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.

<ol>
	<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> Nature Reviews Microbiology. 8 (1), 15-25 (2010).</li><li>Nyholm, S. V., McFall-Ngai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+winnowing:+establishing+the+squid-Vibrio+symbiosis.">The winnowing: establishing the squid-Vibrio symbiosis.</a> Nature Reviews Microbiology. 2 (8), 632-642 (2004).</li><li>D&#246;rr, N. C. D., Blockesh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+type+VI+secretion+system+facilitates+niche+domination.">Bacterial type VI secretion system facilitates niche domination.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (36), 8855-8857 (2018).</li><li>Maclntyre, D. L., Miyata, S. T., Kitaoka, M., Pukatzki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vibrio+cholerae+type+VI+secretion+system+displays+antimicrobial+properties.">The Vibrio cholerae type VI secretion system displays antimicrobial properties.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (45), 19520-19524 (2010).</li><li>Salomon, D., Gonzalez, H., Updegraff, B. L., Orth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrio+parahaemolyticus+type+VI+secretion+system+1+is+activated+in+marine+conditions+to+target+bacteria,+and+is+differentially+regulated+from+system+2.">Vibrio parahaemolyticus type VI secretion system 1 is activated in marine conditions to target bacteria, and is differentially regulated from system 2.</a> PloS One. 8 (4), e61086 (2013).</li><li>Sana, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+Typhimurium+utilizes+a+T6SS-mediated+antibacterial+weapon+to+establish+in+the+host+gut.">Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (34), E5044-E5051 (2016).</li><li>Schwarz, S., 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=Burkholderia+type+VI+secretion+systems+have+distinct+roles+in+eukaryotic+and+bacterial+cell+interactions.">Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.</a> PLoS Pathogens. 6 (8), e1001068 (2010).</li><li>Wenren, L. M., Sullivan, N. L., Cardarelli, L., Septer, A. N., Gibbs, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+independent+pathways+for+self-recognition+in+Proteus+mirabilis+are+linked+by+type+VI-dependent+export.">Two independent pathways for self-recognition in Proteus mirabilis are linked by type VI-dependent export.</a> MBio. 4 (4), (2013).</li><li>Garc&#237;a-Bayona, L., Guo, M. S., Laub, M. T. J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact-dependent+killing+by+Caulobacter+crescentus+via+cell+surface-associated,+glycine+zipper+proteins.">Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins.</a> Elife. 6, 24869 (2017).</li><li>Stubbendieck, R. M., Straight, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+interfaces+of+bacterial+competition.">Multifaceted interfaces of bacterial competition.</a> Journal of bacteriology. 198 (16), 2145-2155 (2016).</li><li>Cornforth, D. M., Foster, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotics+and+the+art+of+bacterial+war.">Antibiotics and the art of bacterial war.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (35), 10827-10828 (2015).</li><li>Shank, E. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+microbial+interspecies+signaling.">New developments in microbial interspecies signaling.</a> Current Opinion in Microbiology. 12 (2), 205-214 (2009).</li><li>Roelofs, K. G., Coyne, M. J., Gentyala, R. R., Chatzidaki-Livanis, M., Comstock, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteroidales+secreted+antimicrobial+proteins+target+surface+molecules+necessary+for+gut+colonization+and+mediate+competition+in+vivo.">Bacteroidales secreted antimicrobial proteins target surface molecules necessary for gut colonization and mediate competition in vivo.</a> MBio. 7 (4), e01055-e01016 (2016).</li><li>Dey, A., Vassallo, C. N., Conklin, A. C., Pathak, D. T., Troselj, V., Wall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sibling+rivalry+in+Myxococcus+xanthus+is+mediated+by+kin+recognition+and+a+polyploid+prophage.">Sibling rivalry in Myxococcus xanthus is mediated by kin recognition and a polyploid prophage.</a> Journal of bacteriology. 198 (6), (2016).</li><li>Danka, E. S., Garcia, E. C., Cotter, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+CDI+systems+multicolored,+facultative,+helping+greenbeards?.">Are CDI systems multicolored, facultative, helping greenbeards?.</a> Trends in Microbiology. 25 (5), 391-401 (2017).</li><li>Willett, J. L., Ruhe, Z. C., Coulding, C. W., Low, D. A., Hayes, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact-dependent+growth+inhibition+(CDI)+and+CdiB/CdiA+two-partner+secretion+proteins.">Contact-dependent growth inhibition (CDI) and CdiB/CdiA two-partner secretion proteins.</a> Journal of molecular biology. 427 (23), 3754-3765 (2015).</li><li>Cianfanelli, F. R., Monlezun, L., Coulthurst, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aim,+load,+fire:+the+type+VI+secretion+system,+a+bacterial+nanoweapon.">Aim, load, fire: the type VI secretion system, a bacterial nanoweapon.</a> Trends in Microbiology. 24 (1), 51-62 (2016).</li><li>Joshi, A., Kostiuk, B., Rogers, A., Teschler, J., Pukatzki, S., Yildiz, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rules+of+engagement:+the+type+VI+secretion+system+in+Vibrio+cholerae.">Rules of engagement: the type VI secretion system in Vibrio cholerae.</a> Trends in microbiology. 25 (4), 267-279 (2017).</li><li>Speare, 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=Bacterial+symbionts+use+a+type+VI+secretion+system+to+eliminate+competitors+in+their+natural+host.">Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (36), E8528-E8537 (2018).</li><li>Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+coculture+to+detect+chemically+mediated+interspecies+interactions.">Using coculture to detect chemically mediated interspecies interactions.</a> Journal of Visualized Experiments. (80), (2013).</li><li>Long, R. A., Rowley, D. C., Zamora, E., Liu, J., Bartlett, D. H., Azam, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+interactions+among+marine+bacteria+impede+the+proliferation+of+Vibrio+cholerae.">Antagonistic interactions among marine bacteria impede the proliferation of Vibrio cholerae.</a> Applied and Environmental Microbiology. 71 (12), 8531-8536 (2005).</li><li>Dunn, A. K., Millikan, D. S., Adin, D. M., Bose, J. L., Stabb, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+rfp-and+pES213-derived+tools+for+analyzing+symbiotic+Vibrio+fischeri+reveal+patterns+of+infection+and+lux+expression+in+situ.">New rfp-and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ.</a> Applied and Environmental Microbiology. 72 (1), 802-810 (2006).</li><li>Sana, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+type+VI+secretion+system+of+Pseudomonas+aeruginosa+strain+PAO1+is+regulated+by+quorum+sensing+and+Fur+and+modulates+internalization+in+epithelial+cells.">The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and Fur and modulates internalization in epithelial cells.</a> Journal of Biological Chemistry. 287 (32), 27095-27105 (2012).</li><li>Bachmann, V., Kostiuk, B., Unterweger, D., Diaz-Satizabal, L., Ogg, S., Pukatzki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salts+modulate+the+mucin-activated+type+VI+secretion+system+of+pandemic+Vibrio+cholerae.">Bile salts modulate the mucin-activated type VI secretion system of pandemic Vibrio cholerae.</a> PLoS. 9 (8), e0004031 (2015).</li><li>Ishikawa, T., Rompikuntal, P. K., Lindmark, B., Milton, D. L., Wai, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum+sensing+regulation+of+the+two+hcp+alleles+in+Vibrio+cholerae+O1+strains.">Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains.</a> PloS One. 4 (8), e6734 (2009).</li><li>Ishikawa, T., 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=Pathoadaptive+conditional+regulation+of+the+type+VI+secretion+system+in+Vibrio+cholerae+O1+strains.">Pathoadaptive conditional regulation of the type VI secretion system in Vibrio cholerae O1 strains.</a> Infection and immunity. 80 (2), 575-584 (2012).</li><li>Pollack-Berti, A., Wollenberg, M. S., Ruby, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+transformation+of+Vibrio+fischeri+requires+tfoX+and+tfoY.">Natural transformation of Vibrio fischeri requires tfoX and tfoY.</a> Environmental Microbiology. 12 (8), 2302-2311 (2010).</li><li>Meibom, K. L., Blockesh, M., Dolganov, N. A., Wu, C. Y., Schoolnik, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin+induces+natural+competence+in+Vibrio+cholerae.">Chitin induces natural competence in Vibrio cholerae.</a> Science. 310 (5755), 1824-1827 (2005).</li><li>Borgeaud, S., Metzger, L. C., Scrignari, T., Blockesh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+VI+secretion+system+of+Vibrio+cholerae+fosters+horizontal+gene+transfer.">The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer.</a> Science. 347 (6217), 63-67 (2015).</li><li>Townsley, L., Mangus, M. P. S., Mehic, S., Yildiz, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Vibrio+cholerae+to+low-temperature+shift:+CpsV+regulates+type+VI+secretion,+biofilm+formation,+and+association+with+zooplankton.">Response of Vibrio cholerae to low-temperature shift: CpsV regulates type VI secretion, biofilm formation, and association with zooplankton.</a> Applied and Environmental Microbiology. 82 (14), 00807-00816 (2016).</li><li>Huang, 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=Functional+characterization+and+conditional+regulation+of+the+type+VI+secretion+system+in+Vibrio+fluvialis.">Functional characterization and conditional regulation of the type VI secretion system in Vibrio fluvialis.</a> Frontiers in microbiology. 8, 528 (2017).</li></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

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...

Watch this Scientific Journal Video about Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates at JoVE.com

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.

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. ...

coincubation-assay-for-quantifying-competitive-interactions-between

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.9K Views.  University of North Carolina, Chapel Hill. 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.

Video: Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

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 ...

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

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

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

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.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

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

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

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

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.

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

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 ...

Biology

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 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 ...

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 ...

Biological Assessment of Thiophenesulfonamides in Quorum Sensing Inhibition

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

Author Spotlight: Advancing Therapeutics to Treat Vibriosis in Humans and Aquatic Organisms

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Biomimetic Materials to Characterize Bacteria-host Interactions

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

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

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

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

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

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors