Name: high throughput co-culture assays for the investigation of microbial interactions
Uploaded: 2019-10-15T14:00:00
Description: Watch this Scientific Journal Video about High Throughput Co-culture Assays for the Investigation of Microbial Interactions at JoVE.com

We thank Daniel May, Marc Chevrette, and Don Hoang for critical reading of the manuscript. This work, including the efforts of Cameron R. Currie, was supported by the University of Wisconsin-Madison, Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation, funding also provided by the National Institutes of Health Centers for Excellence for Translational Research (U19-AI109673-01). Reed M. Stubbendieck was supported by a National Library of Medicine training grant to the Computation and Informatics in Biology and Medicine Training Program (NLM 5T15LM007359). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Informed consent was obtained from the donor&#39;s parents, and the Human Subjects Committee at the University of Wisconsin-Madison approved the study (Institutional Review Board [IRB] approval number H-2013-1044).
1. Sample Culture
NOTE: This procedure is used here for the study of interactions among bacteria isolates from the human nasal cavity. In principle, the following methods are applicable to any culture condition. Brain-heart-infusion broth (...

<ol>
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Antibiotics and other secondary metabolites that mediate microbial interactions are useful for a multitude of applications, including drug discovery. Herein, a protocol for co-culture assays to assess large numbers of microbial interactions is presented. These co-culture interaction assays are a simple, affordable, scalable, and high throughput means to investigate many pairwise combinations of microorganisms in tandem. Target organisms are spotted next to test organisms in a well of a 12 well plate using an inoculation ...

In nature, microorganisms rarely exist in isolation; consequently, they are constantly interacting with other organisms. Therefore, studying how microorganisms interact with each other is essential to understanding a multitude of microbial behaviors1. Microbial interactions can be mutualistic, commensal, or antagonistic. These in teractions can affect not only the microorganisms themselves but also the environments and hosts that the microorganisms colonize1,2.
Many scientists study microbial interactions to identify new antimicrobial molecules. One of the first clinically important antimicrobial molecules was found through the study of microbial interactions. Sir Alexander Fleming observed a contaminating Penicillium spp. isolate that inhibited the growth of a Staphylococcus strain, which led to the discovery of the commonly used antibiotic penicillin3. Characterization of the mechanisms that microorganisms use to antagonize their competitors remains a fruitful resource for the discovery of antimicrobial molecules. For example, it was recently shown that Streptomyces sp. strain Mg1 produces antibiotic linearmycins, which have a lytic and degradative activity against Bacillus subtilis4.
Further, a non-ribosomally synthesized peptide named lugdunin was recently discovered after the observation that nasal commensal Staphylococcus lugdunensis inhibits Staphylococcus aureus5. Studies have also shown that mutualistic interactions between microorganisms are equally as powerful as antagonistic interactions for the discovery of antimicrobial molecules. For example, many fungus-farming ants in the tribe Attini harbor symbiotic bacteria called Pseudonocardia on their exoskeleton that produces antifungal molecules to inhibit an obligate pathogen of their fungal crop6. As the study of microbial interactions has been beneficial for discovering antimicrobial molecules, the use of high throughput screens may result in the discovery of new antimicrobial molecules.
With respect to the cost and ease of performance, the methodologies used to study microbial interactions range from simple to complex. For instance, an agar plug assay is an inexpensive and simple method that can be used to investigate antagonism between multiple microorganisms7. However, an agar plug assay is not an efficient procedure and can be labor-intensive for many pairwise combinations.To assess the effects of microbially produced products on target isolates of interest in a high throughput manner, many laboratories use disk diffusion assays8. These assays are easy and inexpensive and can be scalable to higher numbers of samples7. However, this assay requires the generation of microbial extracts and may produce misleading results for certain combinations of target organisms and antibiotics, such as Salmonella and cephalosporins9.
The preceding approaches rely on isolated components to elicit a response in a target organism, instead of allowing microorganisms to interact with each other. This is of note because interactions between microbes may elicit the production of &#34;cryptic&#34; antimicrobial molecules that are not produced in monoculture. For instance, it was recently shown that the antimicrobial keyicin is only produced by a Micromonospora sp. when co-cultured with a Rhodococcus sp. that is isolated from the same sponge microbiome10. More complex interaction methodologies circumvent this potential monoculture hindrance. For instance, the iChip is useful for isolating rare and difficult to cultivate bacteria from environmental samples and allows for the observation of microbial interactions through growth in situ11. To investigate interactions in detail, matrix assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS) can be used. This approach provides detailed information on the composition and distribution of small molecules and peptides produced by interacting microbial colonies with high spatial resolution. MALDI-TOF-IMS&#160;has also been used in multiple studies of bacterial interactions to characterize the mechanisms of competition12,13,14,15. However, MALDI-TOF-IMS often requires laborious sample preparation, specialized expertise to operate the equipment, and expensive and specialized mass spectrometers. For these reasons, it is a difficult technique to use for high throughput studies. Thus, a simple, scalable, and high throughput co-culture assay for microbial interactions that overcomes many limitations of the above approaches would be beneficial.
Here, a protocol for high throughput microbial co-culture is presented. This assay is simple and easily incorporated into preexisting studies of microbial interactions. In contrast to many commonly used methods for the study of microbial interactions, our method is simple, inexpensive, and is amenable to investigating large numbers of interactions. These assays are not only easy to perform, but the materials are widely available from most laboratory suppliers or public resources (e.g., libraries and makerspaces). Consequently, this assay is advantageous as a first line of investigation to identify and parse interesting patterns among many pairwise combinations of microorganisms, which may be especially useful for the investigation of microbial ecology.

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	<tbody>
		<tr>
			<td>1 &#956;L disposable polystyrene inoculating loops, blue</td>
			<td>VWR</td>
			<td>12000-806</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L disposable polystyrene inoculating loops, yellow</td>
			<td>VWR</td>
			<td>12000-810</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plate, sterile with lid</td>
			<td>Greiner bio-one</td>
			<td>665 180</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL polystyrene round bottom tube, 17 x 100mm style, nonpyrogenic, sterile</td>
			<td>Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 self standing screw cap tubes with caps, sterile</td>
			<td>USA scientific</td>
			<td>1420-9710</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipet</td>
			<td>Cell Treat</td>
			<td>229225B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar, bacteriological</td>
			<td>VWR</td>
			<td>J637</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion Broth</td>
			<td>Dot Scientific</td>
			<td>DSB11000-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate filament, white, 3mm diameter</td>
			<td>Keene Village Plastics</td>
			<td>12.1-3MM-WH-581.2-1KG-R</td>
			<td></td>
		</tr>
		<tr>
			<td>School Glue</td>
			<td>Elmer&#39;s</td>
			<td>EPIE304</td>
			<td></td>
		</tr>
		<tr>
			<td>Taz 6 3D printer</td>
			<td>Lulzbot</td>
			<td></td>
			<td></td>
		</tr>
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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.

Co-culture interaction assays can be used to understand microbial interactions, identify patterns of interest, and uncover microbial isolates with intriguing activities. In these assays, a test organism is monocultured on one side of a 12 well agar plate and incubated for 7 days. Subsequently, a target organism is spotted next to the test organism and the two microbes were co-cultured for 7 days before scoring for the growth phenotype of the target organism. The assays are scored based on...

Watch this Scientific Journal Video about High Throughput Co-culture Assays for the Investigation of Microbial Interactions at JoVE.com

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

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 ...

Our protocol provides a high throughput method for screening a large number of pairwise microbial interactions in tandem to identify interactions of interest which can aid microbiome research and antimicrobial discovery. This method is readily applicable to studying microbial interactions in a variety of systems, as long as the microorganisms are amiable to culture under laboratory conditions. The main advantage of this technique is that it allows users to screen a large number of microbial interactions in a relatively short period of time.New users of this technique should take time, especially when inoculating the bioassay plates, as it is crucial to be careful to minimize overgrowth or contamination. Visual demonstration of this technique is critical, because inoculating the bioassay plates requires precision to ensure that the organisms do not overgrow the wells, and that the co-cultures are not contaminated. Begin by preparing overnight cultures.Use a serological pipette to add three milliliters of BHI broth into 14-milliliter culture tubes. Then use a sterile one microliter inoculating loop to inoculate a bacterial colony into the broth. Swirl the loop to ensure that the clump disperses into the broth.Briefly vortex the culture tubes, and incubate them overnight at 37 degrees Celsius on a shaker at 250 RPM. Once the bacterial cultures reach an OD 600 above one, vortex them to break up the clumps of cells. Prepare PHI media with 1.5%agar, and sterilize it by autoclaving.Then cool the media to 55 degrees Celsius in a temperature-controlled water bath, and dispense three milliliters of molten BHI media into each of the 12 wells on a plate, ensuring that the wells are as even as possible. Then allow the agar to set overnight. Inoculate the test organism on a bioassay plate by inserting a sterile 10-microliter inoculating loop into the overnight culture, and streaking the culture droplet over the left third of a plate well.Repeat until all 12 wells on the plate have been inoculated. Incubate the plates upside down at the appropriate temperature for seven days. If incubating at temperatures greater than or equal to 37 degrees Celsius, or in drier climates, store the plates in a humid container to prevent them from drying out.After the six-day incubation of the bioassay plate, prepare overnight cultures of the target organism as previously described. Prepare the target plate by filling each well of an empty 12-well plate with 1.8 milliliters of BHI, and 200 microliters of the target overnight culture. Place a sterile inoculation stamp into the target plate.Gently swirl the cultures around the wells, ensuring that they do not cross-contaminate neighboring wells. Lift the inoculation stamp, and ensure that there is a droplet of diluted target culture on each stamp tip. Place the inoculation stamp on an uninoculated bioassay plate, and gently rock the stamp so that the culture droplet inoculates each well.Once the stamp is removed, a droplet of culture should be visible in the wells. If any wells are not inoculated with the inoculation stamp, spot three microliters of diluted overnight culture onto the wells using a pipette. Then inoculate the bioassay plates as previously described, but align the stamp tips with the right side of the 12-well plate, ensuring that the stamp does not contact the existing bacterial colony.Carefully remove the stamp, and place it back into the target plate. If the agar has solidified in the wells unevenly, gently rock the stamp back and forth, but be careful not to shift the stamp into the test organism growth. Repeat the inoculation for each bioassay plate until all the plates are inoculated, then incubate the plates upside down at the appropriate temperature for seven days.After co-culturing the test and target organism for one week, score the interactions based on visual assessment. Score the wells with the target organism growth that exhibits indistinguishable growth from the monoculture as zero. Score the wells with diminished growth as one, and no growth as two.The co-culture assays described here were used to assess the inhibitory activity of the actinobacteria test organisms toward the staphylococcus species target organisms isolated from the human nasal cavity. A total of 812 pairwise combinations were tested. Notably, Corynebacterium propinquum strongly inhibited coagulase negative staphylococci, particularly in comparison to other Corynebacteria that either weakly inhibited coagulase negative staphylococci or had no inhibitory effects.Through the use of comparative genomics, a biosynthetic gene cluster for siderophore production was identified in the Corynebacterium propinquum genome. Co-culture interaction assays using standard and iron-supplemented BHI medium determined that the inhibition phenotype between Corynebacterium propinquum and coagulase negative staphylococci was in fact iron-dependent. It is important to be careful when stamping the plate.Double check each well after stamping, and make sure the inoculation stamp is aligned correctly to prevent contamination. After identifying interactions of interest, subsequent studies using microbial genetics and genomics, natural product isolation and characterization, or imaging mass spectrometry can be used to characterize mechanisms underlying these interactions. This protocol has recently been used to uncover siderophore-mediated competition among the human nasal microbiota, and aided in the discovery of a new antifungal molecule called cyphomycin.

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