Name: bacteriophage removal from infected salmonella cultures
Uploaded: 2024-06-28T14:20:00.000+00:00
Duration: 7 min 19 s
Description: Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and ...

We thank Dr. Carmen R. Beuz&#243;n and Roc&#237;o Carvajal-Holguera for helpful discussions and suggestions. This work was supported by the grant PID2020-116995RB-I00 funded by MICIU/AEI/ 10.13039/5011100011033 and the VI Plan Propio de Investigaci&#243;n y Transferencia from the Universidad de Sevilla.

NOTE: Prior to performing the procedure of elimination of phages, we describe the preparation of a Salmonella culture infected with 9NA bacteriophages. In Figure 1, a general representation of the complete procedure for bacteriophage removal in bacterial cultures is illustrated.
1. Preparation of Salmonella cultures infected with bacteriophages
<ol>
	<li>Bacterial culture preparation 
	NOTE: In this study, Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC 14028 opvAB::lacZ (SV8011)13 was used.
	<ol>
		<li>Slowly thaw the vial retrieved from -80 &#176;C that contains the S. enterica strain of interest by placing it on ice.</li>
		<li>Streak out the S. enterica strain from the thawed dimethyl sulfoxide stock onto a Lennox broth (LB) plate (10 g/L tryptone, 5 g/L NaCl, 5 g/L yeast extract, and 15 g/L agar, see Table of Materials). Incubate the plate at 37 &#176;C for 24 h.</li>
		<li>Using an inoculation needle, transfer a colony from the S. enterica plate and inoculate it into 5 mL of liquid LB in a glass tube. Incubate at 37 &#176;C for 24 h with aeration by shaking at 200 rpm.</li>
	</ol>
	</li>
	<li>Bacteriophage preparation 
	NOTE: Bacteriophage 9NA, which belongs to the family Siphoviridae (order Caudovirales)14, was used in this protocol. Notably, other Salmonella enterica bacteriophages that use the O-antigen of the lipopolysaccharide as a receptor can be used.
	<ol>
		<li>Prepare the bacteriophage broth by adding 0.1 mL of concentrated bacteriophage lysate to a mixture of 100 mL of nutrient broth (NB; 5 g/L peptone and 3 g/L yeast extract), 2 mL of 50x E salts (38.78 mM MgSO47 H2O, 520.5 mM Citric acidH2O, 2.87 M K2HPO4 anhydrous and 829 mM NaNH4HPO44 H2O) and 0.4 mL of 50% glucose. Mix thoroughly by inversion.
		<ol>
			<li>A representative example of bacteriophage concentration could be 109 PFU/mL (Plaque Forming Units/mL), but lower concentrations can be used with similar results (see Table of Materials). Estimate bacteriophage concentration following step 1.2.3.</li>
		</ol>
		</li>
		<li>Prepare lysate by following the steps described below.
		<ol>
			<li>Prepare the S. enterica inoculum in 5 mL of LB (10 g/L tryptone, 5 g/L NaCl, and 5 g/L yeast extract). Incubate at 37 &#176;C and 200 rpm for 24 h.</li>
			<li>Mix 4 mL of bacteriophage broth with 1 mL of the overnight culture of S. enterica. Incubate the mix at 37 &#176;C and 200 rpm for 8 h.</li>
			<li>To discriminate between bacteria and bacteriophages, centrifuge the mix at 2,377 x g for 20 min. Recover the supernatant in a glass tube, add 800 &#181;L of chloroform (see Table of Materials), and vortex it. Make sure to only transfer the upper phase when a mixture with chloroform is used. 
			NOTE: It is important to prevent the transfer of residual chloroform, which could affect the efficiency of the procedure. The chloroform step is common in both isolation and propagation protocols as a way of maximizing released phages. However, chloroform may inactivate some phages or destroy their infectivity15. Therefore, this step is only applicable to phages resistant to chloroform.</li>
			<li>Maintain the lysates at room temperature for 2 h and then store them at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Titrate the lysate to quantify the number of bacteriophages/number of PFU per mL present in the prepared lysate. To do this, perform the overlay technique described below.
		<ol>
			<li>Prepare 5 mL of LB of inoculum for S. enterica. Incubate at 37 &#176;C for 24 h with aeration by shaking at 200 rpm.</li>
			<li>Perform serial dilutions of the lysate into LB to reach a countable number of PFU/mL. To determine the necessary dilution factors, identify the initial bacteriophage lysate concentrated. For a representative example of bacteriophage concentration at around 109 PFU/mL, the dilution factors that result in a countable number of plaques are between 106-1010.</li>
			<li>Add 100 &#181;L of the appropriate lysate dilution and 60 &#181;L of the overnight culture of S. enterica prepared the day before to 5 mL of LB soft agar, prepared by mixing liquid LB and LB-agar in a proportion 1:1 and maintaining at 56 &#176;C to avoid solidification. Mix carefully, avoiding bubbles.</li>
			<li>Pour each mix into the top of an LB plate and keep on the bench until the medium solidifies.</li>
			<li>Incubate plates for 24 h at 37 &#176;C. Count the number of plaques on each plate. To determine the number of PFU/mL, multiply the number of plaques of each plate by the dilution factor (FD). To ensure efficient phage infection, 109-1012 PFU/mL are necessary. 
			PFU/mL= No. plaques in the plate x FD</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Infection of Salmonella cultures with bacteriophages
	<ol>
		<li>Dilute an overnight culture of S. enterica to 1:100 in a final volume of 5 mL of LB and add 0.1 mL of lysate (concentrated at 109-1012 PFU/mL). Incubate at 37 &#176;C and 200 rpm for 24 h.</li>
	</ol>
	</li>
	<li>Determination of phage titer as PFU in plate assay
	<ol>
		<li>To quantify the number of bacteriophages in the infected culture, perform plate assays to determine the phage titer as PFU/mL as described below.
		<ol>
			<li>Transfer 1 mL of the culture to a tube. Centrifuge at 17,115 x g for 2 min and pour the supernatant into a new labeled vial.</li>
			<li>Add 100 &#181;L of chloroform to the vial and vortex to remove bacterial debris from the solution. Follow step 1.2.3.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Bacteriophage removal from infected Salmonella enterica cultures (see Figure 2)
NOTE: To monitor the bacteriophage removal process, different aliquots are taken throughout the cleaning protocol for titration. In total, there are eight aliquots to confirm that the number of PFU/mL is decreasing throughout the cleaning process until complete elimination.
<ol>
	<li>Removal of bacteriophage in suspension
	<ol>
		<li>Dilute the bacterial culture containing phages to 1:100 in 5 mL of 0.22 &#181;m-filtered LB in a 50 mL conical centrifuge tube. Titration point 1: This is the first aliquot for titration (the starting infected Salmonella culture).</li>
		<li>Centrifuge the suspension for 10 min at 2,377 x g. Carefully remove the supernatant. Do not use a pipette; pour the tube. Try to leave some volume at the bottom of the conical centrifuge tube to ensure the presence of cells.</li>
		<li>Wash bacterial cells with 10 mL of 0.22 &#181;m-filtered LB medium. Repeat centrifugation and washing steps 3x. Resuspend the pellet in 5 mL of 0.22 &#181;m-filtered LB. Titration point 2: This is the second aliquot for titration.</li>
		<li>Transfer 50 &#181;L of the above bacterial suspension through a sterile 0.45 &#181;m filter (see Table of Materials) using a vacuum filtration system. Rinse the filter with 100 mL of 0.22 &#181;m-filtered LB. 
		NOTE: The head size of bacteriophage 9NA is 0.067 &#181;m. In contrast, Salmonella is 2-5 &#181;m long and 0.5-1.5 &#181;m wide. Thus, if 0.45 &#181;m membranes are used, most bacteria will not pass because they are larger than 0.45 &#181;m, while most phages will pass through the 0.45 &#181;m filter pores.</li>
	</ol>
	</li>
	<li>Removal of bacteriophage contained inside Salmonella cells
	<ol>
		<li>To facilitate phage release from bacteria cells, incubate the 0.45 &#181;m filter containing cells for 2 h at 37 &#176;C inside the incubator without disassembling the system. 
		NOTE: This step of incubation is required to promote the lysis of the infected bacteria and the release of intracellular phages into the filter.</li>
		<li>Wash the 0.45 &#181;m filter using a vacuum filtration system, rinsing the filter with 100 mL of 0.22 &#181;m-filtered LB.</li>
		<li>Recover bacterial cells from the filter in 2 mL of 2x LB (20 g/L tryptone, 10 g/L NaCl, 10 g/L yeast extract; see Table of Materials). To do this, transfer the 0.45 &#181;m filter to a flask using sterile forceps. Then, add 2 mL of 2x LB over the filter and pipette several times to release cells from the filter.</li>
		<li>Centrifuge 1 mL of this suspension for 2 min at 10,354 x g. Discard the supernatant and wash bacterial cells 3x with 1 mL of filtered LB medium. Resuspend cells in 1 mL of 2x LB. Titration point 3: This is the third aliquot for titration. 
		NOTE: The use of a more concentrated LB (2x LB) is highly recommended because, as described below, commercial lipopolysaccharide (LPS) will be added, and this LPS dissolves in water, thus diluting the medium.</li>
	</ol>
	</li>
	<li>Avoid reinfection by deceiving the bacteriophages released from bacterial cells with commercial lipopolysaccharide as described below.
	<ol>
		<li>Incubate 20 &#181;L of the bacterial suspension in the presence of a commercial S. enterica lipopolysaccharide (LPS; see Table of Materials) to a final concentration of 3.75 mg/mL in 1 mL of 0.22 &#181;m-filtered LB for 2 h at 37 &#176;C with shaking at 200 rpm. 
		NOTE: Phages released from cells into the medium may bind to the commercial S. enterica LPS instead of bacterial LPS.</li>
		<li>After 2 h of incubation, transfer 1 mL of culture to a microcentrifuge tube and centrifuge the suspension for 2 min at 10,354 x g. Titration point 4: Use the supernatant as the fourth aliquot for titration.</li>
		<li>Wash pellet 3x with 1 mL of filtered LB medium.</li>
		<li>After washing, resuspend the pellet in 1 mL of 2x LB. Incubate 20 &#181;L of this mix in 1 mL of 0.22 &#181;m-filtered LB with 0.8 mg/mL of commercial LPS for 2 h at 37 &#176;C with shaking at 200 rpm. Titration point 5: Use the rest of the bacterial suspension for the fifth titration.</li>
		<li>After 2 h of incubation, transfer 1 mL of culture to a microcentrifuge tube and pellet by centrifugation for 2 min at 10,354 x g. Titration point 6: Use the supernatant as the sixth aliquot for titration by pouring the supernatant into a new vial.</li>
		<li>Wash bacterial cells 3x with filtered LB. After washing, resuspend collected cells in 1 mL of 0.22 &#181;m-filtered LB.</li>
		<li>Pass 1 mL of bacterial suspension through a sterile 0.45 &#181;m filter using a vacuum filtration system. Rinse the filter using 100 mL of 0.22 &#181;m-filtered LB.</li>
		<li>Recover cells from the 0.45 &#181;m filter in 2 mL of 2x LB medium. To that end, transfer the 0.45 &#181;m filter to a flask using sterile forceps. Then, add 2 mL of 2x LB over the filter and pipette several times to try to release cells from the filter.</li>
		<li>Pellet 1 mL of cells by centrifugation for 2 min at 10,354 x g. Wash 3x with 0.22 &#181;m-filtered LB. After washing, resuspend collected cells in 1 mL of 2x LB. Titration point 7: This is the seventh aliquot for titration. This aliquot will inform about the efficiency of removal of phages in bacterial cultures.</li>
	</ol>
	</li>
</ol>
3. Preparation of bacteriophage-free Salmonella culture after bacteriophage removal
<ol>
	<li>Prepare the phage-free inoculum. Add 100 &#181;L of cells from the bacterial suspension to 900 &#181;L of LB containing 0.45 mg/mL LPS. Incubate at 37 &#176;C for 24 h with shaking at 200 rpm.</li>
	<li>Titration point 8: Prepare the eighth aliquot for titration (the final potential un-infected culture). This point is to confirm the absence of phage re-infection.</li>
</ol>

Using Bacterial LPS to Eliminate Bacteriophages from Salmonella Cultures

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The authors have nothing to disclose.

Diverse strategies are employed by bacteriophages to recognize and infect bacterial hosts. Different molecular structures on the surface of bacteria can act as phage receptors: protein, polysaccharide, lipopolysaccharides (LPS), and carbohydrate moieties20. In Gram-negative bacteria, LPS is a common receptor for phages. In addition, other receptors are outer membrane proteins, pili, and flagella21.
The specific interaction between bacteriophages and bacteria based on the recognition of LPS8 has been exploited in this work for the development of a highly efficient protocol for the elimination of bacteriophages in infected bacterial cultures (Figure 1 and Figure 2). Our protocol does not favor the selection of phage-resistant cells; it only eliminates bacteriophages (Figure 6). Both phage-susceptible and phage-resistant cells remain in bacterial culture after performing this protocol of phage removal.
The traditional standard practice when a phage infection occurs is to attempt to eliminate all the contaminated material, followed by cleaning and sterilization22. The decontamination procedure involves exposing the bacterial culture to stressful conditions, such as high temperatures, in order to partially or completely eliminate the bacterial cells. As described in representative results, the crucial step in this protocol is the incubation of phages-infected bacterial cultures with commercial LPS, a non-harmful substance for bacterial cultures. This helps to preserve the viability of bacterial cultures and offers significant advantages for industrial applications in fermenters and bioreactors.
The incubation time in this protocol is 2 h to ensure sufficient time for phage lysis of bacterial cells. If different bacterial strains and bacteriophages are to be used, this parameter should be considered and defined by the user. In this case, an assay similar to that described in Figure 4 should be performed prior to the experiment.
Interestingly, the efficacy of this cleaning protocol could also be analyzed by employing an assay that monitors the phage content of a given sample. In this sense, epigenetic biosensors are a novel tool for bacteriophage detection23. A well-known phage biosensor able to detect coliphages, which use LPS as a receptor, is the opvAB::gfp system13,18,23,24. This phage biosensor detects an increase in the OpvABON subpopulation in the presence of phages that use O-antigen as a receptor. In this sense, we could use an opvAB::gfp fusion to monitor LPS-binding phages in various steps of this protocol and/or diverse media and conditions. These approaches could be valuable in determining the timing and locations at which an effective protocol may be necessary.
While LPS recognition is common, phages can also utilize a variety of other surface receptors on bacterial cells for attachment and infection. Here, we have used the Gram-negative Salmonella as a representative enterobacteria and the bacteriophage 9NA that uses LPS as a receptor and genome ejection trigger. Other enterobacteria phages (e.g., Escherichia coli T5) bind loosely to LPS and require an outer membrane protein for genome injection. The protocol described is applicable for bacteriophages that recognize and need the O-antigen of LPS for successful infection, such as 9NA, Det7, and P2213,25,26,27. Accordingly, the successful implementation of this protocol for the phage decontamination of bacterial cultures involves determining whether the source of the phage infection requires recognizing LPS in the host.
In conclusion, and despite the potential limitations of the protocol, our representative results clearly demonstrate that this method is a powerful tool to clean bacterial Salmonella cultures of bacteriophages that use LPS as a receptor and genome ejection trigger.

Microbial populations are faced with multiple challenges in natural environments, and an especially severe threat is the potential of infection by bacteriophages, the viruses that infect bacteria1. These viruses are widespread on the planet, exhibiting great diversity and abundance2,3,4,5. Although bacteriophages are diverse in size, morphology, and genomic organization, all share the same structure: a DNA or RNA genome enveloped by a capsid formed by phage-encoded proteins6. Bacteria have evolved a diverse array of defense mechanisms against them7. A key aspect of bacteriophage infection, which is relevant for characterization as well as for detection, is the receptor binding domains present on tail fibers. Bacteriophages have proteins on their surface called receptor-binding proteins or tail fibers for recognizing and binding to specific receptor sites on the surface of the bacterial cell. In the case of Gram-negative bacteria, the recognition of surface structures, such as lipopolysaccharides (LPS), outer membrane proteins, pili, and/or flagella, are involved in phage-bacteria interaction8. This interaction between bacteriophages and bacteria is highly specific and depends mainly on their ability to attach to host surfaces. The O-antigen of lipopolysaccharide is a commonly used receptor9.
The investigation of bacteriophage-bacteria interactions is not only fascinating from a biological standpoint but also has practical applications in areas such as phage therapy and biotechnology. While bacteriophages have valuable roles in various contexts, for example, altering microbial populations10, their presence can be undesirable in certain industrial processes. In pharmaceuticals, biotechnology, and food production, the presence of bacteriophages can impact the quality and safety of the final products, making their removal essential to meet quality standards. In bioprocessing and biomanufacturing, where bacterial cultures are used to produce various compounds (e.g., proteins, enzymes, or antibiotics), the presence of bacteriophages can lead to the disruption of production processes due to their ability to balance the bacterial population in every shared environment. Phages can occasionally turn the industrial microbiologist&#39;s professional life into a nightmare11. The design of effective procedures to remove phages is critical to ensure consistent and reliable production, enhancing process efficiency. Apart from these industrial aspects, in a research laboratory setting, where precision and reproducibility are crucial, the elimination of bacteriophages is essential for obtaining accurate and reliable results. Furthermore, the removal of phages can also be used to simulate varying environments to test different hypotheses12. Removing phages can also be very useful in the research environment since many phage-based studies, such as the enumeration of bacteria following phage application, would benefit from a step to remove phages in order to produce much more reliable viable counts.
Phage removal based on colony isolation would take several days to ensure that colonies are phage-free, whereas the procedure described here allows the generation of phage-free cultures in hours. This protocol permits us to follow the evolution of bacterial cultures without stopping them from isolating colonies. In this sense, it is possible to simulate fluctuating environments (presence and/or absence of phages) to test different hypotheses. Furthermore, this protocol permits the qualitative and quantitative analysis of the presence of phages in a bacterial culture.
In summary, designing cost-effective procedures for the removal of bacteriophages is crucial for maintaining product quality, safety, and process efficiency in various industries and for advances in basic and applied research. Here, we describe a highly effective protocol based on the use of LPS for the removal of bacteriophages, which use LPS as a receptor, from infected Salmonella cultures, which is both time-efficient and requires minimal equipment.

<table><tbody><tr><td>20 mL syringe</td><td>BD Discardit II</td><td>300296</td><td>No special requirements</td></tr><tr><td>50 mL conical tubes</td><td>Avantor</td><td>525-0610</td><td>No special requirements</td></tr><tr><td>90 x 14 mm Petri dishes</td><td>Deltalab</td><td>200209</td><td>No special requirements</td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A1296</td><td>No special requirements</td></tr><tr><td>Bacteriophage lysate</td><td></td><td></td><td>Minimal concentration: 10&lt;sup&gt;9&lt;/sup&gt; PFU/mL</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td>No special requirements</td></tr><tr><td>Chloroform</td><td>Panreac</td><td>131252</td><td>No special requirements</td></tr><tr><td>Citric acid &amp;middot; H2O</td><td>Merck</td><td>1.00247</td><td></td></tr><tr><td>Colony counter</td><td></td><td></td><td>No special requirements</td></tr><tr><td>Evans Blue&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E-2129</td><td></td></tr><tr><td>Flasks</td><td></td><td></td><td>No special requirements</td></tr><tr><td>Fluorescein sodium salt</td><td>Sigma-Aldrich</td><td>F-6377</td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td>No special requirements</td></tr><tr><td>Glass tubes</td><td></td><td></td><td>No special requirements</td></tr><tr><td>Glass tubes for lysate&amp;nbsp;</td><td></td><td></td><td>No special requirements</td></tr><tr><td>Glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>1.05104.1000</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; anhydrous&amp;nbsp;</td><td>Merck</td><td>1.05104</td><td></td></tr><tr><td>Lipopolysaccharide from &lt;em&gt;Salmonella enterica&lt;/em&gt; serotype Typhimurium</td><td>Sigma-Aldrich</td><td>L6511-25 mg</td><td>Dissolved in sterile water</td></tr><tr><td>Membrane 0.45 &amp;micro;m&amp;nbsp;</td><td>MF-Millipore</td><td>HAWP02500</td><td>No special requirements</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; &amp;middot; 7 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.05886</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td>No special requirements</td></tr><tr><td>NaNH&lt;sub&gt;4&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; &amp;middot; 4 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S9506</td><td></td></tr><tr><td>Peptone</td><td>iNtRON</td><td>Ba2001</td><td>No special requirements</td></tr><tr><td>Syringe Filter 0.22 &amp;micro;m</td><td>Millex</td><td>SLGSR33SB</td><td>No special requirements</td></tr><tr><td>Toothpicks</td><td></td><td></td><td>No special requirements</td></tr><tr><td>Tryptone</td><td>Panreac</td><td>403682.1210</td><td>No special requirements</td></tr><tr><td>Vacuum pump</td><td>Thermo Scientific</td><td></td><td>No special requirements</td></tr><tr><td>Yeast extract</td><td>iNtRON</td><td>48045</td><td>No special requirements</td></tr></tbody></table>

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.

Salmonella enterica and other Gram-negative bacteria have an outer membrane containing LPS. The O-antigen of LPS is a commonly used receptor by bacteriophages 9NA to infect Salmonella cultures16,17.
Given the specific affinity of bacteriophages for the O-antigen or core polysaccharide regions of LPS, we wanted to examine whether Salmonella enterica commercial&#160;LPS could be used as a decoy to exclude 9NA bacteriophages. To do it, we mixed known concentrations of the commercial LPS and 9NA lysate, followed by a titration. Commercial LPS and 9NA bacteriophages were mixed in a total volume of 200 &#181;L and incubated for 2 h at 37 &#176;C without shaking. For titration, 100 &#181;L of the LPS-9NA mixture and 60 &#181;L of an overnight culture were added to 5 mL of LB soft agar and poured onto the top of an LB plate. Plates were incubated for 24 h at 37 &#176;C. As displayed in Figure 3, the lysate titer decreases proportionally when the concentration of S. enterica commercial LPS increases. These results indicate that the commercial LPS functions correctly as a decoy for 9NA bacteriophages.
Interestingly, the time required for phage 9NA to lyse Salmonella cells and release phages is facilitated by the number of bacteria and phages (Figure 4). In order to study how this aspect could affect the phage removal protocol, we tested different bacteria: phage ratios (1:1, 1:10, 1:100, and 1:1000). As seen in Figure 4, there is a drop in OD600 nm of the culture at 1.5-2.5 h after the addition of 9NA phage. OD600 nm values nearing zero are indicative that the bacterial culture is being lysed18. For this reason, incubation times in this protocol were defined as 2 h in order to ensure sufficient time for phage contained inside Salmonella cells to lyse bacteria and be released. This time should be estimated for each host-phage system prior to performing this protocol.
Once we determined that commercial LPS works as a decoy and the lysis time for 9NA infected bacteria, we performed the protocol described for cleaning infected Salmonella cultures of the bacteriophages (Figure 1 and Figure 2). In order to monitor the presence of bacteriophages along each step of the protocol, we did plaque assays to calculate the infectivity of the resulting cultures in 100 &#181;L of the resulting mixture at different points (Figure 5). We can observe that repeated washing and filtering are not sufficient for the elimination of bacteriophages from cultures (titration points 1-3); however, the number of phages decreases as soon as we employ a step of incubation with commercial LPS (titration point 4). The crucial step for the complete removal of bacteriophages in a bacterial culture is the second incubation with commercial LPS (titration point 6). This step is essential for successful removal of 9NA bacteriophage in Salmonella cultures.
An interesting aspect of this protocol would be to know the level of phage resistance after phage removal steps. A procedure that separates phages from phage-resistant bacteria has no utility. For this reason, it is crucial to reveal that the bacteria remaining in the culture is phage-susceptible. To demonstrate that susceptible-phage cells remain in the cultures after the procedure, we used Evans Blue Uranine (EBU) plate assay to screen for phage contamination19. EBU plates were made of LB medium supplemented with 10 mL/L K2HPO4 25%, 5 mL/L glucose 50%, 2.5 mL/L fluorescein 1%, 1.25 mL/L Evans Blue 1%, and 15 g/L agar. Cross-streaking on EBU plates with 9NA phage was used to discriminate phage-resistant and phage-sensitive isolates (Figure 6). The bacterial cultures obtained at the end of the cleaning protocol were used to get isolated colonies, which were checked for phage contamination (Figure 6B). We can observe the existence of both resistant and sensitive cells. This protocol does not favor the selection of phage-resistant cells; it only eliminates bacteriophages.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66855/66855fig01.jpg" /> 
Figure 1: Brief outline of the procedure for elimination of bacteriophages in Salmonella cultures. The workflow is divided into different stages: preparation of bacterial culture and lysate, infection of the bacterial culture with bacteriophages, removal of bacteriophages from infected bacterial cultures, and preparation of a phage-free bacterial inoculum. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig01large.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/66855/66855fig02.jpg" /> 
Figure 2: Procedure for bacteriophage removal from infected Salmonella enterica cultures. The process consists of three phases: 1) Removal of bacteriophages in suspension, 2) Removal of phages contained inside bacterial cells, and 3) Avoidance of reinfection. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig02large.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/66855/66855fig03.jpg" /> 
Figure 3: LPS-decoy assay to measure the efficiency of Salmonella enterica commercial LPS to bind bacteriophages. Titration of a 9NA lysate (PFU/mL) at increasing concentrations of Salmonella enterica commercial LPS. The experiment was carried out in triplicate. Mean and standard deviation are presented. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66855/66855fig04.jpg" /> 
Figure 4: Bacteriophage 9NA lysis time in Salmonella cultures. Growth curves of the Salmonella enterica cultures in the presence of bacteriophage 9NA at bacteria:phage ratios of 1:1, 1:10, 1:100, and 1:1000. The experiment was carried out in triplicate. Mean and standard deviation are presented. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66855/66855fig05.jpg" /> 
Figure 5: Plaque assay for infectivity testing during bacteriophage removal in Salmonella cultures. (A) Titration of eight aliquots was taken at different points of the protocol (titration points 1-7 are marked in Figure 2). For this experiment, Salmonella enterica serovar&#160;Typhimurium strain ATCC 14028 opvAB::lacZ (SV8011) and bacteriophage 9NA were used. Experiments were performed in triplicate, and the mean and standard deviations are shown.&#160;(B)&#160;Soft agar plates with Salmonella enterica were obtained following the overlay technique using aliquots from eight titration points. The plates correspond from the left to the right with titration points 1-8. (C) Optical density at 600 nm of bacterial culture at different times of the phage-remove protocol. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66855/66855fig06.jpg" /> 
Figure 6: Testing for phage-resistant bacteria after bacteriophage removal procedure. (A) Schematic diagram of a typical EBU agar plate used for the cross-streak agar assay: the vertical dark region at the center represents the zone of 9NA lysate. The dot represents the location where the tested cells are inoculated at a safe distance from the lysate zone, and the horizontal solid lines represent either the phage-resistant cells that are growing across the lysate zone or the phage-sensitive cells that are not growing beyond the lysate zone. (B) EBU plate assays for testing 11 colonies were obtained at the end of the removal protocol. Control R and S are examples of phage-resistant and phage-sensitive isolates, respectively. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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 ...

bacteriophage-removal-from-infected-salmonella-cultures

Research

JoVE Journal

Biology

774 Views.  Universidad de Sevilla. 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.

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

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

Environment

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Immunology and Infection

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray (ChIP-chip); however, ChIP-seq has become the preferred method through technological advancements, decreasing financial barriers to sequencing, and massive amounts of high-quality data output. Techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections, are described in this protocol. ChIP-seq is performed on Salmonella enterica serovar Typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CsgD, to determine differential binding in the two cell types. Here, we demonstrate the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for cross-linker access, and performing routine ChIP-seq steps to obtain high quality sequencing results.

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a method used to establish interactions between transcription factors and the genomic sequences they control. This protocol outlines techniques for performing ChIP-seq with bacterial biofilms, using Salmonella enterica serovar Typhimurium bacterial biofilm as an example.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

Genetics

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

Bioengineering

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

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.

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 ...

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 ...

Bacteriophage Removal from Infected Salmonella Cultures

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

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