in water [4].…”

Expression stability of 13 housekeeping genes during carbon starvation of Pseudomonas aeruginosa

“…RNAbased genotypic approaches targeting the detection of stress related genes in VBNC has been used in Salmonella spp. in biosolid qRT-PCR, for detecting the VBNC state of Vibrio cholera Escherichia coli, Pseudomonas putida and Legionella pneumophila (Buse et al, 2013;Dunaev et al, 2008;González-Escalona et al, 2006;Gunasekera et al, 2002;Vora et al, 2005;Yaron and Matthews, 2002). To facilitate those studies, several housekeeping genes have been identified for the opportunistic human pathogen P. aeruginosa under various experimental conditions.…”