https://orcid.org/0000-0002-9338-5119
Figures
Abstract
Background
Vibrio cholerae are often infected with pathogen-specific virulent bacteriophages (phages) that may be found associated with V. cholerae or independently from the bacteria, especially in environmental water samples. When detected, these phages can serve as a surrogate for detection of V. cholerae and they also have an important role in the ecology of V. cholerae. Vibriophages can be detected using plaque or molecular (PCR) assays, but these methods are time-consuming and require specialized laboratory resources.
Methodology
To increase the accessibility of phage detection for epidemiologic applications, we developed a simple, rapid, and inexpensive colorimetric assay to detect vibriophage that can be scaled quickly to screen large numbers of samples. The assay uses resazurin and V. cholerae AC6169 that is susceptible to vibriophages ICP1, 2, and 3. Resazurin is a dye that turns from blue to pink when added to a culture broth with growing bacteria. We hypothesized that when a bacteria-free test sample, such as filtered wastewater containing vibriophage is added to culture broth with AC6169 and resazurin, the phages will lyse the bacteria preventing their growth, and the color of the broth will remain blue. However, if there are no vibriophages in the sample, the bacteria will grow rapidly, and the culture broth will turn pink.
Results
We developed the assay using ICP1 spiked samples of environmental water, stool and frozen bile peptone and found it to be sensitive with a limit of detection of 4–40 plaque forming units/ml.
Conclusion
This colorimetric assay provides a convenient method to detect vibriophages on a larger scale than was possible earlier. Its use should help to better understand the role of vibriophage as a surrogate for detecting V. cholerae and better understand their role in the pathogenesis, ecology, and epidemiology of cholera.
Author summary
We developed a simple and inexpensive colorimetric assay to detect virulent vibriophage in samples of wastewater and stool. The assay uses resazurin, a blue dye and a V. cholerae AC6169 that is susceptible to vibriophages. Bacteria-free filtered samples suspected of having vibriophage are incubated for 2 or 3 hours in a broth with AC6169 following which resazurin is added. After a further 30-minute incubation, the color of the broth is observed. If the sample contains vibriophage, the broth will remain blue, but if there are no vibriophage, the broth will turn pink. The change in color from blue to pink is due to the metabolic reaction from the growing bacteria. When vibriophage are present, they lyse AC6169 thus preventing the color change. If vibriophage are not present, the bacteria multiply rapidly, and the metabolic reaction causes the broth to change color to pink. Because the assay procedure is inexpensive and simple, laboratories in cholera endemic areas should find it convenient for their epidemiologic and clinical surveillance activities. Future field research needs to assess the distribution of these phages in cholera endemic areas.
Citation: Luo W, Dinowitz D, Camilli A, Andrews JR, da Silva KE, Debes AK, et al. (2025) A colorimetric method for detecting virulent bacteriophage to Vibrio cholerae in fecal and environmental samples. PLoS Negl Trop Dis 19(12): e0013674. https://doi.org/10.1371/journal.pntd.0013674
Editor: Jeffrey H. Withey, Wayne State University School of Medicine, UNITED STATES OF AMERICA
Received: March 22, 2025; Accepted: October 22, 2025; Published: December 18, 2025
Copyright: © 2025 Luo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the National Institute of Allergy and Infectious Disease to DAS (grant number 5R01AI123422), AC (grant number R37AI055058) and JRA (grant number R01 AI181283). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Cholera, a bacterial diarrheal disease caused by Vibrio cholerae serotypes O1, is characterized by severe watery diarrhea that can quickly lead to severe dehydration and death [1]. This disease continues to be a major public health problem and the World Health Organization (WHO) considers cholera to be an “Ongoing Health Emergency [2].” Currently an estimated 2.9 million cases with 95,000 deaths occur each year [3]. Patients with cholera are sometimes co-infected with virulent bacteriophage (phage) that can specifically prey on V. cholerae. When these phage-infected bacteria are excreted in the patient’s stool, the phages may also be shed [4].. In areas with poor sanitation, they may be co-ingested by the next human host. Thus, infections with V. cholerae can occur either with phage or without phage, but phages cannot persist for a long time without the V. cholerae on which they prey [5].
Because vibriophage exist in a close relationship with V. cholerae, the detection of the phage is an indicator that V. cholerae are present [6]. Therefore, when vibriophages are detected in a stool sample of a patient suspected of having cholera, this may also confirm the diagnosis of cholera. Similarly, when vibriophage are identified in an environmental sample, such as wastewater, this provides evidence that V. cholerae is also being expelled into the environment even if the bacterium itself is not detected in the sample [7]. This concept can be called diagnosis by phage proxy [8].
Most cholera diagnostic efforts are directed toward detecting the V. cholerae by commercialized rapid diagnostic tests (RDT), culture or by PCR. To evaluate vibriophage as a diagnostic proxy when the primary V. cholerae target is not detected, we explored methods to detect vibriophage simply and rapidly in patients’ stools and in environmental samples. Traditional methods to detect phage use plaque assays on soft agar with a lawn of susceptible bacteria [9]. With this approach, one can isolate and screen for pathogen-specific phages. Using plaque assays, three different V. cholerae-specific phages were isolated from stool samples in Bangladesh, named ICP1, ICP2 and ICP3 [10]. These phages were genetically and phenotypically characterized, and molecular methods for scaled detection in clinical samples were developed [11–13] Epidemiologically, ICP1 has been found in Africa in addition to Asia [14]. ICP1 and ICP3 have been detected to date in Southeast Asia [10].
While plaque assays and PCR can readily detect vibriophage, these assays require time, technical laboratory skills and resources. A colorimetric method using the dye resazurin might be adapted to screen fecal and environmental samples more quickly and inexpensively. Such an assay could facilitate large-scale screening efforts in both clinical and environmental settings, ultimately improving cholera surveillance and outbreak response. Similar methods have been used to detect or study phage-specific for E. coli and Salmonellae [15–18]. Resazurin, a blue dye, is converted into pink, fluorescent resorufin in the presence of metabolically active cells; specifically, NADPH dehydrogenase from the bacteria convert resazurin to resorufin. This conversion can be detected through the visual observation of its pink color. Resazurin has also been used to determine bacterial antibiotic susceptibility [19,20] and for screening natural materials for their inhibitory ability against bacteria [21]. We hypothesized that if vibriophages are present in a test sample consisting of bacterial-free filtered feces or environmental water, it will lyse a pan-sensitive V. cholerae test strain. Thus, in the presence of resazurin, the susceptible bacteria in a culture broth mixed with a test sample with phages will fail to grow and the culture broth will remain blue. However, if the test sample does not have phages, the bacteria will grow rapidly, and the broth will turn pink. Ideally, this change would be apparent to the ‘naked eye’; however, the color change could also be objectively determined by measuring the OD600 of the culture supernate. An OD600 is the appropriate wavelength because this is the peak absorbance for the blue dye, compared to the pink dye with a peak of 570 nm [22]. Although ICP1, 2 and 3 were identified in Bangladesh, this manuscript focuses only on ICP1 since it has been identified in Africa where there are multiple ongoing cholera outbreaks [14,23].
Methods
A series of experiments were carried out to explore methods appropriate for phage detection. These methods are modified from methods used to detect phage for Salmonella Typhi [18]. The experiments used varying concentrations of ICP1 which were spiked into samples of environmental water, stool, or bile peptone broth (BP) to determine the sensitivity of detection in these samples.
Materials and preparation of the materials: Environmental water was obtained from the northern area of the Chesapeake Bay (pH was 6.1). Bile peptone (BP) was prepared according to an established recipe of 20g Ox Bile (Millipore-Sigma #70168-100g), 5g Dextrose(Fisher Chemicals #D16-500), 8g Sodium phosphate dibasic (Santa Cruz #c-203277), 10g Peptone from gelatin (Millipore-Sigma,#107284.100), and 2g potassium dihydrogen phosphate (Fisher # P285-500) in one liter with water and autoclaved for 15 minutes at 121°C). Vials of BP were spiked with varying concentrations of phage and were then frozen (-80°C) until tested. Fresh dog stools were diluted 1:5 in PBS to simulate a diarrheal stool.
Vibriophage ICP1_2011_A7 and phage indicator strain V. cholerae strain AC6169 were used in this study. AC6169 is a genetically engineered, non-toxigenic, antibiotic-sensitive, Biosafety Level 1 (BSL-1) derivative of the O1 serogroup, El Tor biotype strain E7946. This strain was constructed using the suicide plasmid pCVD442 to mediate several allelic exchange events as described [24]. First, the CTX prophage and flanking RS1 and TLC mobile elements were deleted (fusion junction sequence 5’-GTGGAGTTCTTTTTT/GCACTGAGGTATTTT-3’). Second, the K139 prophage and its attB site were deleted (fusion junction sequence 5’-TGGGGGTAAAACGC/GAGAGAACCGGGGCTA-3’). Third and fourth, two phase variable O1-antigen biosynthesis genes, manA and wbeL, were locked into their ‘on’ configurations by placing silent A-to-G mutations within their poly-A tracts. This was done to reduce the frequency of appearance of phase-off mutants that no longer make O1-antigen, which is the receptor for vibriophages ICP1 and ICP3. For manA, which has two poly-A tracts, mutations A213G, A216G, A609G, and A612G were made. For wbeL, which has one poly-A tract, mutations A111G and A114G were made. Finally, the streptomycin-resistance point mutation within rpsL was reverted to the wild type sequence. AC6169 was subjected to whole-genome sequencing and the desired mutations confirmed.
To prepare freshly growing V. cholerae AC6169, the bacterial preparation starts two days in advance. From an -80°C stock vial of AC6169, we streaked a Luria-Bertani agar (LA) plate with the bacteria and incubated it overnight at 37°C. The next day, using a single colony, we inoculated a 14-ml culture tube (Falcon cat# 352059) with 5 ml of Luria-Bertani broth (LB) and incubated the tube for 14–16 hours in a shaker incubator at 37°C. From this culture we measured the OD600 and transferred a small volume of the overnight culture broth into a tube with 5 ml LB broth to prepare a broth with an OD600 of 0.05 in the new tube. We then incubated the new tube with shaking at 37°C to reach an OD600 of 0.2.
We prepared a 0.06% solution of resazurin (Invitrogen, cat#R12204) by dissolving it in autoclaved milli-Q water in a 50 ml tube. We vortexed the mixture until the resazurin was completely dissolved. We then filtered it through a 0.22 µm filter (Millipore, cat#SLGVR33RS). We prepared aliquots (550 µl) in 1.5 ml sterile tubes and stored them at -20°C in a foil lined box. When needed, (within 30–60 minutes), we removed an aliquot(s), covered them with foil, and thawed them at room temperature.
To prepare a standard lot of ICP1, we amplified it in the host strain AC6169. First, we streaked an LA plate with frozen AC6169 and incubated the plate overnight at 37°C. The next morning, we picked one colony from the LA plate and inoculated it into 5 ml LB in a 14 ml-culture tube and incubated the tube at 37°C with shaking until the OD600 reached 0.2-0.4. We then added 2.5 µl of ICP1 stock to the tube and continued to shake the tube at 37°C for 3 more hours. We then centrifuged the tube at room temperature at 3500 x g and filtered the supernate through a 0.22 µm Millipore filter to eliminate the bacteria from the supernate. We then stored the filtrate at 4°C until used. To quantify the titer of ICP1 in each lot, we used a standard soft agar overlay method. Briefly, we grew the host strain AC6169 to mid-log phase (OD600 = 0.3-0.5) and diluted it in LB to a OD600 of 0.025. We then serially diluted ICP1 in LB (10-1 to 10-7) and mixed 100 µl of the diluted AC6169 with diluted ICP1 in 96-well microplate wells in duplicate and incubated it at 37°C for 10 minutes to allow phage adsorption. We then added the total 200 µl mixture to a well with 3 ml of 48°C soft agar (0.35% agar in LB) in a 6-well plate. We gently swirled the plate immediately to mix and placed the plate at room temperature until the agar is solidified. We then incubated the plate for 3–4 hours at 37°C until the plaques are observed and quantified. Based on the average number of plaques and the dilution factor, we calculated plaque titer.
Proof of concept. As an initial experiment to establish the proof of concept, we serially (10-fold) diluted ICP1 in 5-ml culture tubes with 0.25 ml LB using concentrations of 4–400 pfu/ml. We then added 250 µl fresh LB and 35 µl of freshly growing AC6169 which had been adjusted to an OD600 of 0.2 and incubated the mixture with shaking at 37°C for 2, 2 ½ or 3 hours. After the incubation, we added 12.5 µl of the resazurin solution (0.06%) and continued the shake incubation for another 30 minutes. We then centrifuged the tubes (3650 x g) and observed the tubes visually to assess their color and photographed the tubes to record their color. To obtain an objective measure of the color change, we diluted 50 µl of the supernate in 950 µl LB broth and measured the OD600. The OD600 of each sample and the controls were recorded and the result was expressed as the ratio of the sample OD600 divided by the OD600 of the negative control sample (without phage). We expect the negative control sample to have a low OD600 and samples with phage should have a significantly higher OD600 than the negative control, with a ratio >2.
Procedure to detect phage in test samples: To detect phage in different sample types spiked with ICP1 (bay water, stool, and bile peptone broth), we evaluated two methods. One method, we call the one-step method, we only amplify the phage once after the sample is made bacteria free. The other method we call the two-step method since we amplify the phage twice.
For the one-step method, for each sample, we centrifuged 1.5 ml of the sample for 5 minutes at 17,000 x g at room temperature to rid the sample of debris. We then prepared a bacteria-free sample by filtering the supernate through a 0.22 Millipore filter or by treating it with chloroform. For filtration we used a 3 ml syringe with the Millipore filter and collected the filtrate in a 1.5ml sterile centrifuge tube. When we used chloroform, we transferred 1 ml of the supernate to a new 1.5 ml tube and added 100 µl of fresh chloroform and vortexed it for 15 seconds. We then centrifuged it at room temperature for 5 minutes at 17,000 x g and then transferred 600 µl from the top of the tube to a new sterile 1.5 ml centrifuge tube. We centrifuged it again for 5 minutes at 17,000 x g and we transferred 250 µl from the top of the tube to a new sterile 5 ml culture tube to be used as the bacteria-free sample for further testing in the same manner as the filtered sample.
Using the bacteria-free sample we proceeded to detect the phage. Using a separate 5 ml culture tube, we mixed 250 µl of the bacteria-free sample with 250 µl LB and 35 µl of freshly growing AC6169 which had been adjusted to an OD600 of 0.2 and incubated the mixture with shaking at 37°C for three hours. After the 3-hour incubation, we added 12.5 µl of the resazurin solution (0.06%) and continued the shake incubation for another 30 minutes. We then centrifuged the tubes for 10 minutes at 3650 x g and observed the tubes visually to assess their color and photographed the tubes to record their color. As was done in the pilot experiment, to obtain an objective measure of the color, we diluted 50 µl of the supernate in 950 µl LB broth and measured the OD600. The OD600 of each sample and the controls were recorded and the final result was expressed as the ratio of the sample OD600 divided by the OD600 of the negative control sample (without phage). The negative control sample will have a low OD600 and those with phage should have a significantly higher OD600 than the negative control, generally the ratio will be > 2.
For the two-step method we amplified the phage as the first step without resazurin. To amplify the phage, we centrifuged the sample for 5 minutes at 17,000 x g at room temperature to rid the sample of debris and mixed 750 µl of the supernate with 675 µl LB and 75 µl of freshly growing V. cholerae AC6169 (OD600 adjusted to 0.2) in a 5 ml culture tube. We then incubated the culture tubes while shaking for 2 hours at 37°C. We then centrifuged the tube at room temperature for 5 minutes at 17,000 x g. We then collected 1 ml of the supernate and rendered it bacteria-free, either by filter sterilizing through a 0.22 µm Millipore filter or by treating with chloroform as described above.
After amplifying the phage, as a second step, we proceeded to detect the phage as was done similarly in the one-step method except that the incubation was for two hours rather than three as in the one-step method before adding the resazurin. The two-step procedure can be done in one day, but it can also be spread over two days by storing the bacteria-free, phage-amplified sample overnight at 4°C. Both the initial amplification (first step) and the detection (second step) need fresh AC6169. A schematic of the procedures for these methods is shown in Fig 1.
To determine the sensitivity of the assay, we spiked the water, bile peptone or stool sample with different concentrations of ICP1 ranging from 4 to 400 plaque forming units (pfu) per ml. For bile peptone broth, after spiking the sample, we froze it at -80°C and later we thawed the sample for testing. For each assay, we included a negative control with AC6169 but without phage, and a second control with neither phage nor AC6169 (dye only control). To confirm that the bacteria-free samples were bacteria-free, we mixed 250 µl of bacteria-free sample with 285 µl of LB and incubated 37°C overnight and observed for growth.
All procedures were conducted in a BSL-2 lab.
Results
The results of the proof-of-concept experiment are shown in Fig 2. The control sample with no phage turned pink as expected due to the growth of AC6169. A clear dose response using the one-step method was observed when the initial incubation was limited to 2 hours, but with longer times of incubation, the bacteria continued to grow and the blue color in each of the tubes increased, obscuring the dose response.
Panel A shows the color reactions using serial dilutions of phage in LB broth with AC6169. Control sample (C1) had no phage and no bacteria. Control sample (C2) is the negative conttrol with no phage but with AC619 bacteria. Test samples were inoculated with serial dilutions of ICP1 with AC6169 from 4 to 400 pfu/ ml (A,B,C). The numbers below each tube are the ratio of OD600 of the test sample compared to the negative control (NC). Panel B shows these results graphically.
To determine if the assay can detect phages in different sample types using the one-step method, we spiked samples of stool, bay water, and bile peptone with serial dilutions of ICP1 and tested the filtrates from a 0.22 Millipore filter or chloroform extraction. Fig 3 shows the OD ratios of the experiments with the different sample types using either Millipore filtration (3a) or chloroform (3b). The limit of detection for these sample types was from 4 to 40 pfu/ml for the Millipore filtration but was less sensitive using the chloroform procedure (40–400 pfu/ml).
Panel A used Millipore filtration and panel B used chloroform to prepare the bacteria-free sample. The red dotted line shows a ratio of 2.0 above which the sample is considered positive.
As shown in Fig 4, we found that prior amplification of phages through a two-step procedure improved the sensitivity of the assay when using chloroform for all three sample types. The limit of detection for these samples was from 4 to 40 pfu/ml.
Panel A shows the colors of the tubes when ICP1 was serially diluted in samples of bay water, stool, and bile peptone broth and incubated with AC6169 and resazurin. The numbers below each tube are the ratio of OD600 of the test sample compared to the negative control (NC). Panel B shows the ratios graphically. The red dotted line shows a ratio of 2.0 above which the sample is considered positive.
Discussion
The study findings show the laboratory feasibility for screening samples of environmental water, stool, and frozen bile peptone broth for virulent vibriophages (phage) using a method that leverages a dye (resazurin) that in the experimental conditions detects bacterial lysis. When the bacterium is allowed to grow, in the presence of resazurin, the color of the broth changes from blue to pink; however, if vibriophages are present, the phage lyse the bacteria, the bacteria cannot activate the dye, and the color remains blue. This assay uses V. cholerae AC6169 to amplify and detect the phage because this strain is receptive to phage specific to V. cholerae (ICP1, 2, and 3). This V. cholerae strain, AC6169, is a non-toxigenic strain which allows for its use as a BSL1 bacteria; furthermore, it is susceptible to each of the known vibrio phages ICP1, 2 and 3. We were able to discern positive samples by comparing the colors of the tubes using the naked eye; however, we found that some samples are not as clear and measuring the OD600 provides a more objective outcome.
In the assay, AC6169 is the only agent in the test condition to convert resazurin. To minimize false positives, the assay requires that the test sample must be devoid of other biologic agents (bacteria) and inorganic sources of NADH oxidase. The bacteria in the test sample can be eliminated either passing the sample through a 0.22 µ filter or by killing the bacteria with chloroform. While filtration may be a gold standard, filters are costly. The cost of a one-step procedure per sample was estimated at $4 using filtration and $1.7 using chloroform. Compared to filtration, use of chloroform does require additional centrifugation steps to reduce the residual leftover of chloroform which adds to the workload.
Initially we determined if filtered water and fecal samples could be tested without the initial step of phage amplification. We assumed that a few phages would be able to attack the target bacterium AC6169 and would be detectable even without prior amplification. However, we found that the two-step assay was more sensitive when the sample is first mixed with AC6169 to amplify the phage before the sample is rendered bacteria-free for the detection step with AC6169 bacteria in LB broth and resazurin. Of note, for both the one and two step methods, we incubated the sample with LB for 2 or 3 hours before adding resazurin and then continued incubation for an addition 30 minutes.
We feel this assay shows promise for screening samples of environmental water for vibriophages, especially wastewater, for evidence of cholera transmission in the area. The bacterium V. cholerae can also occasionally be detected in contaminated water and this assay for phage does not negate the need for detection of the bacteria either by culture or PCR. It simply allows for phage detection as a proxy for V. cholerae when the bacteria are lower in number or degraded. Previously we described a rapid diagnostic test assay for detection of V. cholerae using samples of environmental water enriched in alkaline peptone water, and this method also provides results within a day [25]. The circumstances for which assay for V. cholerae or vibriophage is most appropriate or most sensitive need to be determined. It should be noted that the two-step assay provides for a multi-fold amplification of phages and is expected to be more sensitive compared to other plaque or PCR assays without this amplification.
In addition to environmental samples, we found that this assay can also be used to screen stool samples for evidence of cholera. Currently, confirmation of a cholera case depends on the detection of fecal V. cholerae by RDT, culture or PCR, but detection of the vibriophage in the patient’s stool also provides confirmatory evidence that the patient had cholera. In Bangladesh, the presence of phage as well as antibiotics was found to reduce the sensitivity of stool culture for cholera, presumably because the phage had lysed the V. cholerae that had been present in the intestine [26]. Thus, identifying vibriophage in the stool may increase the ability to detect patients with cholera. Similarly, phage in the environment may also lyse V. cholerae and the identification of phage may also detect cholera environmental contamination by proxy in this setting [27].
We examined the recovery of phage from frozen bile peptone broth because one of our partners in Africa had been collecting Moore swabs from environmental samples to study another pathogen, Salmonella Typhi. [28] This lab had saved frozen samples of Moore swabs that had been incubated in bile peptone broth before freezing. Thus, we wanted to determine if phage would survive and could be recovered from these frozen samples. We have not yet attempted to recover vibriophage from other types of frozen samples, but we found that viable V. cholerae O1 could not be recovered from spiked bile peptone broth.
The strength of this method includes its sensitivity, its low cost, and the rapid turnaround. Results can be available within a few hours, and this rapid turnaround time allows for additional samples to be collected up or downstream from the original sampling site in an attempt to better localize the source and spread of the cholera contamination.
The test does have several limitations. This report used only ICP1 and did not test this assay using ICP2 or ICP3; however, the strain AC6169 is known to be receptive to ICP2 and ICP3. Secondly, while the one-step method using a two-hour initial incubation time might be semi-quantitative but with longer incubation times, it is not quantitative. Clearly, the two-step method is not quantitative. Thirdly, we tested these three sample types, but there may be other sample types that need to be evaluated. Fourthly, we found that the target, non-toxigenic bacteria, AC6169, was very useful but there may be other strains which may be more widely available. Finally, we used the phage ICP1 to develop the assay and to use as a control reagent. As phages are identified in different labs, these labs may want to save their local phages to use as a control for their assay. It should also be noted that this assay does not provide pure phages, but it does amplify the phages making it possible to isolate the phage using plaque assay procedures.
In terms of the next steps, we believe these findings demonstrate the laboratory feasibility of this procedure as a screening test for virulent vibriophage and as a proxy for pathogen detection. However, the method requires further evaluation with clinical and environmental samples in regions with active cholera outbreaks. Positive samples will likely need subsequent confirmation using either PCR or plaque assays [10,29] for phages with paired assays for V cholerae bacteria. While this assay using resazurin appears to provide a practical method for detecting vibriophage in cholera endemic areas, it will need to be compared to alternative assays to determine its relative suitability for these labs. While AC6169 is known to be susceptible to these three phages, there may be other vibriophages to which AC6169 is not susceptible; these phages may be detected using traditional plaque assays using other strains of V. cholerae. Finally, the epidemiology and ecology of virulent phages specific to V. cholerae needs to be further investigated and we feel these methods can be used to better understand the role that vibriophages play in transmission and the epidemiology of cholera.
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