JCM is funded by a BBSRC-IPA award with Unilever Plc (BB/L023040/1). HAG is funded by a Saudi Arabian Ministry of Higher education PhD studentship.

The following method is adapted 7,8 to test competitor strains with inhibitor-producing strains.
NOTE: This protocol involves aerosol generation with bacterial cultures. This can only be performed in a contained space with implementation of locally-approved safety considerations. Spraying of the culture onto the agar plates within a level 2 safety cabinet will minimize contamination of the agar plates and the surrounding environment. This will also protect the researcher from aerosol inhalation of the sprayed bacteria, this is a major concern if the researcher is using containment level 2 organisms. Appropriate personal protective equipment must be worn. The area surrounding the petri dish is best covered with disposable absorbent tissue. Post-spray decontamination using UV light is advised.
1. Preparing the Agar Plates
<ol>
	<li>Prepare brain heart infusion (BHI) agar in accordance to the manufacturer&#39;s instructions.</li>
	<li>Autoclave the BHI agar at 121 &#176;C, 15 psi for 20 min, or according to autoclave manufacturer&#39;s recommendations, to sterilize.</li>
	<li>Pour 15 ml aliquots of sterilized BHI agar into sterile petri dishes and allow it to set. 
	NOTE: Ensure every agar plate to be used within the assay originates from the same stock of agar and that there is the same amount of agar in each plate. This limits the variability in available nutrients and inhibitor diffusion between plates allowing comparability.</li>
</ol>
2. Preparing Sterile Broth
<ol>
	<li>Prepare BHI broth in accordance to the manufacturer&#39;s instructions.</li>
	<li>Add 10 ml aliquots of BHI to 28 ml glass universal bottles.</li>
	<li>Autoclave the BHI broth at 121 &#176;C, 15 psi for 20 min, or according to autoclave manufacturer&#39;s recommendations, to sterilize. 
	NOTE: Ensure all broth used in the assay originates from the same batch of broth autoclaved at the same time to limit variation in nutrients which could cause variation in results.</li>
</ol>
3. Preparing Sterile Vaporizer Bottle(s)
<ol>
	<li>Dismantle the vapourizer bottle.</li>
	<li>Spray 5% surface active cleaning agent through the vapourizer to eliminate internal contaminants, soak the vapourizer bottle, lid and vapourizer in the 5% surface active cleaning agent overnight. 
	NOTE: Gloves and appropriate personal protective equipment should be used when handling surface active cleaning agent.</li>
	<li>Dry the vaporizer, lid and bottles under sterile air flow hood.</li>
	<li>Fill the bottle with 70% ethanol, reattach vapourizer and spray some of the ethanol through the vapourizer.</li>
	<li>Put the lid on the vaporizer and store with the ethanol inside until use.</li>
	<li>Immediately prior to use, aseptically rinse the bottle with sterile BHI broth and spray the broth through the vapourizer. 
	NOTE: This prevents the ethanol affecting the inoculum added to the bottle in section 6.3.</li>
</ol>
4. Preparing the Bacterial Overnight Culture(s)
<ol>
	<li>Streak the competitor and inhibitor strains onto a sterile BHI agar plate and incubate at 37 &#176;C aerobically for approximately 18 hr (overnight). 
	NOTE: This plate produces a stock of bacteria that can be used for multiple replicates within the same week when stored at 4 &#176;C.</li>
	<li>Inoculate 10 ml of sterile BHI broth with a single colony of the required competitor bacterial strain within a 28 ml universal bottle.</li>
	<li>Incubate the universal bottle at 37 &#176;C aerobically overnight by shaking at 250 rpm in an orbital shaker.</li>
</ol>
5. Spot Culture of the Inhibitor-producing Isolate
<ol>
	<li>Prepare the overnight culture(s) as described in section 4.</li>
	<li>Ensure agar plates are completely dry, with no condensation on the lid, prior to inoculation (e.g. by incubating plate for 60 min at 37 &#176;C); this prevents the inoculum from spreading across the plate from the inoculation site.</li>
	<li>Pipette 25 &#181;l of the overnight culture onto the center of the agar plates. Avoid completely depressing the pipette plunger to prevent air bubbles that spray culture across the agar.</li>
	<li>Allow the culture to dry at room temperature to limit spread of the culture. Incubate the agar plates at 37 &#176;C aerobically overnight.</li>
</ol>
6. Preparing the Spray Inoculum of the Test Competitor Strain(s)
<ol>
	<li>Prepare the overnight culture as described in section 4. Dilute the overnight culture 10 fold in BHI broth to yield approximately 4 x 106 CFU ml-1, and a suspension of OD600 0.3 &#177;0.05. 
	NOTE: A 10 fold dilution will not give 4 x 106 CFU ml-1 for all bacterial species, calculate the necessary dilution factor by spread plating serial dilutions between 1 x 10-1-1 x 10-6.</li>
	<li>Pour the diluted culture into a sterile plastic perfume vaporizer bottle. 
	NOTE: The total volume must be more than is necessary for the number of agar plates being sprayed. Approximately 250 &#181;l per plate is suitable for a 9 cm diameter Petri dish.</li>
</ol>
7. Deferred Growth Inhibition Assay
<ol>
	<li>Spray the culture through each of the vaporizer bottles to ensure that the culture is loaded in the spray mechanism prior to spraying onto the agar.</li>
	<li>From a distance of approximately 15 cm above the agar, spray approximately 250 &#181;l of diluted culture (3 sprays of a typical 50 ml vaporizer) over the entire agar surface. Incubate the agar plates at 37 &#176;C aerobically overnight. Repeat for other plates with the same number of sprays.</li>
</ol>
8. Interpreting Deferred Growth Inhibition Assay Results
<ol>
	<li>Measure the growth inhibition zone of the sprayed competitor strain (Figure 1A-C, &#34;X&#34; to &#34;X&#34;) in millimeters, subtracting the diameter of the central spot of the inhibitor-producer (Figure 1A-C, &#34;Y&#34; to &#34;Y&#34;). 
	NOTE: Figure 1E shows how this data could be presented</li>
	<li>Record the clarity score for zones of inhibition. 
	NOTE: This indicates the degree of inhibition observed. Examples are shown in Figure 1A-D, strains that can be used for standardization are suggested in the representative results section. The following scale can be used for clarity scoring: a clarity score of 0 to describe a zone of complete inhibition of the competitor strain&#39;s growth. A score of 1 to describe a zone of almost complete inhibition of growth, with only individual colonies present in the zone of clearing. A score of 2 to describe a visible zone of reduced growth, growth within this zone is confluent or near confluent but reduced in density by &#62;50 %. A score of 3 to describe only minimal growth reduction, growth is confluent and reduced by less than 50 %, a zone is still visible and measurable. A score of 4 to describe no visible inhibition of growth. A score of 5 to describe that the growth of the competitor strain was increased in the proximity of the test inhibitor-producing strain. See Figure 1 for examples. These scores are subjective to the experimenter.</li>
</ol>
9. Replicates
<ol>
	<li>Repeat assays at least in biological triplicate to determine the reproducibility of the results obtained. 
	NOTE: If replicates are performed on different days, take measurements and score plates immediately after removal from incubation, then either store plates at 4 &#176;C or take high quality photographs of plates before discarding them. Storage or photographs of plates enables easy comparison of results between different days, this is particularly important for confirming reproducible scoring across days/experiments. Be aware that storage of the plates has the potential to alter results. 
	NOTE: For clear photographs place plates upright without their lid on a matte black background in an area of diffused light, use a high resolution camera (&#8805;8 MP) capable of focusing on objects close to the lens (macro mode). Alternatively, some gel imagers with trans-illuminator set to white light can produce high quality, highly reproducible images.</li>
</ol>

<ol>
	<li>Bolnick, D. I., 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=Why+intraspecific+trait+variation+matters+in+community+ecology.">Why intraspecific trait variation matters in community ecology.</a> Trends Ecol Evol. 26 (4), 183-192 (2011).</li><li>Christensen, G. J. M., 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=Antagonism+between+Staphylococcus+epidermidis+and+Propionibacterium+acnes+and+its+genomic+basis.">Antagonism between Staphylococcus epidermidis and Propionibacterium acnes and its genomic basis.</a> BMC Genomics. 17 (152), (2016).</li><li>Faust, K., 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=Microbial+co-occurrence+relationships+in+the+human+microbiome.">Microbial co-occurrence relationships in the human microbiome.</a> PloS Comput Biol. 8 (7), (2012).</li><li>Frank, D. N., Feazel, L. M., Bessesen, M. T., Price, C. S., Janoff, E. N., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+nasal+microbiota+and+Staphylococcus+aureus+carriage.">The Human nasal microbiota and Staphylococcus aureus carriage.</a> PLoS One. 5 (5), e10598 (2010).</li><li>Fredheim, E. G., Flaegstad, T., Askarian, F., Klingenberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonisation+and+interaction+between+S.+epidermidis+and+S.+aureus+in+the+nose+and+throat+of+healthy+adolescents.">Colonisation and interaction between S. epidermidis and S. aureus in the nose and throat of healthy adolescents.</a> Eur J Clin Microbiol Infect Dis. 34, 123-129 (2015).</li><li>Hibbing, M. E., Fuqua, C., Parsek, M. R., Peterson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+competition:+surviving+and+thriving+in+the+microbial+jungle.">Bacterial competition: surviving and thriving in the microbial jungle.</a> Nat Rev Microbiol. 8, 15-25 (2010).</li><li>Libberton, B., Coates, R. E., Brockhurst, M. A., Horsburgh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+intraspecific+trait+variation+among+nasal+bacteria+shapes+the+distribution+of+Staphylococcus+aureus.">Evidence that intraspecific trait variation among nasal bacteria shapes the distribution of Staphylococcus aureus.</a> Infect Immun. 82 (9), 3811-3815 (2014).</li><li>Libberton, B., Horsburgh, M. J., Brockhurst, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+spatial+structure,+frequency+dependence+and+resistance+evolution+on+the+dynamics+of+toxin-mediated+microbial+invasions.">The effects of spatial structure, frequency dependence and resistance evolution on the dynamics of toxin-mediated microbial invasions.</a> Evol appl. 8, 738-750 (2015).</li><li>Liu, C. M., 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=Staphylococcus+aureus+and+the+ecology+of+the+nasal+microbiome.">Staphylococcus aureus and the ecology of the nasal microbiome.</a> Sci Adv. 1, e1400216 (2015).</li><li>Nascimento, J. S., Ceotto, H., Nascimento, S. B., Giambiagi-deMarval, M., Santos, K. R., Bastos, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriocins+are+alternative+agents+for+control+of+multiresistant+staphylococcal+strains.">Bacteriocins are alternative agents for control of multiresistant staphylococcal strains.</a> Lett Appl Microbiol. 42, 215-221 (2006).</li><li>Park, B., Iwase, T., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+application+of+S.+epidermidis+prevents+colonization+by+methicillin-resistant+Staphylococcus+aureus+in+mice.">Intranasal application of S. epidermidis prevents colonization by methicillin-resistant Staphylococcus aureus in mice.</a> PLoS One. 6 (10), e25880 (2011).</li><li>Uehara, Y., 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=Bacterial+interference+among+nasal+inhabitants:+eradication+of+Staphylococcus+aureus+from+nasal+cavities+by+artificial+implantation+of+Corynebacterium+sp.">Bacterial interference among nasal inhabitants: eradication of Staphylococcus aureus from nasal cavities by artificial implantation of Corynebacterium sp.</a> J Hosp Infect. 44, 127-133 (2000).</li><li>Wos-Oxley, M. 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=A+poke+into+the+diversity+and+associations+within+human+anterior+nare+microbial+communities.">A poke into the diversity and associations within human anterior nare microbial communities.</a> ISME J. 4, 839-851 (2010).</li></ol>

The authors have nothing to disclose.

A critical step of this bacterial species competition protocol that differentiates it from others, is the overnight incubation of the inhibitor isolate before addition of the competitor isolate. The inhibitor strain requires this growth period to produce inhibitory compounds. As the competitor strain is overlaid on top of the same medium, this method allows the researcher to observe the full potential of the inhibitor strain to interfere with growth of the competitor strain. As a result, this assay reflects what happens with nature; one isolate is fully established in a niche, and the other must be able to counteract any inhibitory variables to be able to successfully invade the niche.
There are two parts to this protocol that are most likely to cause erroneous results if performed incorrectly. Firstly, incomplete sterilization of the vaporizer bottles can introduce contaminating bacteria into the assay. Incubation of the broth used to rinse the vaporizer bottle in step 3.6 in a sterile vessel can be used to confirm bottles were completely sterilized. Additional washes in disinfectant solutions, such as Virkon, can be used prior to the wash in surface active cleaning agent, described in step 3.2, if additional sterilization is required.
A second step that may cause erroneous results is the dilution of the competitor strain (step 6.1). If the dilution is not optimal, clear measurements of inhibition may not be easily determined. This assay was optimized using various nasal isolates as inhibitor-producing strains across a wide range of Gram-positive and Gram-negative species versus S. aureus SH1000 as the competitor strain 7,8. It has subsequently been tested using various coagulase-negative and coagulase-positive staphylococci as competitor and inhibitor-producing strains. When testing genera other than those described above as the competitor strain, some optimization of the dilution for the inoculum may be necessary.
If clarity scores vary significantly between repeats, it might be necessary to standardize the competitor strain inoculum by setting it to a specific optical density. However, this will increase the time the assay takes to set up, and is likely to reduce the number of strains it is possible to process per experiment. Visually adjusting the culture to an appropriate OD using a McFarland standard may provide a rapid alternative.
One limitation of this technique is that it it can only be applied to culturable bacteria. This is why many studies use 16S rRNA sequencing to predict competitive exclusion interactions 3,4,13. However, genetic and metagenomic techniques can only distinguish community members to the species level, and/or do not account for particular intraspecific trait variation. It is possible to screen for the association of a particular gene that encodes a trait with the presence or absence of a bacterial species 5, however this requires pre-knowledge of a gene likely to be associated with exclusion of a species.
A safety consideration of this technique is that, due to the aerosol generation of bacteria, it can only be used in laboratories with a level 2 safety cabinet, or with similar safety precautions. There are alternative techniques to test for competition between isolates that account for intraspecific trait variation and do not generate aerosols of bacteria. One such method is the simultaneous antagonism assay 2. In this method, an inoculum of one isolate is spread onto an agar plate, and a second isolate inoculum is spotted or stabbed on top once the first has dried. This would produce an environment where the strains are in direct competition (simultaneous antagonism), both competing to produce inhibitory compounds and scavenge nutrients first. The advantage of the deferred growth inhibition assay over the simultaneous antagonism assay is that the inhibitor isolate should always have the competitive advantage of being fully established before invasion of the competitor isolate. This is more reflective of what would happen in the environment, as in most niches, one isolate will be established before another strain is added, rather than two isolates being added at the same time 8.
Another alternative to avoid aerosol generation of bacteria would be to add the competitor isolate in top agar, which could be poured rather than sprayed over the inhibitor isolate. However, the volume of top agar added would need to be approximately 3 ml to ensure even coverage of the plate. Within this volume, the competitor isolate would not be growing on the same media as the inhibitor isolate, so would not suffer from reduced nutrients. The competitor would also not be in direct contact with any inhibitory compounds until they diffused into the top agar. Such an assay could not be described as deferred inhibition or simultaneous antagonism.
A similar method has previously been used to assess the levels of bacitracin resistance amongst clinical S. aureus isolates 10. The main difference between the protocol described here and that described by 10, is the latter only classified competitor strains as sensitive (complete inhibition- clarity score 0) or resistant (partial to no inhibition- clarity score 1-5). The deferred growth inhibition assay could therefore also be used to screen for levels of resistance to producers of particular antimicrobials, or even to search for novel antimicrobials active against multi-drug resistant pathogens.
It has been suggested that the host microflora could be manipulated to eliminate undesirable members of the bacterial community 9. Application of corynebacteria or Staphylococcus epidermidis have already been shown to eliminate S. aureus colonization in the nares in a significant proportion of the population 11,12. Studies into specific strains that can competitively exclude undesirable members of the host flora, through techniques such as the one described here, may therefore lead to future therapeutics.

Bacteria living in communities compete at the interspecific and intraspecific level for space and nutrients 6. Competition between microbes can often lead to exclusion of particular species or strains within a community. Exclusion can be due to competition for a particular receptor or nutrient, or the production of an antimicrobial compound by a member of the community 6.
Studies investigating competitive exclusion frequently examine the presence and absence of species using 16S rRNA sequencing 3,4,13. However, intraspecific trait variation, for example the presence or absence of antimicrobial peptide production, can hugely impact the ability of one species to inhibit another 1. Since 16S rRNA sequencing cannot distinguish between strains of the same species, intraspecific trait variation cannot be assessed. The technique described here can be used to assess the potential of individual strains to exclude other bacteria. This method has previously been used to show a positive correlation between the presence of Staphylococcus aureus inhibitory bacteria and the absence of S. aureus in a nasal community 7.
The deferred growth inhibition (competition) assay can identify production of antimicrobial compounds or nutrient competition by the inhibitor strain. It can also detect positive interactions, whereby the presence of the inhibitor strain actually improves the growth of the competitor strain. This assay can be used to create quantitative data (zone of clearing size) and qualitative data (degree of inhibition), both of which can be overlaid onto data of the presence or absence of a species within a community to find correlations between phenotype (trait) and competitive exclusion 7.

<table><tbody><tr><td>Brain heart infusion broth</td><td>Lab M</td><td>lab049</td><td>rich general purpose media</td></tr><tr><td>agar-agar</td><td>Merck Milipore</td><td>101614</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt</td><td>82.1473.001</td><td></td></tr><tr><td>Plastic perfume vaporizer</td><td>not known</td><td>-</td><td>10 cm high with 3.5 cm diameter clear plastic spray bottle with lid, purchased from the travel section of a supermarket</td></tr><tr><td>Universal bottles</td><td></td><td>no longer in production, alternative SLS BOT5006</td><td>28 ml glass cylindrical bottle (~280 mm diameter, 850 mm height), flat bottomed with plastic screw cap</td></tr><tr><td>Decon 90</td><td>Decon&amp;nbsp;</td><td></td><td>surface active cleaning agent&amp;nbsp;</td></tr></tbody></table>

deferred growth inhibition assay to quantify the effect of bacteria-derived antimicrobials on competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The assay results should be observable as a difference in the overlaid competitor strain near the centrally spotted inhibitor-producing strain (Figure 1). These differences can be interpreted as full inhibition (Figure 1A), partial inhibition (Figure 1B and 1C), no inhibition (Figure 1D) or positive association. The widely available lab strains S. epidermidis T&#252;3298 and S. epidermidis Rp62A, and S. epidermidis Rp62A and S. aureus Newman were used in Figures 1B and 1D respectively. We suggest that these strains could be used to test this protocol.
Depending on species tested, full inhibition and partial inhibition are most likely the result of antimicrobial peptides, antibiotics or other diffusible growth inhibitors produced by the inhibitor-producing strain. Partial inhibition has also been linked to reduced nutrient availability for the competitor strain due to uptake or sequestering of nutrients by the inhibitor strain 6.
Comparison of the size of the zone of inhibition between different inhibitor-producing isolates tested against the same competitor strain indicates differences in any of the following: the amount of antimicrobial produced by the inhibitor-producing strain; the ability of the inhibitor to diffuse through the media; the type of inhibitor produced and the resistance level of the competitor strain to the inhibitors being produced. Comparison of the size of the zone of inhibition between different competitor strains tested against the same inhibitor-producing strain is indicative of the resistance levels of the competitor strains to the inhibitor being produced.
Results will vary depending on the inhibitory strain used and the competitor strain used. Repeats using the same inhibitory and competitor strain should ideally have the same clarity score (described in section 8.2), this indicates good technique has been used throughout.
If inconsistent application of bacteria has occurred the plates should not be used. For example, if the test competitor culture is too dilute the zone of inhibition will appear larger and with a less clearly defined edge than is typically observed (Figure 2A); this would affect reliability of any comparative zone measurements. If the inhibitor-producing strain spot was not fully dried before incubation overnight, the inhibitor strain is likely to spread and not form a clearly defined spot (Figure 2B), which typically affects zone measurements but can also affect the clarity scoring. If the competitor strain is not sprayed evenly this can lead to an uneven spread of bacteria (Figure 2C). If the competitor strain is too concentrated it is possible that the competitor strain may grow on top of the inhibitor strain (Figure 2C), in extreme cases this can lead to some inversion of competitor/inhibitor strain roles.
<img alt="Figure 1" src="/files/ftp_upload/54437/54437fig1.jpg" /> 
Figure 1: The range of qualitative and quantitative results that can be obtained from the deferred growth inhibition assay. Results for the growth inhibition can range from (A) complete inhibition (score of 0), (B) partial inhibition (score of 1), (C) partial inhibition (score of 2) to (D) no inhibition (score of 4). Labels indicate the edges of growth of the inhibitor strains (Y) and the outermost edge of the inhibition zones (X). The size of the zone of inhibition range from 0 mm to more than 10 mm, as shown in (E), which shows the measurements for the plates photographed in A, B, C and D. Results are shown for recent staphylococcal isolates (A, C) or common lab staphylococcal isolates S. epidermidis T&#252;3298 (inhibitor) and S. epidermidis Rp62A (competitor) (B), and S. epidermidis Rp62A (inhibitor) and S. aureus Newman (competitor) (D). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54437/54437fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54437/54437fig2.jpg" /> 
Figure 2: Examples of inconsistent results due to poor technique. Results may appear different than expected if (A) competitor strain was too dilute, (B) inhibitor strain was not fully dry before incubation/plates were not dry or (C) if the competitor strain was not sprayed evenly and was too concentrated. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54437/54437fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition at JoVE.com

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

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Biology

14.5K Views.  University of Liverpool. The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Video: Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Immunology and Infection

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

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, ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Source: Smith, S., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62851">Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

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Chapters in this video

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Biomimetic Materials to Characterize Bacteria-host Interactions

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

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

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level