VIABILITY DETECTION AND QUANTIFICATION ASSAY OF WATERBORNE PATHOGENS BY ENRICHMENT

Abstract

A process for detecting viable waterborne pathogens in a water sample, includes enriching a concentrate of bacteria from the sample and taking a first DNA extract at time T.sub.0 and a second DNA extract at time T.sub.2 after an incubation period. Real-time polymerase chain reaction performed on the DNA extracts yields respective cycle threshold values Ct. The change in Ct provides an indication of the targeted pathogen in the sample. An order of magnitude quantification of the sample can also be performed using a serial dilution technique on the T.sub.0 DNA extract. The process has particular application for detecting Legionella, including viable but not culturable cells.

Claims

1. A process for detecting presence of viable targeted waterborne pathogens comprising: (A) obtaining a liquid sample; (B) collecting a concentrate of bacterial cells from the liquid sample; (C) enriching the concentrate of bacterial cells to produce an enriched concentrate of bacterial cells; (D) extracting a T.sub.0 DNA extract from the enriched concentrate of bacterial cells at time T.sub.0; (E) storing the T.sub.0 DNA extract; (F) incubating the enriched concentrate of bacterial cells; (G) extracting a T.sub.2 DNA extract from the incubated enriched concentrate of bacterial cells at time T.sub.2; (H) analyzing the T.sub.0 DNA extract with real-time Polymerase Chain Reaction (PCR) to determine a cycle threshold Ct value for the T.sub.0 DNA extract; (I) analyzing the T.sub.2 DNA extract with real-time Polymerase Chain Reaction (PCR) to determine a cycle threshold Ct value for the T.sub.2 DNA extract; and (J) analyzing a difference in Ct value (Ct) between the T.sub.0 DNA extract and the T.sub.2 DNA extract to determine a qualitative assessment of the presence of viable waterborne pathogens in the liquid sample.

2. The process of claim 1 wherein the qualitative assessment indicates the presence of targeted waterborne pathogens in the liquid sample including viable but not culturable cells of the targeted waterborne pathogen.

3. The process of claim 1 wherein the qualitative assessment comprises determining if the Ct is greater than a cut-off value.

4. The process of claim 3 wherein the cut-off value is Ct is greater than approximately 1.5.

5. The process according to claim 1 comprising: (A) obtaining the liquid sample from a water source; and (B) neutralizing residual oxidants and antimicrobials within the liquid sample to obtain a substantially oxidant-free antimicrobial sample.

6. The process according to claim 5, wherein the sample is neutralized using a reducing agent.

7. The process according to claim 6 wherein the reducing agent comprises at least one of sodium thiosulfate (Na.sub.2S.sub.2O.sub.3) or sodium bisulfite.

8. The process of claim 1 wherein collecting the concentrate of bacterial cells comprises performing a filter concentration on the liquid sample.

9. The process of claim 8 wherein the filter concentration comprises using a membrane in a filtration unit, aseptically removing the membrane from the filtration unit and providing the membrane into an enrichment broth.

10. The process according to claim 9, wherein the membrane is a polycarbonate membrane.

11. The process according to claim 10, wherein the polycarbonate membrane is a track-etched membrane.

12. The process according to claim 11, wherein the polycarbonate membrane has a pore size of less than 0.25 m.

13. The process according to claim 5, wherein the polycarbonate membrane has a diameter of about 47 mm or larger.

14. The process according to claim 1, wherein the concentrate of bacterial cells is enriched in a solution of growth medium.

15. The process according to claim 14, wherein the growth medium is a Buffered Yeast Extract media comprising Vancomycin, Polymyxin, Cycloheximide, Glycine, Iron pyrophosphate, L-cysteine, and Bovine serum albumin.

16. The process according to claim 1, wherein the T.sub.0 DNA extract is stored at about 20 C.

17. The process according to claim 1, wherein the concentrate of bacterial cells is incubated at a temperature from 30 C. to 37 C.

18. The process according to claim 1, wherein the concentrate of bacterial cells is shaken at 50 rpm during the incubation period.

19. The process according to claim 1, when the Ct value of T.sub.2 DNA extract is equal to or greater than the Ct value of T.sub.0 DNA extract, the sample is assessed to not contain viable targeted waterborne pathogens.

20. The process according to claim 1, when the Ct value of T.sub.2 DNA extract is less than the Ct value of T.sub.0 DNA extract, the sample is assessed to contain viable targeted waterborne pathogens.

21. The process according to claim 1 comprising: (A) diluting the T.sub.0 DNA extract; (B) detecting PCR amplification by performing real-time PCR on the T.sub.0 DNA extract dilution; (C) repeating the diluting and detecting until no PCR amplification is detected; (D) wherein the highest dilution of the T.sub.0 DNA extract at which PCR amplification is detected provides an order of magnitude quantification of the viable targeted waterborne pathogen in the liquid sample.

22. The process according to claim 21, when only the T.sub.0 DNA extract shows PCR amplification, the quantification of the liquid sample is 1 Colony Forming Units (CFU)/ml.

23. The process according to claim 21, wherein the dilution is a serial dilution of 10-fold to 100-fold.

24. The process according to claim 23, wherein when only both the T.sub.0 DNA extract and a 1:10 dilution of the T.sub.0 DNA extract show PCR amplification, the quantification X of the liquid sample is 1<X10 CFU/ml.

25. The process according to claim 23, wherein when each of the T.sub.0 DNA extract, the 1:10 dilution of the T.sub.0 DNA, and a 1:100 dilution of the T.sub.0 DNA show PCR amplification, the quantification X of the liquid sample is 10<X100 CFU/ml.

26. The process according to claim 1, wherein the targeted pathogen is Legionella.

27. A process for detecting presence of viable targeted waterborne pathogens comprising: (A) step for obtaining a liquid sample; (B) step for collecting a concentrate of bacterial cells from the liquid sample; (C) step for enriching the concentrate of bacterial cells to produce an enriched concentrate of bacterial cells; (D) step for extracting a T.sub.0 DNA extract from the enriched concentrate of bacterial cells at time T.sub.0; (E) step for storing the T.sub.0 DNA extract; (F) step for incubating the enriched concentrate of bacterial cells; (G) step for extracting a T.sub.2 DNA extract from the incubated enriched concentrate of bacterial cells at time T.sub.2; (H) step for analyzing the T.sub.0 DNA extract with real-time Polymerase Chain Reaction (PCR) to determine a cycle threshold Ct value for the T.sub.0 DNA extract; (I) step for analyzing the T.sub.2 DNA extract with real-time Polymerase Chain Reaction (PCR) to determine a cycle threshold Ct value for the T.sub.2 DNA extract; and (J) step for analyzing a difference in Ct value (Ct) between the T.sub.0 DNA extract and the T.sub.2 DNA extract to determine a qualitative assessment of the presence of viable waterborne pathogens in the liquid sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 illustrates flowchart of a process for determining a presence of target pathogens in a liquid sample;

[0022] FIG. 2 is substantially a schematic process of the flowchart of FIG. 1;

[0023] FIG. 3 is substantially a flowchart of an order of magnitude quantification method based on serial dilution;

[0024] FIG. 4 substantially shows real-time PCR amplification curves for Legionella pneumophila (ATCC 33823) analyzed with PVT-VIABLE;

[0025] FIG. 5 substantially shows spread plates for Legionella pneumophila (ATCC 33823) analyzed with PVT-VIABLE;

[0026] FIG. 6 substantially shows real-time PCR amplification curves for an environmental water sample analyzed with PVT-VIABLE;

[0027] FIG. 7 substantially shows spread plate counts for an environmental sample analyzed with PVT-VIABLE; and

[0028] FIG. 8 substantially shows real-time PCR amplification curves for an environmental water sample analyzed with PVT-VIABLE and Amoeba co-culture.

DESCRIPTION OF CERTAIN EMBODIMENTS OF THE PRESENT INVENTION

[0029] In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings, which form a part of this application. The drawings show, by way of illustration, specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

[0030] PVT-VIABLE means Phigenics Validation Test-Viability Identification Assay by Legionella Enrichment.

[0031] (LOD) means Limit of Detection.

[0032] (VBNC) means Viable But Not Culturable.

[0033] (CFU) means Colony-Forming Unit.

[0034] (Ct) means the Cycle Threshold values. Cq may be used interchangeably with Ct to also refer to the Cycle Threshold value.

[0035] (PCR) means Polymerase Chain Reaction.

[0036] T.sub.0 DNA extract means a neat DNA extraction that occurs before the time of incubation.

[0037] (PBS) means Phosphate Buffered Saline.

[0038] T.sub.2 DNA extract means DNA extractions after a period of incubation.

[0039] T.sub.0/T.sub.2 represents a period of incubation between the T.sub.0 DNA extract and the T.sub.2 DNA extract, commonly, though not exclusively, a 40 h-48 h time gap.

[0040] EB7 means a modified Buffered Yeast Extract (BYE) media. The EB7 constitutes the following, Vancomycin, Polymyxin B, Cycloheximide, Glycine, Iron pyrophosphate, L-cysteine hydrochloride, and bovine serum albumin.

[0041] Deoxyribonucleic acid (DNA) synthesis is a cellular function common to all living organisms during cell division. With every cell division the amount of DNA doubles and this process only occurs when cells divide (Huisman & D'Ari, (1981)). DNA can be detected via polymerase chain reaction (PCR), a technique that amplifies a segment of DNA to reach detectable limits. This reaction uses a polymerase to add nucleotides to the strand starting at a specific primer sequence (Saiki et al., (1988)).

[0042] Traditional PCR requires post reaction analysis to determine a positive or negative reaction. In contrast, real-time PCR (RT-PCR) monitors the reaction as it progresses using fluorescent probes.

[0043] Clinical microbiology laboratories have been revolutionized by real-time PCR technology enabling human microbial infection diagnostic capabilities. The real-time PCR testing method utilizes a combination of PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel (U.S. National Library of Medicine National Institutes of Health, Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing (2006)).

[0044] When using real-time PCR, a positive signal is produced when the fluorescent signal crosses the calculated threshold value. The more DNA present in the sample at the start of the reaction, the fewer cycles it takes to cross the threshold. Thus, a lower Cycle threshold Ct value depicts a higher starting concentration of DNA. A higher starting concentration of DNA is indicative of a higher starting concentration of cells. Equipment for real-time PCR procedures is known in the art. In one particular embodiment, the PCR equipment may include the Bio-Rad CFX 96 Deep Well Touch.

[0045] Legionella

[0046] A gram-negative bacillus was isolated from infected patients of the 1976 American Legion convention in Philadelphia, Pa. This bacillus would later be identified as Legionella, which is also characterized as non-acid fast, heterotrophic, aerobic and catalase positive (McCoy, (2005)). Legionella is a very common waterborne pathogenic bacterium, for example, a study done in Ontario, Canada showed that 55% of building water tested positive for Legionella pneumophila (Dutka & Walsh, (1984)). These bacteria proliferate in host cells like Acanthamoeba and human alveolar macrophages during infection (Percival & Williams, (2014)). There are over 60 species within the genus Legionella and 70 serogroups (Cunha, 2010a; Morita et al., (2017)). Legionella bacteria are the cause of legionellosis, an atypical form of pneumonia caused aspiration of contaminated water by susceptible populations. Pontiac Fever, also caused by Legionella, is a mild, flu-like form of legionellosis that can occur in healthy populations (Cunha, 2010b).

[0047] Enrichment of Target Organisms

[0048] Most standard detection methods for foodborne pathogens (Bacteriological Analytical Methods, BAM) require a pre-enrichment step with non-selective or selective growth media. This is true for Shiga toxin producing E. coli, Listeria monocytogenes, Salmonella spp., and others; Myint, Johnson, Tablante, & Heckert, (2006)). These enrichment procedures are used to increase the bacterial load in a sample to a detectable level for further analysis. The analysis steps are usually composed of a mixture of traditional culture assays, biochemical assays, and molecular assays that are specific to the target organism, but they all have one factor in common, the analysis of the sample takes place after the enrichment period.

[0049] For the present invention the test hypothesis was whether two technologies could be combined to analyze building water samples for viable Legionella. The two technologies in question were the theory of enrichment of a sample and the analysis of the concentration of DNA, via RT-PCR, at two timepoints during the enrichment period in liquid media.

[0050] The above described strategy presented multiple complications, the first being the selective enrichment of Legionella from water that was possibly contaminated with highly competitive background microbiota. This was accomplished by titrating the typical antibiotics used for Legionella culture on solid media, glycine, vancomycin, polymyxin B, and cycloheximide.

[0051] Surprisingly, the titration of these selective agents allowed for the selective enrichment and targeting of Legionella while decreasing the growth of the background microbiota. For highly contaminated samples the background microbiota was not eliminated. Additional steps are needed to address water samples with a high bacterial load. The second issue that needed to be resolved was to determine the positive predictive value of this assay when compared to the traditional culture methods. Curiously, the data show that the PPV of PVT-VIABLE when compared to the traditional ISO 11731 method is low. Many more positive samples were observed using the PVT-VIABLE assay. This is attributed to the unanticipated ability of PVT-VIABLE to resuscitate VBNC cells.

[0052] A flowchart 100 of a PVT-VIABLE process in accordance with an embodiment of the present application is depicted in FIG. 1. The process 100 is depicted schematically at 200 in FIG. 2. The process 100 includes obtaining a liquid sample (step 102). In a practical real-world analysis, obtaining the sample may include sampling a water source and shipping the sample to a laboratory or test site where the analysis is to be conducted. To simulate the water sampling process within the laboratory, e.g. for generating calibration and comparative data, a water sample obtained within the laboratory may be allowed to stand at room temperature for a period of time, e.g. 24 hours, to simulate shipping.

[0053] At the laboratory or testing site, the process 100 includes collecting a concentrate of bacterial cells from the liquid sample (step 104) and enriching the concentrate of bacterial cells to produce an enriched concentration of the targeted bacterial cells (step 106). As shown in FIG. 2, the concentrate may be obtained by filter concentration of the sample 204 followed by inoculation of the sample concentrate into a nutrient rich growth environment 206 to produce the enriched concentrate of the bacterial cells. In alternative embodiments, collection of the bacterial concentrate may be completed by centrifugation, or any other method known in the art provided that cell damage is limited through the collection process.

[0054] A T.sub.0 DNA extract 208 is extracted (step 108) from the enriched concentrate of bacterial cells at time T.sub.0 and stored 110. The remaining enriched concentrate is then incubated 112 for an incubation period. At time T.sub.2 after the incubation period, a T.sub.2 DNA extract 214 is extracted from the enriched concentrate (step 114). Real-time PCR is then performed on the T.sub.0 DNA extract and T.sub.2 DNA extract to determine a cycle threshold Ct for each of the DNA extracts (step 116).

[0055] After completion of the RT-PCR process the data from the report is analyzed. The Ct values of each timepoint for each sample are assessed and compared. A decrease in Ct value from T.sub.0 to T.sub.2 corresponds to an increase in nucleic acid concentration. An increase in Ct value indicates a decrease in nucleic acid concentration. Therefore, any significant decrease in Ct value indicates growth of the cell population that was inoculated into the broth. The indication of cell growth may be an indicator of the presence of viable Legionella or other targeted pathogen.

[0056] At step 118, the difference in the Ct value (Ct) between the T.sub.0 DNA extract and T.sub.2 DNA extract is analyzed to provide a qualitative assessment 218 of the presence of the targeted waterborne pathogens for the sample, such as viable Legionella.

[0057] An order of magnitude quantification of the targeted pathogens (step 120) can optionally be performed using a serial dilution technique described in more detail below.

[0058] Samples of both potable and non-potable water can be analyzed for Legionella and other waterborne pathogens using this method. In one embodiment, sample size is between 100-1000 ml in volume and collected in sterile containers containing sodium thiosulfate to neutralize any residual disinfectant in the sample. Prior to shipping, the sample may be treated to neutralize residual oxidants and antimicrobials to provide a substantially oxidant-free sample. It should be noted that all collection containers should be sterilized in a manner that is consistent with degradation of any DNA. Simple sterilization to prohibit the growth of living organisms may be insufficient. Prompt delivery of the samples to the laboratory or testing site is beneficial to ensure consistency and accuracy of results.

[0059] In one embodiment, the filter concentration process may include filtering a one hundred milliliter sample using a filtration unit having a 47 mm diameter track etched polycarbonate membrane, with a porosity of less than 0.4 m and preferably around 0.2 m. After the entire 100 ml has been filtered, the sample membrane is aseptically removed from the filter apparatus using sterile forceps. The membrane is placed into a cell culture flask containing 10 ml of an enrichment broth and is shaken by hand for 30 seconds to transfer the bacteria from the membrane into the enrichment broth.

[0060] The enrichment broth may be EB7 as defined above. In one embodiment, the EB7 is a modified BYE media comprising Vancomycin 0.25-0.4 g, Polymyxin 15,000-40,000 IU, Cycloheximide 15-40 mg, glycine 2-4 g as well as Iron pyrophosphate, L-cysteine hydrochloride, and bovine serum albumin. In one specific embodiment, the EB7 media includes Vancomycin (0.3375 mg), Polymyxin B (27,000 IU), Cycloheximide (27 mg), Glycine (3.0125 g), Iron pyrophosphate (0.25 g), L-cysteine hydrochloride (0.4 g), and bovine serum albumin (10 g). Alternative enrichment media may include R2A or 1395 broth, both of which are known in the art.

[0061] In one embodiment, the incubation period may be 40-48 hours. Greater or lesser periods may be used depending on various factors including type of pathogen under analysis, accuracy of results required, etc. During the incubation period, the cell culture flasks may be kept at a temperature from about 30 C. to about 37 C., preferably about 35 C. with shaking at 50 rpm.

[0062] In one embodiment, the T.sub.0 and T.sub.2 DNA extraction comprise taking a 2.0 ml aliquot of the broth and processing the aliquot via any appropriate DNA extraction method that gives PCR quality DNA. For example, the DNA extraction should yield acceptable 260/280 and 260/230 ratios and have an acceptable DNA concentration for PCR. In a particular embodiment, a proprietary DNA extraction method referred to as the Phigenics Ultra-Rapid DNA Extraction (P.U.R.E.) may be used.

[0063] FIG. 3 shows a flowchart 300 of a method for order of magnitude quantification of the targeted pathogen using a serial dilution technique. At step 302, the T.sub.0 DNA extract is diluted and then a real-time PCR is performed on the dilution 304. If the dilution shows PCR amplification (decision 306), then the process returns to step 302 where further dilution occurs. Once the dilution ceases to show PCR amplification, the level of pathogen may be quantified 308 by analyzing the highest dilution that showed PCR amplification. That is, the highest dilution of the T.sub.0 DNA extract at which PCR amplification is detected provides an order of magnitude quantification of the viable targeted waterborne pathogen in the liquid sample.

[0064] In one embodiment, the first dilution may be 10-fold to yield a 1:10 dilution. The second dilution may be a further 10-fold to yield a 1:100 dilution.

[0065] If only the original T.sub.0 DNA extract shows PCR amplification, then the concentration X of targeted pathogen in the sample may be considered to be 1 CFU/ml. If the 1:10 dilution is the highest dilution that shows PCR amplification, then the X may be considered to be within the range 1<X10 CFU/ml. If the 1:100 dilution is the highest dilution that shows PCR amplification, then the X may be considered to be within the range 10<X100 CFU/ml.

[0066] Greater resolution may be produced in the quantification if required by smaller serial dilutions.

Example 1Pure Culture Tests

[0067] The PVT-VIABLE protocol was tested on four species of pure culture Legionella including lab strains L. pneumophila (ATCC 33823) and L. longbeachae (ATCC 33462), and environmentally isolated L. micdadei and L. anisa. Cell suspensions of each organism were prepared in sterile phosphate buffered saline (PBS) to an OD.sub.600 of 0.05. Then each suspension was serially diluted to 10.sub.3. Next, 10 l of each dilution was added to 100 ml of PBS to simulate a water sample. Each 100 ml sample was analyzed by the PVT-VIABLE protocol.

[0068] FIG. 4 shows the real-time PCR amplification curves for the 10.sub.2 dilution of L. pneumophila analyzed by the PVT-VIABLE process. A dilute cell suspension of Legionella pneumophila was made in sterile PBS and analyzed with the PVT-VIABLE protocol. DNA was extracted at T.sub.0 and T.sub.2. The T.sub.0 DNA extract was stored at 20 C. to preserve the state of the DNA in the T.sub.0 DNA extract until T.sub.2 when the extracts were analyzed by real-time PCR. The difference (Ct) between the T.sub.0 Ct (filled squares) and the T.sub.2 Ct (open squares) was 14.62. In FIG. 4, a positive control is indicated by the filled circles and a negative control is indicated by the filled triangles.

[0069] For comparative analysis and to prove the feasibility of the PVT-VIABLE process, traditional plating of the sample may also be conducted. FIG. 5 shows the corresponding spread plates for Legionella pneumophila (ATCC 33823) for the sample analysis using the PVT-VIABLE process illustrated in FIG. 4. Spread plates were prepared for the T.sub.0 extract (A. in FIG. 5) and for the T.sub.2 extract (B. in FIG. 5).

[0070] As stated, the Ct was 14.62 which was very high, but normal for a lab strain sample that did not have any competing microorganisms. Similarly, the spread plates for this sample (FIG. 5) show a great deal of growth in the broth culture. All species tested grew well in EB7 and had Ct greater than 1.5.

Example 2Environmental Sample Tests

[0071] PVT-VIABLE was tested on 301 environmental water samples. Water samples were collected from multiple building water systems and analyzed with the PVT-VIABLE protocol. DNA was extracted at T.sub.0 and T.sub.2. The T.sub.0 DNA extract was kept at 20 C. until T.sub.2 when the extracts were analyzed by real-time PCR.

[0072] FIG. 6 shows an example of the real-time PCR amplification curves for one PVT-VIABLE positive sample (filled squaresT.sub.0, open squares T.sub.2, filled circles-positive control, filled triangles-negative control). This sample had a Ct of 3.45 and the sample was also culture positive.

[0073] FIG. 7 shows the spread plate counts corresponding to the real-time PCR data shown in FIG. 6 plated at T.sub.0 (A. in FIG. 7) and T.sub.2 (B. in FIG. 7). FIG. 7 shows the culture results for this sample increased in CFU/ml from 4 to >300.

TABLE-US-00001 TABLE 1 PVT-VIABLE beta testing results for over 300 samples. 301 potable water PVT-VIABLE Beta Test Statistics VIABLE+ and Culture+ 30 VIABLE+ and Culture 45 VIABLE and Culture+ 2 VIABLE and Culture 194 Culture+ 32 Culture 239 VIABLE+ 80 VIABLE 220 Total # of Samples 301 % culture+ 10.63% % VIABLE+ 26.58% Sensitivity 97.56% Positive Predictive Value 40.00% Negative Predictive Value 98.98%
samples were analyzed by PVT-VIABLE and compared to the spread plating method on GVPC agar. VIABLE positive (VIABLE+) means the sample had a Ct1.5 between the two real-time PCRs. That is, a Ctapproximately 1.5 between the T.sub.0 and T.sub.2 DNA extracts is considered to indicate the presence of the viable waterborne pathogen in the sample. Culture positive (culture+) means 1 colony was detected on the T.sub.0 spread plate or the T.sub.2 spread plate. For the PPV and NPV statistics, the culture result was set to true.

[0074] The results in table 1 show that PVT-VIABLE works well on real water samples and is surprisingly more sensitive than the traditional culture method. The sensitivity of the assay is very high at 97.56%; only 2 samples were false negative (VIABLE/culture+) and these results were likely due to competing microbiota overgrowth. The NPV was 98.98% due to the 2 false negatives mentioned above. The PPV (set to culture=true) of the assay was strikingly low at 40% compared to ISO 11731 spread plates culture (40% of the samples that are VIABLE positive were also culture positive) and this is due to the 45 samples that were PVT-VIABLE positive and culture-negative (tentative culture method false negatives).

[0075] This statistic was calculated using spread plate culture result=true. The ability of PVT-VIABLE to detect more viable Legionella samples than the traditional method, can be explained in two ways: 1.) PVT-VIABLE detected both viable and VBNC Legionella, therefore, many more positive results were obtained compared to spread plate cultures which detect only those bacteria that can form colonies on the plates, 2.) PVT-VIABLE detected viable Legionella at a lower limit of detection than the traditional spread plate methods. In reality then, the PVT-VIABLE method revealed that spread plate cultures returned many false-negative results because of the reasons discussed above. VIABLE positive, culture-negative samples are preliminary confirmation that VBNC cells can be resuscitated in the enrichment broth. An increase in DNA over the 40-48 h incubation time conclusively demonstrates cellular growth and is a more accurate and sensitive indication of the viability of an organism compared to the traditional definition of bacteria being able to form visible colonies on solid growth media.

Example 3VBNC Resuscitation Experiment

[0076] A side-by-side experiment was designed to show the ability of PVT-VIABLE to resuscitate VBNC cells in comparison to resuscitation via co-culture with amoebae, the natural host for Legionella. Twenty-eight potable water samples from a chloraminated water system were analyzed by PVT-VIABLE and with amoeba co-culture in three separate experiments. In short, the same collection and preparation as PVT-VIABLE was done on the water sample, then the filter membrane was added to a flask containing 10 ml of PYG media and 10.sub.3 to 10.sub.4 cells/ml of Acanthamoeba castellanii (a natural host for Legionella) All samples were spread plated at T.sub.0 and T.sub.2. The same DNA extractions and real-time PCR were performed on the amoeba flasks and the PVT-VIABLE flasks. The Ct values for each method were compared. FIG. 8 shows the real-time PCR amplification curves for an environmental water sample analyzed with PVT-VIABLE (A. in FIG. 8) and Amoeba co-culture (B. in FIG. 8) (filled squaresT.sub.0, open squares T.sub.2, filled circlespositive control, filled trianglesnegative control). A positive Ct for PVT-VIABLE and for amoeba co-culture with no CFUs on the spread plate at T.sub.0 shows a resuscitation, growth, and infectivity of VBNC cells from an environmental water sample. According to the real-time PCR results there were seven VIABLE+/Amoeba+, two VIABLE/Amoeba+, four VIABLE+/Amoeba, and 15 VIABLE/Amoeba samples. The seven environmental samples that were VIABLE+/Amoeba+ and culture negative show VBNC resuscitation by PVT-VIABLE that was confirmed by amoeba co-culture.

[0077] The PVT-VIABLE approach described herein offers an innovative method for the determination of viable Legionella due to enrichment aspect and to the approach of measuring viability through the increase in nucleic acid concentration. This tandem approach has not been applied to Legionella and this approach differs from currently available technologies. This invention also improves upon existing technology by significantly decreasing the time it takes for a viable Legionella diagnostic. The current timeline is 10 days-14 days using prior art techniques. With the method as described herein, results will be available approximately 96 hours (4 days) after samples are taken from a facility. This method also improves on the prior art by resuscitating and detecting injured VBNC cells that would have otherwise gone undetected using traditional prior art techniques.

[0078] In one embodiment, results may be given as positive or negative for viable Legionella samples at a limit of detection (LOD) of 1 viable cell/100 ml (10 viable cells/L). Note that the volume of sample filtered determines the LOD. For example, if 1 L of sample is filtered, then the LOD is 1 viable cell/L of sample.

[0079] It can be seen from the foregoing examples that the Ct value between the T.sub.0 and T.sub.2 extracts determined by the described PVT-VIABLE method can be indicative of the presence of pathogens within a water sample, including viable but non-culturable cells. In the present examples, a Ct greater than approximately 1.5 provides a threshold requirement for indicating the presence of waterborne pathogens in the sample, specifically viable Legionella. The present inventors have conducted experiments to determine an appropriate value for Ct.

[0080] Out of 49 samples, 14 were positive for Legionella. There were 11 Legionella species and three L. pneumophila serogroup 2-14. Samples were taken from the following; 40 samples from bathroom sinks, 1 sample from a kitchen sink, five samples taken from showers and three samples taken from drinking fountains, which were all tested for the presence of Legionella. All 14 positives were detected as viable on the PCR with a Ct greater than 1.5. All but one of the 14 positive samples were detected as viable with the PCR with a Ct of 1.

TABLE-US-00002 TABLE 2 Samples analyzed using the Ct 1 cutoff value. SAMPLE STATISTICS 1 # True Positive (1 Ct) 14 False positive (N ISO, 1 Ct) 1 False negative (P ISO, but 1 Ct) 0 True Negative (N ISO and 1 Ct) 34 Total # of +ISO plate 14 Total # of ISO plate 35 Total # of Samples 49 ISO+ AND VIABLE 4 ISO AND VIABLE 28 Total # Shower 5 Total # Bathroom Sink 40 Total # Kitchen Sink 1 Total # Drinking Fountain 3 PREVALENCE OF Legionella positives 28.57% SENSITIVITY 100.00% POSITIVE PREDICTIVE VALUE 93.33% (1 Ct correlates to P ISO) Negative Predictive Value 87.50% (N PCR correlates to N ISO)

TABLE-US-00003 TABLE 3 Samples analyzed using the Ct 1.5 cutoff value. Increasing the cut off to 1.5 Ct the PPV was 100% and it retained 100% sensitivity SAMPLE STATISTICS 1.5 # True positive (1.5 Ct) 14 False positive (N ISO, 1.5 Ct) 0 False negative (P ISO, but 1.5 Ct) 0 True Negative (N ISO and 1.5 Ct) 35 Total # of +ISO plate 14 Total # of ISO plate 35 Total # of Samples 49 ISO+ AND VIABLE 4 ISO AND VIABLE 28 Total # Shower 5 Total # Bathroom Sink 40 Total # Kitchen Sink 1 Total # Drinking Fountain 3 PREVALENCE OF Legionella positives 28.57% SENSITIVITY 100.00% POSITIVE PREDICTIVE VALUE 100.00% (1.5 Ct correlates to P ISO) Negative Predictive Value 87.50% (N PCR correlates to N ISO)

TABLE-US-00004 TABLE 4 Samples analyzed using the Ct 2 cutoff value. Increasing the cut off to 2 Ct, one PVT-VIABLE positive was missed, lowering the sensitivity to 92.86%. SAMPLE STATISTICS 2 # True Positive (2 Ct) 13 False positive (N ISO, 2 Ct) 0 False negative (P ISO, but 2 Ct) 1 True Negative (N ISO and 2 Ct) 35 Total # of +ISO plate 14 Total # of ISO plate 35 Total # of Samples 49 Biplate positive 4 Legionella species 28 Total # Shower 5 Total # Bathroom Sink 40 Total # Kitchen Sink 1 Total # Drinking Fountain 3 PREVALENCE OF Legionella positives 28.57% SENSITIVITY 92.86% POSITIVE PREDICTIVE VALUE 100.00% (2 Ct correlates to P ISO) Negative Predictive Value 87.50% (N PCR correlates to N ISO)

[0081] It can be seen from Tables 2-4 that setting the cut off value of Ct can have an impact on the usefulness of the results. If the value of Ct is set too low, then the sensitivity is high, but false positives may be recorded. Increasing the cut-off value of Ct above 1 reduces the false positives but increasing the cut-off value of Ct excessively leads to reduced sensitivity and the introduction of false negatives. Through experiment, the present inventors have found that a cut-off value for Ct should be in the range 1<=Ct>=2. Preferably, the cut-off value should be approximately Ct=1.5.

[0082] While the specification makes specific reference to detecting Legionella bacteria, the PVT-VIABLE technique, based on enrichment of targeted pathogens, may be used to determine the presence of other types of waterborne pathogens. Targeting of alternative pathogens may be achieved through selection of different filtering membranes, different enrichment cultures and experimental determination of appropriate incubation parameters, Ct cut-off values, etc. Other pathogens of note would be non-tuberculosis Mycobacteria, Pseudomonas aeruginosa, Salmonella, E. coli, among others. The Ct cutoff value of 1.5 is appropriate for Legionella and for an incubation period of 40-48 hours. Other waterborne pathogens may have a different derived Ct than Legionella but the principle of the matter (the change in DNA concentration) remains the same. That is, the Ct cutoff may be different for different organisms. For example, the Ct cut-off value may be larger in some samples that grow very well in the media. Alternatively, or in addition, incubation periods may be altered to account for the different growth rate of the target organism in the medium. For example, the target organism is Pseudomonas (another waterborne pathogen) then the incubation could be shorter because the doubling time for Pseudomonas is shorter.

[0083] The membrane filter should be 0.2 m in order to capture the bacteria. 0.45 m can also be used, but some bacteria may be missed. The sample size shouldn't be less than 100 ml but can be higher than that if it is determined that it is necessary.

[0084] Many modifications and other implementations of the disclosure set forth herein will come to mind to one skilled in the art to which this disclosure pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosure is not to be limited to the specific implementations disclosed, and that modifications and other implementations are intended to be included within the scope of the appended claims. Moreover, although the foregoing descriptions and the associated drawings describe example implementations in the context of certain example combinations of elements and/or functions, it should be appreciated that different combinations of elements and/or functions may be provided by alternative implementations without departing from the scope of the appended claims. In this regard, for example, different combinations of elements and/or functions than those explicitly described above are also contemplated as may be set forth in some of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.