Validation of the PhageDx™ Listeria Assay for Detection of Listeria Spp. on Stainless Steel and Ceramic Environmental Surfaces AOAC Performance Tested MethodSM 102005

Abstract Background The PhageDx™ Listeria Assay is a simple, specific, and sensitive assay based on the infection of Listeria spp. by selected bacteriophages and the resultant expression of a luciferase reporter gene. Results are generated in as little as 24.5 h for stainless steel and ceramic environmental surfaces. Objective An AOAC Performance Tested MethodsSM (PTM) study was conducted to validate the PhageDx Listeria Assay for the detection of Listeria on stainless steel and ceramic surfaces. Method The performance of the PhageDx method was compared to that of the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM) Ch. 10. Inclusivity/exclusivity, product consistency and stability, and robustness testing also were conducted. Results Inclusivity testing demonstrated that the reporter bacteriophages were specific for Listeria ssp. and detected 58/61 Listeria strains tested, including all 34 L. monocytogenes strains. The reporter bacteriophage also was shown to not detect 46/47 non-Listeria bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least 6 months. Matrix studies on stainless steel and ceramic surfaces showed that there was no significant difference between the PhageDx Listeria Assay and the FDA/BAM Chapter 10 reference method. Conclusions and Highlights The validation study demonstrates that the PhageDx Listeria Assay is an effective method for the detection of Listeria spp. on stainless steel and ceramic environmental surfaces and meets the qualifications for AOAC PTM status.


General Information
Listeria monocytogenes is a bacterial pathogen that can be found in moist environments such as soil, water, and decaying vegetation and when ingested, most commonly through eating contaminated food, can cause listeriosis. In the United States, listeriosis is estimated to affect approximately 1600 people resulting in about 260 deaths annually. Symptoms of infection include diarrhea, vomiting, nausea, headaches, stiff neck, confusion, loss of balance, convulsions, fever, and muscle aches. Populations that are particularly susceptible to the effects of listeriosis are pregnant women and their newborns, older adults (65þ years) and people with weakened immunity, where infections can result in death. Infections in pregnant women can also result in miscarriage, stillbirth, premature delivery, or life threatening infant infections (3,4).
Common food sources of Listeria contamination include deli meats, dairy products, and produce. Listeria spp. can survive in a wide range of conditions typically used in food preservation such as extremes in temperature and pH and high salt concentrations. Food contamination commonly occurs via transfer from contaminated surfaces that the food comes in contact with (3,4). Thus, an essential measure to prevent Listeria contamination is to monitor sanitation of food preparation areas.
The PhageDx TM Listeria Assay provides all the reagents necessary for sample infection, lysis, and luciferase detection. After a 20-24 h enrichment of the test portions in buffered Listeria enrichment broth (BLEB) at 35 6 1 C for stainless steel and ceramic surfaces (4 Â 4" test area), a portion of the enriched sample is placed in a well of a 96-well break-away plate and infected with the PhageDx Listeria Recombinant Phage at 30 C for 4 h. Then, lysis/luciferase substrate master mix is added to the infected sample and read on a luminometer for signal measurement. Results are expressed as relative light units (RLU). Total sample handling time is approximately 30 min.

Principle
The PhageDx Listeria Assay is a simple, specific, and sensitive diagnostic test for Listeria on ceramic and stainless-steel environmental surfaces. The kit is designed to be used with a Promega luminometer and the clear results indicate which samples contain viable Listeria. The assay consists of five basic steps; sample collection, enrichment, phage infection, substrate addition, and signal read. Presumptive results are available in 24.5 h which includes a 20-24 h enrichment step and a 4 h infection step. The PhageDx Listeria Assay is designed to be performed by qualified laboratory personnel in laboratories performing microbiological analysis.
The PhageDx Listeria Assay is based on the infection of Listeria spp. by bacteriophages and replication of the infecting bacteriophages within their specific hosts. Bacteriophages demonstrate a high specificity for their bacterial host and are capable of replicating within their host quickly to high numbers. The recombinant phages used in the PhageDx Listeria Assay also express a luciferase reporter during replication. The presence of Listeria spp. is determined by incubating the lysate with the appropriate luciferase substrate and detecting emitted light in a luminometer. An absence of detected light above background indicates that no viable Listeria are present in that sample.

Definitions
(a) Probability of detection (POD).-The proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte level or concentration. POD is concentration dependent. Several POD measures can be calculated: POD R (reference method POD), POD C (confirmed candidate method POD), POD CP (candidate method presumptive result POD), and POD CC (candidate method confirmation result POD). (b) Difference of probabilities of detection (dPOD).-Difference of probabilities of detection is the difference between any two POD values. If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level (2).

General Preparation
(a) Prepare BLEB media according to manufacturer's instructions. (b) Before using the reagents, flick or spin the tube to collect all of the solution at the bottom of the tube. (c) Before adding the pre-warmed BLEB to the sample, confirm that the media and incubator are warmed to 35 6 1 C. Do not allow the pre-warmed media to cool before adding to the sample. Maintain the media at 35 6 1 C in an incubator or water bath if preparing multiple samples. It is important to maintain the temperature of the sample and BLEB media used in the enrichment incubation. (d) Prepare the Promega luminometer by following the manufacturer's cleaning procedure and make sure there are no signal "hot spots" that will affect the sample results by reading an empty plate. All signals should be less than 20 RLU. Set up the luminometer to read the appropriate wells on the plate using the pre-programmed PhageDx Assay protocol or, alternatively, set up a read protocol with a 3 min or 180 s delay between starting the program and the start of the signal reads and two plate read runs with a 1 s integration per sample.

Interpretation and Test Result Report
(a) The luminometer program will display the results on the screen as RLU values corresponding to the well positions of the break-apart plate. (b) Samples positive for Listeria will have a reading value of 300 RLU or greater. Negative samples will be less than 300 RLU. (c) Once all of the samples have been run and analyzed, remove the plate from the luminometer and follow the manufacturer's instructions for cleaning the instrument and shut down. Note: In some cases, the PhageDx Listeria Assay will generate a very high signal and result in adjacent wells reading as a false positive due to a bleed-over signal from a well with a high signal. If a sample well is positive and has a 1000Â lower signal than the adjacent sample well with a higher signal (typically in the range of 300-2000 RLU), this could be a result of bleed-over signal. In these cases, we recommend that the contents of the potential false positive well (lower RLU sample) be transferred to a new well at least a 2-3 well distance from the high signal well or to a new strip and re-read to confirm that the signal is from the sample and not a result of bleed-over signal.

Confirmation
We recommend that presumptive positives from the phage assay be confirmed.  . Method developer studies were conducted in the laboratories of Laboratory Corporation of America Holdings, and included the inclusivity/exclusivity study, product consistency and stability studies, and robustness testing. The independent laboratory study was conducted by Q Laboratories, Inc., and included inclusivity and exclusivity studies for selected strains and matrix studies for all claim matrixes.

Method Developer Studies
(a) Inclusivity and exclusivity studies.-Inclusivity (Listeria) and exclusivity strains were obtained from academic, governmental, and commercially available sources. A total of 61 Listeria strains were tested for the inclusivity study, 54 strains by LabCorp and an additional seven by Q Laboratories. For inclusivity testing done at LabCorp, 54 Listeria species covering L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3a, 4a, 4b, 4c, 4d, and 4e, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi strains were examined. Each strain was grown at 35 6 1 C for 20-24 h in BLEB medium. The cultures were then diluted to approximately 100 times the LOD 50 of the PhageDx Listeria Assay and transferred to a 96-well plate. A total of 47 non-Listeria strains were tested for the exclusivity study, 45 strains by LabCorp and three by Q Laboratories (Enterococcus faecalis ATCC 29212 tested by both LabCorp and Q Laboratories). For exclusivity testing done at LabCorp, 45 different strains of non-Listeria species and strains were grown under optimal enrichment conditions suitable to the strain type (medium and temperature) for a minimum of 20-24 h. One hundred microliters of the undiluted cultures were transferred to 96-well plates.
For both inclusivity and exclusivity studies, samples were infected with 10 mL of PhageDx Listeria phage reagent at 30 6 1 C for 4 h. After infection, 65 mL of lysis/luciferase master mix was added to the wells and samples were read on a luminometer. Samples with a signal of !300 RLU were considered positive.
(b) Product consistency (lot-to-lot) and stability studies.-Three separate production lots of PhageDx Listeria recombinant phage were prepared according to written manufacturing documents and tested according to quality control procedures. Quality control procedures verified that each lot when diluted to working concentration had the same titer, background, and level of detection. Recombinant phage reagents were aged between 0 and 6 months when assayed for stability.
Consistency and stability were done according to AOAC guidance, where a sample was inoculated with L. monocytogenes (ATCC 7302) to give fractional positives. Ten replicates were run in the PhageDx Assay, and the RLU values analyzed. A set of stability studies was also conducted using the non-target bacterium Enterococcus faecalis (ATCC 29212). Overnight cultures of E. faecalis were used directly in the assay.
(c) Robustness study.-Three parameters were varied to demonstrate assay robustness: infection time (630 m), recombinant phage concentration (620%), and lysis/luciferase master mix amount (65 mL). Briefly, stainless-steel surfaces were inoculated with L. monocytogenes (ATCC 19115) at a low level to generate partial positives or 100Â E. faecalis (ATCC 29212) and allowed to dry and sit for 16-24 h at room temperature (20-25 C). The PhageDx Listeria Assay protocol was followed with the variations in infection time, recombinant phage concentration, and lysis/ substrate master mix amounts as indicated in Table 4. Samples with RLU values of 300 or greater were considered positive. Samples were confirmed by allowing samples to enrich for a total of 24-28 h and plating on RAPID' L. monocytogenes plates and incubating at 37 6 1 C for an additional 24-48 h. The presence of blue (pale blue, grey-blue, or dark blue) colonies on selective plates indicated a positive result for L. monocytogenes.

Independent Laboratory Validation Study
(a) Inclusivity and exclusivity.-The inclusivity and exclusivity study evaluated two strains of L. grayi, three strains of L. seeligeri, two strains of L. welshimeri, one strain of Enterococcus faecalis, one strain of Enterococcus faecium, and one strain of Streptococcus pyogenes. All cultures evaluated were propagated from a stock culture stored at À70 C to tryptic soy agar (TSA) with 5% sheep blood agar (SBA) and incubated for 24 6 2 h at 35 6 1 C. After incubation of the target organisms, a single colony from SBA was transferred to a 9 mL aliquot of BLEB and incubated for 20-24 h at 35 6 1 C. Exclusivity strains were transferred to non-selective media and incubated under conditions for optimal growth. Inclusivity and exclusivity testing was done in the same manner as method developer's inclusivity and exclusivity studies. All inclusivity and exclusivity strains were blind coded and randomized. Tests results were decoded and reported as either positive or negative. (c) Organism preparation and inoculation.-For ceramic surfaces, 4 Â 4" areas were inoculated with 0.25 mL of diluted L. innocua (ATCC 43547) culture and sampled using sampling sponges premoistened in letheen broth. For stainless steel surfaces, 4 Â 4" areas were inoculated with 0.25 mL of diluted L. monocytogenes 4a (CWD 1554). In addition to the L. monocytogenes culture, stainless-steel surface was co-inoculated with a competitor organism, Enterococcus faecalis (ATCC 29212), at 10Â the level of the target organism. All cultures utilized were propagated from a stock culture stored at À70 C to SBA and incubated for 24 6 2 h at 35 6 1 C. After incubation, a single colony from SBA was transferred to a 9 mL aliquot of brain heart infusion (BHI) broth for 20-24 h at 35 6 1 C. Each culture was then diluted to the target concentration by performing serial dilutions using 0.1% peptone water as the diluent. For the uninoculated test portions, sterile 0.1% peptone water was applied to the test area. Each surface was dried for 16-24 h at room temperature (24 6 2 C) prior to sampling. To determine the inoculation level for the environmental surfaces, aliquots of each inoculum were plated onto TSA in triplicate.
(d) FDA/BAM Chapter 10.-For environmental samples, sponges were pre-moistened with 10 mL of Dey-Engley (D/E) neutralizing broth. Following addition of D/E neutralizing broth surfaces were swabbed using firm and even pressure 10 times vertically and horizontally. All environmental samples were then stored at 22 6 2 C for 2 h 6 15 min. Swabs were enriched with 90 mL of BLEB, massaged by hand for 2 min and incubated at 30 6 1 C for 4 h. Following 4 h of incubation, selective supplements acriflavine (10 mg/L), sodium nalidixate (40 mg/L), and cycloheximide (50 mg/L) were added to each test portion and samples were re-incubated for 24-48 h.
After 24 h of total incubation, the enriched samples were streaked to modified Oxford (MOX) agar plates and incubated at 35 6 1 C for 24-48 h. In addition to MOX each enriched sample was streaked to the chromogenic selective agar brilliance Listeria agar (BLA) and incubated at 37 6 1 C for 24 6 2 h. The enriched samples were re-incubated for an additional 24 h at 30 6 1 C and then streaked to a second MOX and BLA agar plate which was incubated for 24-48 h at 35 6 1 C and 37 6 1 C for 24 6 2 h, respectively. MOX and BLA agar plates were examined     (e) PhageDx Listeria Assay.-Sponges from ceramic (4 Â 4" test area) and stainless steel (4 Â 4" test area) swabbed surfaces were enriched according to the protocol described in Organism preparation and inoculation. PhageDx Listeria Assay was performed as described in Sample preparation. Briefly, using a polyurethane sponge premoistened with letheen, surfaces were swabbed and the sponge placed into the sample bag and held at 22 6 2 C for 2 h 6 15 m. Swabs were enriched with 20 mL of pre-warmed BLEB, massaged by hand for 10-20 s and incubated at 35 6 1 C for 20 h. Following enrichment, 150 mL sample aliquots were transferred to a 96-well plate. Ten microliters of phage reagent was added to each sample and samples were incubated at 30 6 1 C for 4 h. After infection, 65 mL of lysis/luciferase substrate master mix was added to the samples and then read on a luminometer. All samples were culturally confirmed by the FDA/BAM Chapter 10 reference method. All samples were also confirmed by an alternate confirmation described previously in Confirmation. Final confirmation for all samples was obtained by Bruker MALDI Biotyper following AOAC Method 2017.09.

Results
Inclusivity studies for the PhageDx Listeria Assay demonstrated that of the 61 Listeria inclusivity strains tested, 58 were detected and three were not detected ( Table 1). All 34 L. monocytogenes strains tested were detected, while those strains that were not detected included one strain each of L. innocua (ATCC BAA-680), L. seeligeri (ATCC 51335), and L. welshimeri (ATCC 43551; Table 1). Those strains that were not detected can be divided into two categories. The first category includes those that would be detected with higher cell numbers. L. innocua (ATCC BAA-680) and L. welshimeri (ATCC 43551) were detected with 20 h enrichment, or within the recommended enrichment time frame for environmental samples (data not shown). L. seeligeri (ATCC 51335), however, remained negative with extended enrichment times.
Exclusivity studies showed that of the 47 exclusivity strains examined, 46 of the 47 strains were negative (Table 2).
One strain, Lactobacillus plantarum ATCC 14917, was reported as positive; however, the signal generated was significantly lower than that of Listeria strains (data not shown).
Lot-to-lot studies showed that the PhageDx Listeria recombinant phages can be manufactured consistently and are stable for at least 6 months when stored at 4 C. Manufactured lots were made on 2/20/20, 4/20/20, and 8/20/ 20 according to written manufacturing documents. Each lot produced similar results when tested according to QC tests for bacteriophage concentration, background signal, and LOD. Performance tests to determine the stability of each lot were performed to determine the shelf life of the recombinant phage. These tests demonstrated that lots produced 0 months prior to testing showed no significant difference from lots produced at least 6 months prior to testing. Additionally, no variation in exclusivity was observed with these three recombinant phage lots in tests with E. faecalis. Results shown in Table 3.
Robustness testing of the PhageDx Listeria Assay demonstrated that variations in infection time, recombinant phage concentration, and lysis/luciferase master mix amounts do not alter the results compared to the standard protocol. Infection times of 3.5 and 4.5 h, recombinant phage volumes of 8 and 12 mL, and lysis/luciferase master mix volumes of 60 and 70 mL produced identical results to the standard protocol of 4 h infection, 10 mL of recombinant phage, and 65 mL of lysis/luciferase master mix in both uninoculated and low inoculum test samples (Table 4). These results indicate that these deviations from the PhageDx Listeria Assay protocol did not alter the final results.
Matrixes studies were performed as per criteria outlined in Appendix J of the Official Methods of Analysis Manual, and fractional positive results were obtained for the PhageDx Listeria Assay (6). For ceramic and stainless steel (4Â4" test areas) environmental samples, the inoculum recovered for the target L. innocua and L. monocytogenes strains fell within the targeted levels, while the competitor organism was at a level at least 10 times higher. The probability of detection (POD) was calculated as the number of positive outcomes divided by the total number of trials (6). Comparisons of the PhageDx presumptive results to the confirmed results using the FDA/BAM Chapter 10 reference method showed no significant difference between the two (Table 5). No significant difference was found when comparing the results of the PhageDx presumptive results and FDA/ BAM Chapter 10 results in an unpaired study (Table 6). There was also no significant difference in the PhageDx presumptive results and results of the PhageDx confirmation method (Table 7). Finally, there was no difference between the presumptive PhageDx results confirmed using the PhageDx confirmation method and the FDA/BAM reference method (Table  8). In summary, POD analysis between the PhageDx Listeria Assay method and the reference method indicated that there was no significant difference at the 5% level between the number of positive results by the two methods for both matrixes. The POD analysis between the PhageDx Listeria Assay presumptive and confirmed results indicated that there was no significant difference at the 5% level for the method for both matrixes. A summary of POD analyses (8) are presented in Tables 5-8.

Discussion
The results of this validation study show that the PhageDx Listeria Assay is an effective alternative to the FDA/BAM Chapter 10 for the detection of Listeria on stainless steel and ceramic surfaces. In inclusivity and exclusivity testing, the method was shown to be specific for Listeria, correctly identifying 58/61 Listeria target strains including all 34 L. monocytogenes strains tested. The PhageDx Listeria Assay was unable to detect three strains within the inclusivity panel, one strain each of L. innocua (ATCC BAA-680), L. seeligeri (ATCC 51335), and L. welshimeri (ATCC 43551). It is unclear exactly why these strains failed to generate a positive result since the PhageDx Listeria Assay was able to detect other members of the species. One possible explanation is that these strains do not have the receptor(s) or the optimal receptors required for efficient recognition by the phage (9). Possessing a receptor that was sufficiently similar to Listeria also may explain why Lactobacillus plantarum generated a false positive result. Another possibility for a false negative may be that the strain  has a mechanism that prevents the phage from producing luciferase. These mechanisms may include, but are not limited to, preventing phage absorption, preventing phage DNA entry, or the cutting of phage nucleic acids (10). The recombinant phage can be produced consistently and is stable for 6 months when stored appropriately. Robustness testing of the PhageDx Listeria Assay indicated that the method works well when the assay parameters (infection time, recombinant phage concentration, and substrate amount) were varied from the stated protocol.
Independent laboratory testing demonstrated that the PhageDx Listeria Assay was able to detect Listeria at low levels and in the presence of competing bacteria on stainless steel and ceramic surfaces. These studies also demonstrated that the performance of the PhageDx Listeria Assay was not statistically different from that of FDA/BAM Chapter 10 for environmental surfaces. Additionally, the PhageDx confirmation procedure was shown to produce results identical to the FDA/BAM Chapter 10 reference method confirmation procedure.
The PhageDx Listeria Assay has a number of advantages over the FDA/BAM Chapter 10 reference method. In addition to being a specific assay, the results are easy to interpret as an RLU endpoint is used to determine the outcome of the assay. Another advantage is that PhageDx provides a presumptive positive result in as little as 24.5 h compared to 72þ h in the case of the FDA/BAM Chapter 10 reference method. PhageDx is also a simple test that involves only five basic steps: enrichment, sampling, infection, substrate addition, and signal readout. Finally, PhageDx Assay is a rapid method that offers a considerable time savings compared to the FDA/BAM Chapter 10 reference method.

Conclusion
Results of this validation study support the claim that the PhageDx Listeria Assay is a specific, sensitive, fast, and simple method for the detection of Listeria on stainless steel and ceramic surfaces and is statistically comparable to the FDA/BAM Chapter 10 reference method. By using a luciferase-expressing recombinant bacteriophage, the PhageDx Assay was able to detect a positive sample after 20 h enrichment and a 4 h infection for environmental surfaces. The PhageDx Listeria Assay thus offers a significantly shorter time to results compared with the FDA/BAM Chapter 10 reference method.

Submitting Company
Laboratory Corporation of America