Name: 'bioluminescent' reporter phage for the detection of category a bacterial pathogens
Uploaded: 2011-07-08T12:00:00
Description: Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

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).

1. Y. pestis plate inoculation
<ol>
	<li>Streak Y. pestis A1122 (BeiResources # NR15) stock cultures onto Luria-Bertani (LB) agar (Miller). Use sterile technique and perform all Y. pestis manipulations in a class II type A biosafety cabinet. Y. pestis A1122 is a BioSafety Level (BSL) 2 excluded select agent strain. It lacks both the 75 kb pair low-calcium response (Lcr) virulence plasmid, and the pgm locus that are required for virulence. Grow Y. pestis at 28&deg;C ...

<ol>
	<li>Darling, R. G., Catlett, C. L., Huebner, K. D., Jarrett, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threats+in+bioterrorism.+I:+CDC+category+A+agents.">Threats in bioterrorism. I: CDC category A agents.</a> Emerg Med Clin North Am. 20, 273-309 (2002).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory manual of plague diagnostic tests.. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (1999).</li><li>Inglesby, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+as+a+biological+weapon,+2002:+updated+recommendations+for+management.">Anthrax as a biological weapon, 2002: updated recommendations for management.</a> JAMA. 287, 2236-2252 (2002).</li><li>Schuch, R., Fischetti, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+genomic+analysis+of+the+Wbeta+and+gamma+phages+infecting+Bacillus+anthracis:+implications+for+evolution+of+environmental+fitness+and+antibiotic+resistance.">Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.</a> J Bacteriol. 188, 3037-3051 (2006).</li><li>Garcia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Yersinia+pestis+bacteriophage+phiA1122+reveals+an+intimate+history+with+the+coliphage+T3+and+T7+genomes.">The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.</a> J Bacteriol. 185, 5248-5262 (2003).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioluminescent+phage+for+detection+of+Yersinia+pestis.">Diagnostic bioluminescent phage for detection of Yersinia pestis.</a> Journal of Clinical Microbiology. 47, 3887-3894 (2009).</li><li>Schofield, D. A., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-mediated+bioluminescent+detection+of+Bacillus+anthracis.">Phage-mediated bioluminescent detection of Bacillus anthracis.</a> Journal of Applied Microbiology. 107, 468-478 (2009).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CDC: Basic laboratory protocols for the presumptive identification of Yersinia pestis. , 1-19 (2001).</li><li>Gunnison, J. B., Larson, A., Lazarus, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+between+Pasteurella+pestis+and+Pasteurella+pseudotuberculosis+by+action+of+bacteriophage.">Rapid differentiation between Pasteurella pestis and Pasteurella pseudotuberculosis by action of bacteriophage.</a> J Infect Dis. 88, 254-255 (1951).</li><li>Lazarus, A. S., Gunnison, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Action+of+Pasteurella+pestis+Bacteriophage+on+Strains+of+Pasteurella,+Salmonella,+and+Shigella.">The Action of Pasteurella pestis Bacteriophage on Strains of Pasteurella, Salmonella, and Shigella.</a> J Bacteriol. 53, 705-714 (1947).</li><li>Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., Filippov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+detection+of+Yersinia+pestis+using+amplification+of+plague+diagnostic+bacteriophages+monitored+by+real-time+PCR.">Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.</a> PLoS One. 5, e11337-e11337 (2010).</li></ol>

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 ...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>Difco LB agar, Miller</td>
			<td>VWR</td>
			<td>90003-346</td>
		</tr>
		<tr>
			<td>Difco LB broth, Miller</td>
			<td>VWR</td>
			<td>90003-350</td>
		</tr>
		<tr>
			<td>17 x 100 mm culture tubes</td>
			<td>USA Scientific</td>
			<td>1485-0810</td>
		</tr>
		<tr>
			<td>n-Decanal</td>
			<td>Sigma</td>
			<td>D7384</td>
		</tr>
		<tr>
			<td>Veritas microplate luminometer</td>
			<td>Turner Biosystems</td>
			<td>9100-001</td>
		</tr>
		<tr>
			<td>Microlite microtiter 96-well plate</td>
			<td>VWR</td>
			<td>62402-984</td>
		</tr>
	</tbody>
</table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

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&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;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 &#34;light-tagged&#34; 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 &#934;A1122 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 W&#946;::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.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
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.

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

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

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Research

JoVE Journal

Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Bioluminescent Bacterial Imaging In Vivo

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...