Name: Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates (Video) | JoVE Journal
Uploaded: 2019-07-22T13:00:00.000+00:00
Duration: 7 min 43 s
Description: Learn a robust coincubation assay to quantify competitive interactions between Vibrio fischeri isolates using fluorescent markers and antibiotic resistance. This method reveals how interbacterial competition shapes population structure.

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We would like to thank reviewers for their helpful feedback. A.N.S. was supported by the Gordon and Betty Moore Foundation through Grant GBMF 255.03 to the Life Sciences Research Foundation.

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</ol>

The authors have nothing to disclose.

The coincubation assay described above provides a powerful method to discover interbacterial competition. This approach allowed for the identification of intraspecific competition among V. fischeri isolates and characterization of the competitive mechanism19. Although the method described was optimized for the marine bacterium V. fischeri, it can be easily modified to accommodate other bacterial species including clinical and environmental isolates. It is important to note that c...

This manuscript outlines a culture-based method to determine whether two bacterial isolates are capable of competitive interactions. When studying mixed populations, it is important to assess the extent to which the bacterial isolates interact, particularly whether isolates are directly competing through interference mechanisms. Interference competition refers to interactions where one population directly inhibits the growth or kills a competitor population1. These interactions are important to identify because they can have profound effects on a microbial community&#8217;s structure and function2,3.
Mechanisms for microbial competition have been discovered broadly in genomes of bacteria from diverse environments including both host-associated and free-living bacteria4,5,6,7,8,9. A variety of competition strategies have been described10,11 including diffusible mechanisms, such as bactericidal chemicals1,12 and secreted antimicrobial peptides13, as well as contact-dependent mechanisms that require cell-cell contact to transfer an inhibitory effector into target cells9,14,15,16,17,18.
Although culture-based coincubations are commonly used in microbiology5,8,19, this manuscript outlines how to use the assay to characterize the mechanism of competition, as well as suggestions for adapting the protocol for use with other bacterial species. Furthermore, this method describes multiple approaches for analyzing and presenting the data to answer different questions about the nature of the competitive interactions. Although the techniques described here were used previously to identify the interbacterial killing mechanism underlying intraspecific competition between symbiotic strains of coisolated Vibrio fischeri bacteria19, they are suitable for many bacterial species including environmental isolates and human pathogens, and can be utilized to evaluate both contact-dependent and diffusible competitive mechanisms. Steps in the protocol may require optimization for other bacterial species. Given that more model systems are expanding their studies beyond the use of isogenic organisms to include different genotypes10,16,20,21, this method will be a valuable resource for researchers seeking to understand how competition impacts multi-strain or multi-species systems.

<table><tbody><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Fisher</td><td>05-408-129</td><td></td></tr><tr><td>10 &amp;mu;L multichannel pipette</td><td></td><td></td><td></td></tr><tr><td>100 &amp;mu;L multichannel pipette</td><td></td><td></td><td></td></tr><tr><td>300 &amp;mu;L multichannel pipette</td><td></td><td></td><td></td></tr><tr><td>10 &amp;mu;L single channel pipette</td><td></td><td></td><td></td></tr><tr><td>20 &amp;mu;L single channel pipette</td><td></td><td></td><td></td></tr><tr><td>200 &amp;mu;L single channel pipette</td><td></td><td></td><td></td></tr><tr><td>1,000 &amp;mu;L single channel pipette</td><td></td><td></td><td></td></tr><tr><td>24-well plates</td><td>Fisher</td><td>07-200-84</td><td>sterile with lid</td></tr><tr><td>96-well plates</td><td>VWR</td><td>10062-900</td><td>sterile with lid</td></tr><tr><td>Calculator</td><td></td><td></td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td>stock (20 mg/mL in Ethanol); final concentration in media (2 &amp;mu;g/mL LBS)</td></tr><tr><td>Fluorescence dissecting microscope with camera and imaging software</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td>Fisher</td><td>08-880</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>Fisher</td><td>BP906-5</td><td>stock (100 mg/mL in water, filter sterilize); final concentration in media (1 &amp;mu;g/mL LBS)</td></tr><tr><td>Nitrocellulose membrane (FS MCE, 25MM, NS)</td><td>Fisher</td><td>SA1J788H5</td><td>0.22 &amp;mu;m nitrocellulose membrane (pk of 100)</td></tr><tr><td>Petri plates</td><td>Fisher</td><td>FB0875713</td><td>sterile with lid</td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td></td></tr><tr><td>Semi-micro cuvettes</td><td>VWR</td><td>97000-586</td><td></td></tr><tr><td>TipOne 0.1-10 &amp;mu;L starter system</td><td>USA Scientific</td><td>1111-3500</td><td>10 racks</td></tr><tr><td>TipOne 200 &amp;mu;L starter system</td><td>USA Scientific</td><td>1111-500</td><td>10 racks</td></tr><tr><td>TipOne 1000 &amp;mu;L starter system</td><td>USA Scientific</td><td>1111-2520</td><td>10 racks</td></tr><tr><td>Vortex</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;LBS media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 M Tris Buffer (pH ~7.5)</td><td></td><td></td><td>50 mL 1 M stock buffer (62 mL HCl, 938 mL DI water, 121 g Trizma Base)</td></tr><tr><td>Agar Technical</td><td>Fisher</td><td>DF0812-17-9</td><td>15 g (Add only for plates)</td></tr><tr><td>DI water</td><td></td><td></td><td>950 mL</td></tr><tr><td>Sodium Chloride</td><td>Fisher</td><td>S640-3</td><td>20 g</td></tr><tr><td>Tryptone</td><td>Fisher</td><td>BP97265</td><td>10 g</td></tr><tr><td>Yeast Extract</td><td>Fisher</td><td>BP9727-2</td><td>5 g</td></tr></tbody></table>

coincubation assay for quantifying competitive interactions between vibrio fischeri isolates

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.

In order to assess competitive interactions between bacterial populations, a coincubation assay protocol was developed and optimized for V. fischeri. This method utilizes stable plasmids that encode antibiotic resistance genes and fluorescent proteins, allowing for differential selection and visual discrimination of each strain. By analyzing the data collected from the coincubation assay, the competitive outcome of an interaction and the mechanism of the interaction can be identi...

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. ...

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JoVE Journal

Immunology and Infection

Source: Speare, L., et al. Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates. J. Vis. Exp. (2019)This video demonstrates a co-incubation assay used to study how bacterial strains compete in a shared environment. It is widely applied in microbiology to identify mechanisms, such as contact-dependent or contact-independent killing, that drive these interactions.

Coincubation Assay for Studying Competitive Interactions Between Bacterial Strains

Source: Brumfield, K. D., et al. Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates. J. Vis. Exp. (2017)This video demonstrates a soft agar assay using triphenyltetrazolium chloride to assess and compare the motility of Vibrio cholerae strains by visualizing metabolic activity through red zone formation around actively migrating cells.

Motility Assay for Comparing Vibrio cholerae Strains Using Triphenyltetrazolium Chloride

Source: Brumfield, K. D., et al., Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates. J. Vis. Exp. (2017).This video demonstrates the Voges-Proskauer (VP) test to differentiate among three biotypes of Vibrio cholerae: classical, wild-type El Tor, and El Tor variant. Each culture is inoculated into a glucose-containing medium, incubated, and treated with α-naphthol and potassium hydroxide to detect the presence of acetoin. A...

Voges-Proskauer Assay for Differentiating Biotypes of Vibrio cholerae

Source: Nag, D., et al. Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model. J. Vis. Exp. (2018)This video demonstrates a protocol to quantify the intestinal bacterial load in adult zebrafish infected with an antibiotic-resistant strain of Vibrio cholerae. Following euthanasia and dissection, the intestinal tract is isolated, homogenized with glass beads, and processed to release the bacteria. The resulting homogenate is plated onto a selective medium, and...

Quantifying Intestinal Bacterial Load in Zebrafish Infected with Vibrio cholerae

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.

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

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.

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

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.

Biomimetic Materials to Characterize Bacteria-host Interactions

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

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

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Learn a robust coincubation assay to quantify competitive interactions between Vibrio fischeri isolates using fluorescent markers and antibiotic resistance. This method reveals how interbacterial competition shapes population structure.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

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