Name: bacteriophage effectiveness for biocontrol of foodborne pathogens evaluated via high-throughput settings
Uploaded: 2021-08-19T14:00:00.000+00:00
Duration: 7 min 22 s
Description: Watch this Scientific Journal Video about Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings at JoVE.com

This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC Discovery Grant, RGPIN-2019-04384), the Canada Foundation for Innovation (Project # 38710), and Major Innovation Fund, Alberta. We thank Dr. John Kastelic for editing the manuscript.

1. Preparation of buffer and reagents
<ol>
	<li>Make 500 mL of tryptic soy broth (TSB) (15 g of TSB powder and 500 mL of ultrapure water) and autoclave. 
	NOTE: This can be stored at room temperature for up to 3 months or at 4 &#176;C for up to 6 months.</li>
	<li>Make 500 mL of tryptic soy agar (TSA) (20 g of TSA powder and 500 mL of ultrapure water) and autoclave. 
	NOTE: This can be stored at 4 &#176;C for up to 3 months.</li>
	<li>Make 500 mL of Phosphate Buffered Saline (PBS; 4 g of NaCl, 0.1 g of KCl, 0.77 g of Na2HPO4, and 0.12 g of KH2PO4, 500 mL of ultrapure water), measure and adjust the pH to 7.2 &#177; 0.2 at 25 &#176;C and autoclave. 
	NOTE: This can be store at 4 &#176;C for up to 6 months.</li>
	<li>Make 200 mL of 1 M MgSO4 (49.294 g and 200 mL of ultrapure water) and sterilize via 0.22 &#181;m polyethersulfone filter.</li>
	<li>Make 500 mL of TSB with 10 mM MgSO4 (mTSB; 15 g of TSB powder, 500 mL of ultrapure water, and 5 mL of 1 M MgSO4) and autoclave; let the autoclaved media cool to room temperature. Apply the aseptic technique. Using a serological pipette, carefully transfer 5.0 mL of 1 M MgSO4; swirl gently to mix.</li>
</ol>
2. Preparation of bacterial culture
<ol>
	<li>To prepare Bacterial Research Laboratory Culture (BRLC), remove glycerol stock vial(s) from the freezer inventory and transfer to the laboratory.</li>
	<li>Use a disposable inoculation loop or equivalent, dip into the vial, remove a scraping of inoculum (frozen slush), and streak onto a TSA plate or equivalent within a level II biological safety cabinet. Incubate the plate at 37 &#177; 2 &#176;C for 15&#8722;18 h.</li>
	<li>Bag the prepared BRLC plates and store at 4 &#176;C. 
	NOTE: General expiry period for BRLC: 14 days at 4 &#176;C after generation.</li>
	<li>Inoculate a single colony of each E. coli O157 strain from the BRLC plates to be tested in 10 mL of TSB and statically incubate at 37 &#176;C for 18 h to reach 9 log10 CFU/mL.</li>
	<li>Prepare a serial dilution of the overnight cultures (the following morning), each E. coli O157 strain using mTSB containing 10 mM of MgSO4 to achieve 4-5 log10 CFU/ml (or other desired inoculum level).</li>
	<li>Mix an equal volume of an overnight culture of each strain to achieve 4-5 log10 CFU/mL in total (or as desired) to prepare the bacterial mixture.</li>
	<li>Immediately place the diluted bacterial culture at 4 &#176;C for pending use.</li>
	<li>Dilute the inoculum culture 10 or 100-fold and plate 0.1 mL aliquots of these dilutions on the TSA plates to obtain the isolated colonies.</li>
</ol>
3. Preparation of phage working solutions
<ol>
	<li>Propagate high-titer working stocks for each phage to be screened (&#8805;108 PFU/mL) by following the standard methods4. 
	NOTE: The general expiry period for the phage stocks is 3 months in a plastic bottle at 4 &#176;C after generation.</li>
	<li>To achieve the desired titer of, e.g., ~108 PFU/mL for lysis kinetics, dilute individual phage preparations with mTSB containing 10 mM of MgSO4. Prepare phage cocktails by mixing equal volumes of each working stock with the same titer in all the possible combinations.</li>
</ol>
4. Preparation of in-vitro lysis kinetics for individual phage and phage cocktails
<ol>
	<li>Prepare serial 10-fold dilutions of each phages in Columns 1-8 in sterile 96-well microplates to set up the microplate assay. 
	NOTE: Four phages against one bacterial strain can be tested in duplicate, in adjacent columns, on each plate. The remaining four columns are for controls, which are without phage, as well as mTSB blank (Figure 1).</li>
	<li>Place 180 &#181;L of mTSB to wells from Columns 1 to 12&#160;of the 96-well microplate.</li>
	<li>Add 20 &#181;L of diluted individual phages or phage cocktails (~108 PFU/mL) to wells 1-8 of the top row of the microplate (Row A).</li>
	<li>For the phage-free and Blank control, add 20 &#181;L of mTSB to the top well of Columns 9, 10, and 11.</li>
	<li>With a 12-channel pipette, dilute down the plate. Transfer 20 &#181;L from row to row. Mix the well contents by gentle, repeated aspiration, ejection (at least five times), and changing tips between dilutions. Remove 20 &#181;L from the last row (Row H). 
	NOTE: Using tips with filters is recommended to prevent cross-contamination.</li>
	<li>Set up a reservoir for each strain, use 1 mL pipette to transfer 2-3 mL of the diluted culture into the reservoir. For columns 1-10, use a multichannel pipette to add 20 &#181;L of the diluted bacterial culture to each well. Change tips between each addition.</li>
	<li>Cover and incubate the microplate at desired conditions (e.g., 37 &#176;C for 10 h or 22 &#176;C for 22 h).</li>
	<li>At 2 h or other desired intervals, remove the microplates from the incubator.</li>
</ol>
5. Determination of optical density
<ol>
	<li>Examine for optical density at 600 nm (OD600nm) using a Microplate Reader. 
	NOTE: It is advised to set up and save the assay protocol in the program before test plate preparation.</li>
	<li>Turn on the microplate reader and open program.</li>
	<li>Select the Simple mode in the Startup Options window and select Create a New Protocol in Task Manager (Supplementary Figure 1a,b).</li>
	<li>From the Select Plate Type window, choose 96 Well Plate from the drop-down list (Supplementary Figure 2).</li>
	<li>Select Absorbance for detection method, the Endpoint/Kinetic option for read type, and Monochromators for optics type. Click on OK (Supplementary Figure 3).</li>
	<li>For Read Step, enter 600 nm for wavelengths and select Normal for read speed. Click on OK (Supplementary Figure 4).</li>
	<li>To set the temperature for the assay, click on the Incubate checkbox and select Incubator On. Enter the desired temperature in the Temperature box. Click on OK. To prevent condensation on the plate lid during incubation, set a temperature gradient by entering a value (1-3) in the Gradient box. Click on OK (Supplementary Figure 5a). 
	NOTE: Click on the Preheat Before Continuing With the Next Step checkbox for the 37 &#176;C incubation temperature. This step is not required for the test condition at 22 &#176;C (RT).</li>
	<li>Next, click on the Kinetic checkbox to open the Kinetic Setup. Set the run time for 10 h for 37 &#176;C incubation or 22 h for RT. Enter 2 h for reading interval. Click on OK (Supplementary Figure 5b).</li>
	<li>Click on the Shake checkbox in the Procedure window to set up the shake condition, if needed, for each kinetic read (Supplementary Figure 6a).</li>
	<li>Select Linear for Shake Modes and change Duration to 30 s. Set Linear Frequency value to 731 cpm (2 mm), and then click on OK (Supplementary Figure 6b).</li>
	<li>In the Protocol Summary Dialogue window, click on Plate Layout and select Blanks, Assay Controls, and Samples. Click on Next (Supplementary Figure 7). 
	NOTE: Enter 1 in Number of Different Control Types for negative control.</li>
	<li>After defining the settings for each well type, click on Finish.</li>
	<li>Select a Well ID in the left-hand side interface, and then assign it to the matrix shown in the plate layout widow. Click on OK (Supplementary Figure 8).</li>
	<li>Click on the Read Plate button and save the protocol as a .prt file and click on Save (Supplementary Figure 9).</li>
	<li>Insert the plate and click on OK.</li>
	<li>Once the experiment is completed, save the experiment as a .xpt file and click on Save (Supplementary Figure 10).</li>
	<li>Export data to Excel by clicking on the Yes button in the message window box for further analysis (Supplementary Figure 11).</li>
	<li>Click on the Save Yes/No selection and the Don&#39;t Ask Again checkbox to save preference.</li>
</ol>
6. Data analyses
<ol>
	<li>Repeat at least two independent experiments as described above. Compile results from all the independent trials. Calculate the average and standard deviation of the OD600 from each phage-treated and -free culture.</li>
	<li>Perform square root of the OD values at 600 nm and analyze them using an appropriate statistical model for each bacterial strain and temperature. 
	NOTE: For SAS software, the MIXED model and the least-squares to differentiate means (P &#60; 0.05) are selected. For each strain, assign panels A&#8722;G to each phage treatment of which overall anti-O157 efficacy across time and MOIs differed (P &#60; 0.05).</li>
	<li>Define superior phage efficacy based on OD600nm value &#8804; 0.01 corresponding to no detectable bacterial growth (limit of detection:300 CFU/mL).
	<ol>
		<li>Analyze effects of time, incubation temperature, E. coli O157 strains, MOIs, and phage types on phage efficacy using an appropriate statistical model. 
		NOTE: For SAS software, use GLIMMIX with random measures.</li>
		<li>Calculate Odds Ratios to compare superior efficacy for different environmental and biological factors of interest.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Carvalho, C., Costa, A. R., Silva, F., Oliveira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophages+and+their+derivatives+for+the+treatment+and+control+of+food-producing+animal+infections.">Bacteriophages and their derivatives for the treatment and control of food-producing animal infections.</a> Critical Reviews in Microbiology. 43 (5), 583-601 (2017).</li><li>Czaplewski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatives+to+antibiotics-a+pipeline+portfolio+review.">Alternatives to antibiotics-a pipeline portfolio review.</a> The Lancet. Infectious Diseases. 16 (2), 239-251 (2016).</li><li>Cisek, A. A., Dabrowska, I., Gregorczyk, K. P., Wyzewski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+therapy+in+bacterial+infections+treatment:+one+hundred+years+after+the+discovery+of+bacteriophages.">Phage therapy in bacterial infections treatment: one hundred years after the discovery of bacteriophages.</a> Current Microbiology. 74 (2), 277-283 (2017).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Escherichia+coli+O157+on+beef+at+37,+22+and+4&#176;C+by+T5-,+T1-,+T4-and+O1-like+bacteriophages.">Control of Escherichia coli O157 on beef at 37, 22 and 4&#176;C by T5-, T1-, T4-and O1-like bacteriophages.</a> Food Microbiology. 51, 69-73 (2015).</li><li>Moye, Z. D., Woolston, J., Sulakvelidze, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+applications+for+food+production+and+processing.">Bacteriophage applications for food production and processing.</a> Viruses. 10 (4), (2018).</li><li>Ly-Chatain, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+factors+affecting+effectiveness+of+treatment+in+phages+therapy.">The factors affecting effectiveness of treatment in phages therapy.</a> Frontiers in Microbiololgy. 5, 51 (2014).</li><li>Dy, R. L., Rigano, L. A., Fineran, P. 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-based+biocontrol+strategies+and+their+application+in+agriculture+and+aquaculture.">Phage-based biocontrol strategies and their application in agriculture and aquaculture.</a> Biochemical Society Transactions. 46 (6), 1605-1613 (2018).</li><li>Bourdin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coverage+of+diarrhoea-associated+Escherichia+coli+isolates+from+different+origins+with+two+types+of+phage+cocktails.">Coverage of diarrhoea-associated Escherichia coli isolates from different origins with two types of phage cocktails.</a> Microbial Biotechnology. 7 (2), 165-176 (2014).</li><li>Niu, Y. D., Liu, H., Johnson, R. P., McAllister, T. A., Stanford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+individual+bacteriophages+does+not+predict+efficacy+of+bacteriophage+cocktails+for+control+of+Escherichia+coli+O157.">Efficacy of individual bacteriophages does not predict efficacy of bacteriophage cocktails for control of Escherichia coli O157.</a> Frontiers in Microbiololgy. 12, 616712 (2021).</li><li>Niu, Y. D., Liu, H., Johnson, R. P., McAllister, T. A., Stanford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+bacteriophage+T5virus+on+growth+of+Shiga+toxigenic+Escherichia+coli+and+Salmonella+strains+in+individual+and+mixed+cultures.">Effect of a bacteriophage T5virus on growth of Shiga toxigenic Escherichia coli and Salmonella strains in individual and mixed cultures.</a> Virology Journal. 17, 3 (2020).</li><li>Br&#252;ssow, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+needed+for+phage+therapy+to+become+a+reality+in+Western+medicine.">What is needed for phage therapy to become a reality in Western medicine.</a> Virology. 434 (2), 138-142 (2012).</li><li>Chan, B. K., Abedon, S. T., Loc-Carrillo, 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+cocktails+and+the+future+of+phage+therapy.">Phage cocktails and the future of phage therapy.</a> Future Microbiology. 8 (6), 769-783 (2013).</li><li>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=Bacteriophage+therapy:+developments+and+directions.">Bacteriophage therapy: developments and directions.</a> Antibiotics. 9 (3), 135 (2020).</li><li>Niu, Y. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+range+and+lytic+capability+of+four+bacteriophages+against+bovine+and+clinical+human+isolates+of+Shiga+toxin-producing+Escherichia+coli+O157:H7.">Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7.</a> Journal of Applied Microbiology. 107 (2), 646-656 (2009).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This protocol described a robust approach for systemically evaluating phage efficacy against foodborne pathogens, including STEC9 and Salmonella10. One critical step is when diluting overnight culture of bacteria, using pre-chilled medium and manipulating the dilution with an ice bucket are recommend to minimize potential bacterial growth. In addition, phage dilution was prepared before diluting bacterial culture. The enumeration step 2.8 provided actual numbers of bacterial inoculum for calculation of the final MOI applied. For phage preparation, crude phage lysates prepared by filter phage infected in 4-6 h of bacterial culture is generally used. The critical step associated with phage infectivity is always to use phage work stocks prepared within 3 months. Extremely accurate pipetting (particularly when using a multichannel pipette) and uniformity of approach are also essential to obtain comparable and interpretable results. Modified TSB supplemented with 10 mM of Mg2+ was used to dilute phages, bacterial culture, and base medium to optimize adsorption and infection of phages.
As bacteria proliferate during the log phase, even below incubator temperature, it is recommended to use diluted overnight culture instead of log-phase culture, to minimize potential bacterial growth.
The proposed protocol has limitations. First, because microplate can only hold 200 &#181;L, prolonged incubation may cause substantial evaporation and is not recommended. In this case, the assay may not be suitable for slow-growing bacteria. Second, the proposed protocol was not able to monitor the amplification of phages. Third, this protocol could not monitor the development of phage resistance over time, a critical factor that determines the outcome of phage treatment11,12. Follow-up experiments are required to assess the further performance of the most influential cocktail in the screening in preventing the emergence of anti-phage mutants in an extensive broth culture system and other biological matrices.
In contrast to conventional antimicrobials, the biological nature of phages affects the complexity of biocontrol and therapeutic use in practical settings. Conventionally, rational selection of phage cocktails is primarily based on lytic activity and the host range of phages. Phage candidates with the strongest lytic activity and broadest host range are often recommended13,14. However, based on the current study, phages such as rV5 and T1, although alone not as virulent as T4 and T5, greatly facilitated overall biocontrol outcome when combined with T4 and/or T5. Consequently, to achieve superior efficacy of phage cocktails, systemic screening of antibacterial activity of potential phage combinations against targeted host strains under desired environmental conditions is recommended. In addition, the determination of receptors for phage candidates and the inclusion of phages with various receptors may prevent competition for host attachment, thwart rapid development of anti-phage mutants, and improve biocontrol outcomes13.
This method enabled accurate quantification of phage lysis kinetics in a high-throughput format. Furthermore, it allowed systematic evaluation of various biological and environmental factors on the antibacterial efficacy of an assortment of phages, thereby facilitating the formulation of phage cocktails with optimized outcomes. The method&#39;s future applications and development are assumed to involve in situ monitoring the efficacy of each phages within phage cocktails by fluorescence labeling of phages. In addition to the proposed protocol, understanding genetic determinants that promote synergistic and facilitated effects between phages when co-infecting one host would facilitate the formulation of appropriate phage cocktails with superior efficacy.

Bacteriophages (phages) are viruses that naturally invade bacterial cells, disrupting bacterial metabolism and causing lysis of the bacterium. In contrast to conventional antimicrobials (e.g., antibiotics), phage host spectrums are relatively narrow, only capable of infecting a targeted set of bacterial species or strains and thus should minimize collateral effects on microbiota that benefit animal and human health. With the rise of antimicrobial resistance (AMR), phages and their derivatives lead to alternative antimicrobials to control bacterial infectious diseases, including AMR bacterial infections in humans and animals1,2. Phages have confirmed therapeutic potential against &#62;20 bacterial pathogens that cause superficial infections and infections of the upper respiratory system and gastrointestinal tract of humans3.
In agricultural settings, a phage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens. Phage biocontrol is well accepted as a green technology, effective at decontaminating foodborne pathogens (e.g., Shiga-toxin producing Escherichia coli (STEC), Salmonella, and Listeria) in various foods4,5. In addition, phages can be used as sanitizers to disinfect food processing surfaces and animal hides, which can be integrated into conventional antimicrobial systems (e.g., chemicals, steam, and hot water pasteurization) to enhance desired outcomes and reduce environmental impacts. The use of phages to reduce zoonotic bacteria in animals is also promising1. However, there is a need to address the technical challenges to improve outcomes from the phage biocontrol approach to be popularly applied in diverse food production systems. The main challenge is the impaired effectiveness of phages due to the development of bacteria-resistant mutants5 and changes in bacterial physiology due to exposure to environmental stressors6.
For minimizing the risk of phage resistance, phage cocktails (i.e., a combination of multiple phages) are proposed and have improved biocontrol potency in agriculture and aquaculture settings7. However, from several studies, it has been proved that phage cocktails did not always offer better effectiveness than the administration of a single phage. For instance, a cocktail of 3 T4-like phages had a narrower host range against E. coli strains8. Furthermore, AKFV33, a member of Tequintavirus, had greater efficacy than a cocktail of four phages in removing E. coli O157 from beef, despite the incubation temperatures applied4. Recently, it has been reported that the effectiveness of individual phages does not predict the efficacy of phage cocktails for control of O1579, as interactions among multiple phages can alter efficacy. Most importantly, numerous factors, such as phage genera and combinations, targeted strains and MOIs, and incubation temperatures and times, may impact interactions between phages. Therefore, carefully screening combinations of phages against specific bacteria to assess phage synergy or facilitation, or at least to ensure minimal phage antagonism under specific environmental conditions, is vitally important for optimal outcomes. Here, a method is described to systematically assess the efficacy of various phage combinations against foodborne pathogens under a range of environmental conditions. The benefit of this approach is to enable screening of all possible biotic and abiotic factors predicted to affect the antibacterial efficacy of phages in natural settings. In the protocol, STEC&#160;O157 and their infecting phages are employed as an example.

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Essential supplies, reagents, and equipment&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Inoculating loops</td><td>VWR</td><td>12000-806</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>1374361</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H2O</td></tr><tr><td>Petri Dishes with Clear Lid</td><td>Fisher</td><td>FB0875713</td><td>Diameter: 100 mm, sterile</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Fisher</td><td>10010023</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-1000XLS+</td><td>METTLER TOLEDO</td><td>17014382</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-300XLS+</td><td>METTLER TOLEDO</td><td>17014405</td><td></td></tr><tr><td>Pipet-Lite Multi Pipette L12-20XLS+</td><td>METTLER TOLEDO</td><td>17013808</td><td></td></tr><tr><td>Pipet-Lite Pipette, Unv. SL-20XLS+</td><td>METTLER TOLEDO</td><td>17014412</td><td></td></tr><tr><td>Pipette Tips RT LTS 1000&amp;micro;L FL 768A/8-low retention</td><td>METTLER TOLEDO</td><td>30389213</td><td></td></tr><tr><td>Pipette Tips SR LTS 20&amp;micro;L F 960A/5</td><td>METTLER TOLEDO</td><td>17005860</td><td></td></tr><tr><td>Pipette Tips SR LTS 300&amp;micro;L 768A/4</td><td>METTLER TOLEDO</td><td>17005867</td><td>no filter</td></tr><tr><td>Reservoir</td><td>METTLER TOLEDO</td><td>89094-662</td><td></td></tr><tr><td>Sterile, clear, 96-well flat-bottom polystyrene microplates with lids</td><td>Fisher</td><td>168055</td><td></td></tr><tr><td>Tryptic soy agar (TSA)</td><td>Sigma</td><td>105458-0500</td><td></td></tr><tr><td>Tryptic soy broth (TSB)</td><td>Sigma</td><td>105459-0500</td><td></td></tr><tr><td>T-Shaped Cell Spreaders</td><td>VWR</td><td>76299-566</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Instruments&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Analog Vortex Mixer</td><td>Fisher</td><td>02-215-414</td><td></td></tr><tr><td>Compact Microbiological Incubators</td><td>Fisher</td><td>50125590H</td><td></td></tr><tr><td>Magnetic Stirrer Hotplates</td><td>FIsher</td><td>13-889-335</td><td></td></tr><tr><td>Polygon Stir Bars</td><td>FIsher</td><td>14-512-125</td><td>length: 20 mm</td></tr><tr><td>Synergy Neo2 Hybrid Multi-Mode Reader</td><td>Fisher</td><td>BTNEO2M</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Software&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SAS</td><td>SAS Institute</td><td>9.4</td><td></td></tr></tbody></table>

bacteriophage effectiveness for biocontrol of foodborne pathogens evaluated via high-throughput settings

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant bacteria, novel antibacterial agents, are urgently needed. A bacteriophage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens in agricultural settings. Phage biocontrol is increasingly accepted as a sustainable technology, effective at decontaminating foodborne pathogens. To ensure effective biocontrol outcomes, systematic screening of phage combinations against targeted bacteria under required environmental conditions is crucial. Antibacterial efficacy of phage cocktails may be affected by phage genera and combination, targeted bacterial strains, the multiplicity of infection, temperature, and time. To formulate a phage cocktail with superior efficacy, the proposed method was to systematically evaluate the effectiveness of individual phages and phage cocktails in killing foodborne bacterial pathogens under targeted conditions. Bacterial killing efficacy was monitored by measuring optical density at desired temperatures and durations. Superior phage efficacy was determined by complete inhibition of bacterial growth. The proposed method is a robust, evidence-based approach to facilitate formulating phage cocktails with superior antibacterial efficacy.

Following the protocol, a comparison of the anti-O157 phage efficacy with various phage combinations, temperatures, times, and MOIs was performed. Impacts of incubation temperature and times, MOIs, phages, and bacterial strains used on enhancing anti-E. coli O157 efficacy is presented in Table 1 (odds ratio table)9. The percentage (%) of optical turbidity measurement &#8804; 0.01 yielded was analyzed by each phage preparation under each experimental condition. Based on this analysis, anti-O157 phage efficacy was maximized after 14, 16, or 18 h of incubation at 22 &#176;C (P &#60; 0.001) and MOI = 1000 (P &#60; 0.001), with 75% and 89% of the growth of phage-treated culture being completely inhibited, respectively. In general, among 11 phage preparations, a cocktail of T1, T4, and rV5 was most effective (P &#60; 0.05) against O157. In addition, across incubation temperatures, times, MOIs, and phages tested, sensitivity to phages varied, with O157 strains tested with strain CO281-31N being most sensitive (P &#60; 0.001) and 3081 (P &#60; 0.001) being least sensitive to phages.
For understanding the phage-killing kinetics of each bacterial strain tested, OD600 value was plotted against each sampling time, MOIs, and phage treatment at each incubation temperature. Representative results from the efficacy of phages against E. coli O157 3081 at 37 &#176;C are shown in Figure 2. It is ensured that the growth curve of phage-free control culture is normal before assessing inhibition of growth curve of phage-treated culture. According to Figure 2, the inclusion of T4 completely inhibited bacterial growth at 37 &#176;C, at each sampling time at an MOI as low as 1. To overview how phage inhibited bacterial growth over time at various temperatures, irrespective of MOIs, the mean OD600nm value averaged from all the MOIs (MOI &#62; 0) was plotted against each sampling time and phage treatment (Figure 3). Compared to 37 &#176;C, particular phage killing efficacy, e.g., phages T4 and T1 + T4, was enhanced over 22 &#176;C. Another approach to summarize and compare overall anti-O157 effectiveness among phages is shown in Table 2 and Table 39. OD600 value was averaged from each sampling time and MOI and ranked phage efficacy from highest (A) to lowest (F: 37 &#176;C and D: 22 &#176;C). This table facilitates the identification of the best phage treatment. For example, for strain 3081, phages T4 and T5 + T4 (Panel A) were the most effective treatment at 37 &#176;C, whereas in addition to phage T4, phages T1 + T4, T1 + T4 + rV5, and 4 phage cocktails (Panel A) were the most effective treatment at 22 &#176;C.
This protocol also enabled us to compare phage efficacy against various bacterial strains targeted and customize phage preparation. For example, when selecting phages, at 37 &#176;C, excluding strain 3081, phage T1 + T4 + rV5 had the most significant effectiveness against all other strains, regardless of their phage types. At 22 &#176;C, the 4-phage cocktail was the most effective against all strains tested. The best MOIs resulted in complete lysis (OD600&#160;&#8804; 0.01), which warranted potential superior efficacy under experimental conditions (Table 2 and Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62812/62812fig01.jpg" /> 
Figure 1: Suggested layout of 96-well microplate assay. Serial 10-fold dilutions of each phage are prepared in Columns 1-8 of sterile 96-well microplates. Four phages against one bacterial strain can be tested in duplicate, in adjacent columns, on each plate. The remaining columns are for controls. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62812/62812fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62812/62812fig02.jpg" /> 
Figure 2: Growth curves of E. coli O157 strain 3081 at 37 &#176;C treated and not treated with phages at each MOIs. The data were compiled from two independent trials. Bars present standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62812/62812fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62812/62812fig03.jpg" /> 
Figure 3: Growth curves of E. coli O157 strain 3081 strains. (A) Growth curves at 37 &#176;C. (B) Growth curves at 22 &#176;C. Each curve represents individual and mixed cultures, treated and not treated with phages across MOIs. The data were compiled from two independent trials. Bars present the standard error of the mean (adapted from Reference9 with permission). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62812/62812fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Odds ratios comparing the likelihood of superior phage efficacy against E. coli O157 at incubation temperatures, incubation times, MOIs, phages, and strains of E. coli O157 (adapted from Reference9 with permission). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62812/62812_Table 1_Final_.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Overall phage efficacy against E. coli O157 at 37 &#176;C (adapted from Reference9 with permission). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62812/62812_Table 2_Final_.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Overall phage efficacy against E. coli O157 at 22 &#176;C (adapted from Reference9 with permission). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62812/62812_Table 3_Final.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Figures 1-11: Snapshots of the microplate reader operation and procedures. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62812/Niu_Supplementary Information.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings at JoVE.com

Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant ...

bacteriophage-effectiveness-for-biocontrol-foodborne-pathogens

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.7K Views.  University of Calgary. Dit eenvoudig uit te voeren protocol evalueert de antibacteriële werkzaamheid van diverse bacteriofaagcombinaties, ook bekend als faagcocktails, onder verschillende omstandigheden in een high-throughput setting. De belangrijkste voordelen van de techniek zijn het vermogen om verschillende biotica en abiotische factoren, zoals temperatuur, te streamen die de faageffectiviteit onder verschillende omgevingsomstandigheden kunnen beïnvloeden. Deze methode kan het ontwerp van geschikte faagcockta...

Video: Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

Setting Up PRANCE: A Comprehensive Guide for Robotics-Accelerated Evolution

A Practical Guide to Phage- and Robotics-Assisted Near-Continuous Evolution

Robotics-accelerated Evolution techniques improve the reliability and speed of evolution using feedback control, improving the outcomes of protein and organism evolution experiments. In this article, we present a guide to setting up the hardware and software necessary to implement Phage- and Robotics-assisted Near-continuous Evolution (PRANCE). PRANCE combines fast phage-based molecular evolution with the ability to run hundreds of independent, feedback-controlled evolution experiments simultaneously. This paper will describe the hardware requirements and setup for PRANCE, including a liquid-handling instrument, a plate reader, auxiliary pumps, heaters, and 3D-printed containers. We describe how to configure the liquid handling robot to be compatible with Python-based open-source software. Finally, we provide suggestions for the first two experiments that can be conducted with a newly constructed PRANCE system that exercises its capabilities and validates that the system is ready to conduct multiplexed evolution. This guide is intended to serve as a handbook for navigating the considerable equipment setup associated with conducting robotics-accelerated evolution.

Author Spotlight: Advancing Protein Engineering &#8211; Harnessing Evolution Through PRANCE and Lab Automation

Phage- and Robotics-assisted Near-continuous Evolution (PRANCE) is a technique for rapid, robust protein evolution. Robotics allows the parallelization of experiments, real-time monitoring, and feedback control.

Robotics-accelerated Evolution techniques improve the reliability and speed of evolution using feedback control, improving the outcomes of protein and ...

Bioengineering

Exploring Gut Phage-Bacteria Dynamics in Murine Models Using T4 Phage

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host&#39;s health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli,&#160;in the context of the murine gastrointestinal tract. The methods described here outline&#160;how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels.&#160;This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

Author Spotlight: Investigating Bacteriophage-Induced Immune Responses in Gnotobiotic Mice

Bacteriophages (phages), viruses that infect bacteria, are an integral component of the gut microbiome. Though these symbiotic inhabitants drive bacterial fitness and population dynamics, little is understood about how they impact gut homeostasis and disease. This protocol studies isolated T4 phages within a mouse model, adaptable to other phage-bacterial pairs.

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across ...

Characterizing Actinobacteriophage Infection Using an Adapted Optical Density Assay

An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual phages interact with slow-growing bacterial hosts such as actinomycetes, an easy and reliable method for quantifying long-term bacterial growth in the presence of phages is needed. Spectrophotometric microplate readers allow for high-throughput repeated measurements, but incubating a small volume for an extended time can present technical challenges. This procedure adapts a standard 96-well microplate to allow for the co-culturing of phages and bacteria without sub-sampling for 96 h, with the bacterial growth recorded every 8 h using spectrophotometric absorbance values. These optical density values are analyzed using R to yield infection metrics, including the percent growth inhibition, relative virulence, and the Stacy-Ceballos index. The methods outlined here provide an effective way to conduct and analyze extended-duration microplate growth curve experiments and includes modifications to reduce evaporation and lid condensation. These protocols facilitate microplate-based assays of interactions between slow-growing bacterial hosts and their bacteriophages.

Author Spotlight: An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection  

An optical density-based microplate method is described to quantify long-term bacterial growth in the presence of bacteriophages, suitable for actinomycetes and other slow-growing bacteria. This method includes modifications to reduce evaporation and lid condensation and R code to calculate virus infection metrics, including the area under the curve, growth maximum, and relative virulence.

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual ...

Biology

Understanding the Impact of Temperate Bacteriophages on Their Lysogens Through Transcriptomics

Temperate phages are found integrated as prophages in the majority of bacterial genomes. Some prophages are cryptic and fixed in the bacterial chromosome, but others are active and can be triggered into a replicative form either spontaneously or by exposure to inducing factors. Prophages are commonly associated with the ability to confer toxin production or other virulence-associated traits on their host cell. More recent studies have shown they can play a much bigger role in altering the physiology of their hosts. The technique described here has enabled us to investigate how prophages affect gene expression in the opportunistic bacterium Pseudomonas aeruginosa.
In this work, the growth of the wild-type P. aeruginosa strain PAO1 was compared with that of isogenic lysogens carrying different combinations of prophages from the Liverpool Epidemic Strain (LES) LESB58. In a lysogen culture, a proportion of bacterial cells will be supporting lytic bacteriophage replication (spontaneous induction) with a high level of expression per cell of&#160;late phage genes, such as those associated with the assembly of phage particles,&#160;thus masking the low-level gene expression associated with lysogen-restricted gene&#160;expression. The impact of spontaneous induction can thus obscure prophage gene expression across a lysogen population.
Growth profiling experiments were used to identify spontaneous induction, which was minimal during the early exponential growth phase. This study reports how to prepare sample cultures during the early exponential growth phase and how to set up adequate controls despite low cell numbers. These protocols ensure the reliable and reproducible comparison of wild-type and lysogenic bacteria under various conditions, thus improving the transcriptomic profiling of prophage genomes and aiding in the identification of previously unrecognized prophage functions.

This protocol enables the impact of prophages on their hosts to be revealed. Bacterial cultures are synchronized using conditions that best support the lysogenic state, limiting spontaneous induction. RT-qPCR unequivocally distinguishes prophage-restricted genes and those uncoupled from phage control from those that are expressed during the lytic replication cycle.

Temperate phages are found integrated as prophages in the majority of bacterial genomes. Some prophages are cryptic and fixed in the bacterial chromosome, ...

Using Bacterial LPS to Eliminate Bacteriophages from Salmonella Cultures

Bacteriophage Removal from Infected Salmonella Cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Author Spotlight: Efficiently Eliminating Bacteriophages from Infected Salmonella Cultures Using Lipopolysaccharides

Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and ...

Detection of Bacteriophages in Environmental Samples

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Alex Wassimi
Viruses are a unique group of biological entities that infect both eukaryotic and prokaryotic organisms. They are obligate parasites that have no metabolic capacity, and in order to replicate, rely on host metabolism to produce viral parts that self-assemble inside host cells.
Viruses are ultramicroscopic&#8212;too small to be viewed with the light microscope, visible only with the greater resolution of the electron microscope. A viral particle consists of a nucleic acid genome, either DNA or RNA, surrounded by a protein coat, known as a capsid, composed of protein subunits or capsomers. In some more complex viruses, the capsid is surrounded by an additional lipid envelope, and some have spike-like surface appendages or tails.
Viruses that infect the intestinal tract of humans and animals are known as enteric viruses. They are excreted in feces and can be isolated from domestic wastewater. Viruses which infect bacteria are known as bacteriophages, and those which infect coliform bacteria are called coliphages (Figure 1). The phages of coliform bacteria are found anywhere coliform bacteria are found.
<img alt="Figure 1" src="/files/ftp_upload/10190/10190fig1.jpg" /> 
Figure 1. Coliphage T2.

Plaque Assay: Phage Enumeration in Environmental Samples

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Alex Wassimi
Viruses are a unique group of ...

Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video