Gene Expression Monitoring for Detection and Quantification of Viable but Non-Culturable Pathogens in Biosolids

Despite stringent monitoring, an increasing number of studies have recently showed evidence of pathogen survival in stabilized biosolids. The magnitude of such pathogen reactivation exceeded the current regulatory limits and it seems that the phenomenon is not limited to a few underperforming facilities. The viable but non-culturable (VNC) state of pathogens has been proposed within the last few decades as a survival mechanism for certain prokaryotic microorganisms, including pathogens, when they encounter unfavorable environmental conditions (Koch, A, 1971; Oliver, J.D., 1993). Several of the recent studies attributed the regrowth of indicator organisms in biosolids to the possible VNC state that microorganisms assume under unfavorable conditions during stabilization or exposure to chemical disinfectants (Higgins et al., 2007; Iranpour and Cox, 2006; Jolis, 2006; Manios, et al., 2006; Qi et al., 2007). The objective of this research is to review the current literature on pathogen regrowth in biosolids and to investigate the relationship between the expression of stress-related genes in Salmonella and cell viability. The study is based on the fact that the quantity of messenger ribonucleic acid (mRNA) in living organisms is proportional to their metabolic activities; i.e., nutritional state, and therefore the mRNA is expected to decrease significantly when organisms are under stress. A list of shock-response and housekeeping genes for Salmonella has been identified by a literature review. Several different protocols and readily available kits for the extraction of mRNA from the thermally treated biosolids have been studied for the best extraction yield. The next step of the research is to expose biosolids samples to heat stress and monitor changes in mRNA concentrations as a function of time. Detection of mRNA will be accomplished through first reverse transcription of mRNA pieces into complementary DNA (cDNA) and amplification of cDNA through q-PCR. Finally, the quantity of genes expressed will be correlated to cell viability by determining the number of cells that maintained cell integrity and ability to grow. The preliminary results suggest that gene expression has the potential to provide accurate and quantitative estimates of viable cells in biosolids. However, extraction of clean and intact mRNA from a complex matrix such as biosolids remains a challenging step for routine use of this novel approach.