1BioSciences Division, Guild Associates, Inc., 2Department of Molecular Genetics and Microbiology, University of Texas at Austin, 3Department of Craniofacial Biology, Medical University of South Carolina
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Schofield, D. A., Molineux, I. J., Westwater, C. 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens. J. Vis. Exp. (53), e2740, doi:10.3791/2740 (2011).
Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases' is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant "light-tagged" reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage ΦA1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage Wβ::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.
1. Y. pestis plate inoculation
2. Y. pestis liquid media inoculation
3. Y. pestis outgrowth, reporter phage addition, and bioluminescent detection
4. Representative results:
A representative time course experiment of reporter phage mediated detection of Y. pestis is depicted in Figure 2. The negative controls of 1) phage alone (no cells), or 2) cells alone (no phage) provide baseline levels of bioluminescence of approximately 20 RLU throughout the 60 min incubation (baseline levels are luminometer specific). In contrast, an increase in bioluminescence for the test samples (reporter phage and cells) is evident at 15 min after phage addition. The signal strength should increase steadily over 60 min. Incubations for prolonged time periods (over 80 min), will result in a signal that will decline from the peak signal due to phage mediated lysis of host cells. Similar results are obtained at incubation temperatures of 37°C even though the optimum temperature for Y. pestis growth is 28°C 9.
Figure 2. Phage-mediated bioluminescent detection of Y. pestis. At time 0, reporter phage and cells were mixed, incubated at 28°C, and bioluminescence (RLU) was monitored over time. A significant increase in RLU (* Students t-test, p<0.05) is evident within 15 min.
This method demonstrates the ability of the reporter phage to rapidly detect Y. pestis since the reporter phage can transduce a bioluminescent signal response to cultured Y. pestis cells within 20 min after phage addition. The reporter phage is also capable of directly detecting Y. pestis in clinical matrices, without the prerequisite of isolation and subsequent cultivation 7. Compared to the standard phage lysis assays which generally require 48 h for completion, this significantly decreases the time to detection.
Previous studies have demonstrated that the wild-type ΦA1122 phage can lyse nearly all natural Y. pestis isolates, and is 'specific' for Y. pestis 6,10,11; however, some Y. pseudotuberculosis strains have been shown to be ΦA1122 susceptible when grown at temperatures above 20°C 6,10,12. The reason for the temperature-sensitive differential susceptibility is unknown, but presumably due to temperature-dependent changes in the cell surface layers/composition. Therefore, a potential caveat of the reporter phage detection system is the possibility of a false-positive response with strains from the closely-related species Y. pseudotuberculosis. Performing the reporter phage assay at the restrictive temperature (20°C) will prevent a false positive signal in samples that may contain Y. pseudotuberculosis. Thus, specificity can be strictly controlled when using isolated cultures grown at a specific temperature.
In summary, rapid detection and diagnosis of Y. pestis is essential for a positive prognosis since the plague, especially pneumonic plague, is nearly always fatal if treatment is not administered within the first 24 h after symptom onset. This method has the potential to meet these needs for the confirmed identification of cultured isolates or within clinically relevant matrices.
No conflicts of interest declared.
This research was supported by the Small Business Innovation Research program of the National Institutes of Health (NIAID, 1R43AI082698-01) and the USDA National Institute of Food and Agriculture (NIFA, 2009-33610-20028).
|Difco LB agar, Miller||VWR international||90003-346|
|Difco LB broth, Miller||VWR international||90003-350|
|17 x 100 mm culture tubes||USA Scientific, Inc.||1485-0810|
|Veritas microplate luminometer||Turner Biosystems||9100-001|
|Microlite microtiter 96-well plate||VWR international||62402-984|