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 for 48 h in a static temperature controlled air incubator. The growth rate of Y. pestis is slow with a generation time of 1.25 h 9.</li>
</ol>
2. Y. pestis liquid media inoculation
<ol>
	<li>Transfer a single Y. pestis colony into a sterile 17 x 100 mm culture tube containing 2 mL of LB broth using a sterile metal inoculation loop. 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. Vortex tube briefly and grow culture at 28&deg;C for approximately 20 h in a shaking incubator (225 rpm).</li>
</ol>
3. Y. pestis outgrowth, reporter phage addition, and bioluminescent detection
<ol>
<li>Dilute the overnight Y. pestis culture 1:20 into fresh LB broth in a 50 mL falcon tube (e.g., 500 &mu;L of cells into 9.5 mL of medium) and grow at 28&deg;C with shaking (225 rpm). Grow until an optical density at 600 nm (A600) of 0.2 is reached (approximately 5 h). 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 7.</li>
<li>Generate the substrate necessary for the bioluminescent reaction by preparing a 2% n-decanal solution. Mix 200 &mu;L of n-decanal with 9.8 mL of LB broth. Vortex vigorously for 5 s. Prime the microplate luminometer injector with 2 mL of the 2% n-decanal solution. Pre-set the luminometer to autoinject 67 &mu;L of the decanal solution to each microplate well and then immediately read the sample for 10 s.</li>
<li>Dispense 1 mL aliquots of LB broth into 3 culture tubes. Add 20 &mu;L of the reporter phage stock solution (stock of 5 x 109 plaque forming units [PFU]/mL) to each tube; the phage and media alone samples serves as a negative control. In the absence of Y. pestis cells, the addition of the reporter phage should not elicit a bioluminescent response.</li> 
<li>Dispense 1 mL aliquots of the Y. pestis culture (from step 3.1) into 6 culture tubes. Add 20 &mu;L of the reporter phage stock to 3 of the cultures (test cultures). The remaining 3 cultures serve as a 'cells alone' negative control (background autobioluminescence). Mix cultures by vortexing briefly, and harvest 200 &mu;L from each sample (for the time 0 read) and dispense into a white 96-well microtiter plate. Incubate the remaining culture at 28&deg;C with shaking (225 rpm).</li>
<li>Measure the time 0 samples for bioluminescence (relative light units, RLU) using the microplate luminometer.</li>
<li>Harvest 200 &mu;L of each sample after 10, 20, 30, 40, 50, and 60 min post-reporter phage addition, and measure the sample for bioluminescence. If Y. pestis cells are present, the reporter phage will specifically bind to the cell, inject its DNA, and use the hosts transcriptional and translational machinery to transcribe and translate the luxAB reporter genes (Figure 1).</li>
<li>The strength of signal and the signal response time is proportional to the number of cells present. At high concentrations of cells (105-108 CFU/mL), a significant increase in RLU compared to the controls should be evident within 20 min. At lower cell concentrations, (102-104 CFU/mL), a significant increase in RLU should be evident within 40-60 min.</li>
<li>Alternatively, to expedite the detection process without the need for Y. pestis outgrowth, a colony from a freshly grown plate may be mixed (by vortexing) directly in a 1.5 mL eppendorf tube with 200 &mu;L of LB broth harboring the reporter phage. Dispense the cell/phage mixture into a well of a microtiter plate. Incubate the microtiter plate at 28&deg;C and read the sample for bioluminescence after 60 min (single time point only).</li>
</ol>
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&deg;C even though the optimum temperature for Y. pestis growth is 28&deg;C 9.
<img src="/files/ftp_upload/2740/2740fig2.jpg" alt="Figure 2"/> Figure 2. Phage-mediated bioluminescent detection of Y. pestis. At time 0, reporter phage and cells were mixed, incubated at 28&deg;C, and bioluminescence (RLU) was monitored over time. A significant increase in RLU (* Students t-test, p&lt;0.05) is evident within 15 min.

<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>

No conflicts of interest declared.

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

&#039;Bioluminescent&#039; 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.

'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. ...

bioluminescent-reporter-phage-for-detection-category-bacterial

Research

JoVE Journal

Immunology and Infection

13.3K Aufrufe.  Guild Associates, Inc.. Eine einfache Methode zur Identifizierung der prioritären bakteriellen Erregern ist gentechnisch Reporter Phagen verwenden. Diese Reporter-Phagen, die spezifisch für ihre besonderen Wirtsarten sind, sind in der Lage schnell Transduktion eines Biolumineszenz-Signal Antwort auf Wirtszellen. Hier beschreiben wir die Verwendung von Reporter Phagen für den Nachweis von Yersinia pestis.

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

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 infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

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

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

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 initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter 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 interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

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

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 bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

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 ...

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 to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

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 combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

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 ...

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 spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

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

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

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

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.