Molecular detection of viable bacterial pathogens in water by ratiometric pre-RNA analysis

The use of PCR in detecting bacteria in clinical or environmental samples is limited in part by false positive detection (PCR can detect non-viable cells) and free DNA in environmental samples. Today, I happened to read a very interesting article on molecular detection of viable bacterial pathogens in water. The article instantly reminded me of a journal club presentation by my friend, Chhavi on detection of viable cells by PCR. So, I did some searches, and came across many articles on use of novel techniques to selectively detect live bacteria.
First, let me elaborate viability in terms of bacteria. Bacteria were traditionally considered viable when they could be cultured. However, today's viability concept is based on the presence of some form of metabolic activity, responsiveness, RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane (Meschke et al., 2010). The presence of an intact membrane has been the focus of recent approaches to limit detection of intact cells using ethidium monoazide or propidium monoazide. These are DNA intercalating dyes and they can selectively enter those cells that have compromised cell walls and membranes. Within these cells, they can covalently link with the DNA. This linked DNA then can not be amplified by PCR, thus, allowing selective amplification of DNA only from live cells.
Recently, Dr. John S. Meschke and his group (University of Washington, Seattle) have developed a new method for detection of viable bacterial pathogens in water. The article is published in the February, 2010 issue of applied and environmental microbiology.
Here is a brief summary of the article:
One approach to detect viable cells is to detect microbial RNA, which is less stable than DNA and as already quickly degrades after cell death. However, mRNA can be difficult to detect, while mature rRNA is stable within inactivated cells. The authors have suggested that microbial-rRNA precursors (pre-rRNA) could be an alternative RNA target. These pre-rRNA molecules have leader and tail sequences that are enzymatically removed during rRNA maturation. These sequences are also phylogenetically specific, so one can detect them in complex samples. It is assumed that once the growth of bacteria ceases, the rRNA processing in bacterial cells continues, while pre-rRNA synthesis stops and thus pre-rRNA conc. decreases. Therefore, the pre-rRNA concentrations could be indicative of the physiological activity of micro-organisms.
In the present work, the authors exploited the replenishment of pre-rRNA that occurs immediately upon nutritional stimulation of nutrient limited bacterial cells. Thus, species-specific pre-rRNA was measured in environmental samples after brief exposure to culture medium by qPCR. The values obtained were compared with those seen in un-exposed control samples. The values that exceed those of control indicate the presence of viable cells. The authors called this approach ratiometric pre-rRNA analysis (RPA) and tested it on rapidly growing Aeromonas hydrophila and slowly growing Mycobacterium avium.

Methodology and Results:

 To assess the time course of pre-rRNA replenishment upon nutrient stimulation: For this, early stationary phase A. hydrophila ATCC7966 cells were washed, re-suspended in autoclaved tap water (ATW) and incubated for seven days with aeration. Similarly, early stationary phase M. avium strain HMC02 cells were washed, re-suspended in ATW, and then incubated for 14 days. The purpose of these experiments was to create nutritionally deficient cells and to drain pre-rRNA pools in simulated water supply environments. To conduct RPA, these cultures were divided into two parts and centrifuged. One pellet was re-suspended in the culture medium (nutritional stimulation) and other in ATW (control). After various time periods of incubations, these cells were lysed, RNA was extracted and qPCR for pre-rRNA was performed from cDNA. The ratios of RT-qPCR values in stimulated and control samples were calculated following normalization to genomic DNA standard curves. It was observed that pre-rRNA stimulation was very rapid in both organisms. For A. hdrophila, 15 minutes and for M. avium, 4 hrs were adequate for near maximum stimulation.

 To assess the specificity of RPA for viable cells: For these experiments, sodium hypochlorite was used to generate suspension with various ratios of viable and inactivated cells. These cell suspensions were incubated for specific periods, plated on medium and post-treatment percent viability was determined by RPA. In addition, the authors also quantified the DNA concentration of the samples by qPCR with the same primers used for RPA. It was observed that samples with no detectable viable cells (0% viability, 0 CFU/ml) exhibited pre-RNA stimulation ratios of one or less. All other sample showed pre-rRNA stimulation ratios of more than or equal to 2. In contrast, qPCR detection of A. hydrophila genomic DNA was strongly positive in all samples. Also, the pre-rRNA stimulation ratios were significantly lower in samples with no detectable CFUs in comparison to samples with detectable CFUs. However, no such correlation of CFU with no. of genomic DNA copies obtained by qPCR was observed.


 Field test of RPA for A. hydrophila: Three fresh water samples and one salt water sample were collected from various sites in Seattle, WA. These water samples were concentrated by filtration, diluted two fold in nutrient broth (stimulated) and in ATW (control). After incubation, these were tested by RPA. Viable counts were also obtained by plating these samples onto ampicillin-dextrin agar with vancomycin. The freshwater sample yielded vales between 280 to 798 CFU/ml A. hydrophila and the RPA results ranged from 4.8 to 39.8. The salt water sample yielded 6CFU/ml, but no pre-rRNA. Therefore, the authors suggest that in its current form, RPA applied to natural samples has a detection limit between 6 to 280 CFU/ml.
In practical terms, RPA can be conducted by dividing a sample into two aliquots. One aliquot is made nutritionally stimulated while other is re-suspended in water. After stimulation for less than or equal to one generation time, species specific pre-rRNA levels can be determined by RT-qPCR and compared with un-stimulated sample. Ideally a threshold pre-stimulation rRNA ratio of one would indicate the presence of viable cells. At present RPA is not quantitative and optimization and standardization of diluted samples may give quantitative results.

Future applications and conclusions:
In the present work, the authors show that RPA can be used to specifically detect viable cells in environmental samples. The method was compared to traditional PCR and it was observed that it reduced the number of false positives obtained by amplification of dead cells. It may prove highly useful in food and water safety analysis.