'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yesinia Pestis and Bacillus Anthrax are category A bacterial pathogens that are the causative agents of the plague and anthrax respectively. Although the natural occurrence of both diseases is now relatively rare, the possibility of terrorist groups using these pathogens as a bio weapon is real. This video demonstrates a method to detect and identify yersinia pestis.

First, the microorganism is grown on agri plates and in liquid media. Next, a y pesti specific reporter pH is added to the culture. The reporter pH harbors the genes encoding bacterial luciferase, luxe AB in its genome.

If Y pesti cells are present, the reporter phage will specifically bind to the cell, inject its recombinant DNA, carrying the luxe AB genes, and will use the host's transcriptional and translational machinery to transcribe and translate the luxe AB reporter genes Results are obtained that show the samples have acquired a bioluminescent phenotype, indicating the unknown microorganism has been positively identified as why pestis. The main advantage of this technique over the existing methods, like the traditional phage lysis assay, is that the time required to detect and identify your senior pestis is minutes instead of days. This method can help to rapidly identifying, detect the presence of Yesenia pesti using isolated cultures or directly from complex clinical or environmental samples.

The implications of this technique extend towards a diagnosis of the plague because the disease is usually fatal, if not treated within the first 24 hours of symptom onset. Thus, the timely identification of Yesenia pests is essential for a positive prognosis. Since phages exist for nearly all bacterial species, the methodology can potentially be adapted for any species that is medically, environmentally, or agriculturally to important Y pestis.

A 1122 is a biosafety level two excluded select agent strain. It lacks both the 75 Kilobase Perlo calcium response or LCR virulence plasmid, and the PGM locus that are required for virulence. Begin this procedure under a class two type a biosafety cabinet using sterile technique streak Y pestis, A 1122.

Stock cultures onto LB agar as shown here. Place the plates upside down in a static temperature controlled air incubator at 28 degrees Celsius for 48 hours. The growth rate of Y pestis is slow with a generation time of 1.25 hours after two days.

Use a sterile metal inoculation loop to pick a single y pestis colony. Select a colony that is uniform in size, is indicative of the other colonies, and is clearly separated from the rest of the colonies on the plate. Transfer the colony to a sterile 17 by 100 millimeter culture tube containing two milliliters of LB broth.

Cap the tube vortex briefly, then place it in a shaking incubator at 225 RPM at 28 degrees Celsius for approximately 20 hours. For this assay, actively growing cells are preferable for detection. Since the ability of the phage to transduce, a bioluminescent response is correlated to the viability and fitness of the host bacteria.

Nevertheless, the detection system has been shown to be compatible with cells harvested at all stages of the growth cycle. After 20 hours of growth, dilute the Y pestis culture one to 20 in a 50 milliliter conical tube by adding 500 microliters of cells to 10 milliliters of lb broth. Place the culture back in the shaking incubator at 28 degrees Celsius.

Allow the culture to grow until an optical density at 600 nanometers of 0.2 is reached. This should take approximately five hours to generate the bioluminescent reaction substrate. Prepare a 2%N decanal solution by combining 200 microliters of N decanal with 9.8 milliliters of LB broth.

Vortex vigorously for five seconds. Next to prime the Microplate Luminometer injector. Place one line of the injector tubing and a 13 milliliter tube containing 2%end decal solution and the other end of the injector tubing in a waste container.

Using the equipment software, instruct the machine to prime the injector line with two milliliters of the decanal solution. The line should fill with a substrate once completed, place the end line of the injector back into its set position ready for injecting into the wells. Next, instruct the luminometer to autoinject 67 microliters of the deel solution to each microplate.

Well then immediately measure the level of bioluminescence emitted for 10 seconds. Next, to prepare each sample and triplicate label three culture tubes for each condition. Here, phase only controls and cells only controls will be prepared in addition to a test sample.

So nine tubes are needed to prepare phage only control samples, dispense one milliliter aliquots of lb broth into three culture tubes. Add 20 microliters of the reporter phage. Stock solution contending five times 10 to the ninth plaque forming units or PFU per milliliter to each tube.

In the absence of Y pesti cells, the addition of the report of phage should not elicit a bioluminescent response. Next, to prepare test samples, dispense one milliliter aliquots of the Y pesti culture into three culture tubes. Add 20 microliters of the reporter phage stock to three of the cultures.

Mix the cultures by vortexing briefly to prepare cells alone. Negative controls, which are used to determine the background auto bioluminescence dispense one milliliter aliquots of the Y pestis culture into three additional culture tubes. Once the samples have been prepared, transfer 200 microliters from each culture into the wells of a white 96 Well microtiter plate For the time zero read.

Place the remainder of each culture in the shaker at 28 degrees Celsius using a microplate luminometer. Measure the time. Zero samples for bioluminescence or relative light units.

Take bioluminescence measurements by harvesting 200 microliters from each culture and performing readings in the Microplate Luminometer at 20, 30, 40, and 60 minutes. After adding the reporter phage to expedite the detection process without the need for y pestis outgrowth, transfer a colony from a freshly grown plate to a micro fuge tube containing 200 microliters of lb, harboring the reporter phage. Mix the sample by vortexing, then dispense the cell phage mixture into a well of a microtiter plate.

Incubate the microtiter plate at 28 degrees Celsius for 60 minutes. Then read the sample for bioluminescence. The assay demonstrated in this video detects the presence of Y pesti cells as described in the introduction.

When y pestis is present, the reporter phage binds to a specific receptor on the cell, injects its DNA, and uses the host's transcriptional and translational machinery to transcribe and translate the luxe. A reporter genes. The strength of signal and the signal response time is proportional to the number of cells present at high concentrations of cells, 10 to the fifth to 10 to the eighth CFUs per milliliter.

A significant increase in RLU compared to the controls should be evident within 20 minutes at lower cell concentrations, 10 to the two to 10 to the fourth CFU per milliliter. A significant increase in RLU should be evident within 40 to 60 minutes. A representative time course experiment of reporter, phage mediated detection of Y pestis is shown here at times zero.

Reporter phage and cells were mixed incubated at 28 degrees Celsius and bioluminescence RLU was monitored over time. An increase in RLU evident within 12 minutes, which was significant with a P value of P less than 0.05, the phage only or cells only negative controls provide baseline levels of bioluminescence of approximately 20 RLU throughout the 60 minute incubation. Note that baseline levels are luminometer specific.

In contrast, an increase in bioluminescence for the test samples reporter phant cells is evident at 12 minutes after phia edition. The signal strength should increase steadily over 50 minutes, although not shown here, incubations for time periods over 80 minutes 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 degrees Celsius, even though the optimum temperature for Y pestis growth is 28 degrees Celsius.

After watching this video, you should have a good understanding of how to use the recombinant reportage for the detection. Identification of Yesenia Pestis once mastered, this technique can be performed in 30 minutes using S Golden Colonies as initial inoculum. While attempting this procedure and using phage in laboratory, it's important to remember that it's very easy to contaminate your bacterial stock cultures with the phage to reduce the possibility of contamination.

Use disposable plastic wear by proppe tips and change your gloves regularly. The ability of the reportage to elicit a Blum response is correlated to the fitness of the cell. Thus following the identification of Yesenia Pesta, the reportage may be used to determine antibiotic susceptibility of the isolate.

For example, Yesenia pesta cells incubated with inhibitory concentration of the antibiotic will either be unable to or be severely curtailing its ability to produce a bioluminescent signal. In contrast, Yesenia pesta cells, which are antibiotic resistant, will not be compromised by the presence of the antibiotic and be able to elicit a bioluminescent response that is analogous to uninhibited cells. Thus, a profile of the pathogen susceptibility to antibiotics may be rapidly determined.

For illustration purposes, we used an attenuated Yesenia pesta strain. In this experiment, Yesenia pests is a category, a bacterial pathogen. When performing this technique with samples that are inspected to Harbor Yesenia Pestis, appropriate biosecurity and biohazard precautions must be followed.