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 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.
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 "cryptic" 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 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.
Informed consent was obtained from the donor'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 (BHI) is used for general propagation of nasal bacteria. All plates are solidified using 1.5% agar. For this study, samples are taken from saline solution flushed into a donor's nose (nasal lavage), transferred into microcentrifuge tubes, and frozen at -80 °C.
2. 3D Printing Stamps
NOTE: Polycarbonate was selected as the stamping material due to its high glass transition temperature (147 °C) that exceeds standard autoclave temperatures (121 °C), which minimizes the potential for deformation after repeated uses.
3. Preparation of Overnight Cultures
4. Preparation of Bioassay Plates
NOTE: Bioassay plates are prepared in a laminar flow hood to maintain sterility.
5. Inoculating Bioassay Plates with the Test Organism
NOTE: A test organism refers to the organism for which the production of inhibitory activity (e.g., antibiotic production) is determined using the co-culture interaction assay. For this experiment, the test organisms are Actinobacteria isolated from nasal lavages samples.
6. Preparation of Target Organisms
NOTE: A target organism refers to the organism whose inhibition status is determined using the co-culture interaction assay. For this experiment, the target organisms are Staphylococcus spp. isolated from nasal lavages samples.
7. Target Organism Inoculation
NOTE: After overnight incubation, ensure that the cultures are turbid (OD600 ≥ 1). Some bacterial cultures may flocculate at the bottom of the culture tube. Vortex the culture tubes to disperse clumps and assess the culture turbidity.
8. Scoring
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 a visual analysis of the growth or inhibition of the target organism.
These co-culture assays were recently used to assess the inhibitory activity of Actinobacteria (test organisms) toward Staphylococcus spp. (target organisms) isolated from the human nasal cavity (Figure 2)16. The co-culture assays were used to identify specific inhibition patterns between Actinobacteria (n = 21) and Staphylococcus isolates (n = 39) and showed that Actinobacteria isolates showed variation in their ability to inhibit coagulase-negative staphylococci (CoNS). A total of 812 pairwise combinations were tested. In particular, Corynebacterium propinquum strongly inhibited CoNS (Figure 2D), especially compared to other Corynebacterium that weakly inhibited CoNS (Figure 2C) or had no effect on CoNS (Figure 2B), and compared to the monoculture control (Figure 2A)16.
Using comparative genomics, a biosynthetic gene cluster for siderophore production was identified in C. propinquum genomes that was absent in the genomes of other Corynebacterium isolates16. Siderophores are chelators produced by microorganisms to scavenge iron from the environment17. Siderophore production by C. propinquum was confirmed and the siderophore was identified as dehydroxynocardamine16. This result led to the hypothesis that inhibition of CoNS was due to siderophore-mediated iron depletion. Subsequently, by performing co-culture interaction assays between C. propinquum and CoNS on both standard and iron-supplemented BHI medium, it was determined that the inhibition phenotype was iron-dependent (Figure 2E). Together, these results suggested that siderophore-mediated iron depletion was responsible for the strong inhibition of CoNS by C. propinquum16.
Figure 1: Photograph of the bioassay inoculation stamp. Please click here to view a larger version of this figure.
Figure 2: Co-culture interaction assays uncover siderophore-mediated inhibition of CoNS by Corynebacterium propinquum. (A) Monoculture of CoNS (target organism) inoculated on a BHI bioassay plate. (B,C,D) Co-cultures between different strains of Corynebacterium spp. (test organisms, left) and the same strain of CoNS (target organism, right) inoculated on BHI bioassay plates. Each panel is a representative image showing interactions with (B) no inhibition (score = 0), (C) weak inhibition (score = 1), or (D) strong inhibition (score = 2). (E) Comparison of interactions between Corynebacterium pseudodiphtheriticum (siderophore non-producer) or Corynebacterium propinquum (siderophore producer) with the same strain of CoNS on BHI media (BHI) and BHI media supplemented with 200 µM FeCl3 (BHI + iron). Please click here to view a larger version of this figure.
Supplementary Data File. Please click here to download this file.
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 stamp, and inhibition of the target organisms is scored based on visual inspection of the target organism's phenotype. The majority of materials used in these assays are readily available through most laboratory suppliers or through public resources. Therefore, the assays can be easily tailored to many laboratory environments. In the laboratory, these co-culture assays have been successful in investigating the interactions of microorganisms associated with many hosts from a variety of environments.
While there are many benefits to this technique, there are also a few limitations. The first notable limitation is that patterns from the co-culture assays are difficult to interpret without other metadata. In two recent papers that employed these co-culture assays16,18, inhibition patterns of interest were only identified in conjunction with other metadata, such as the taxonomic identity of the test organisms. Nevertheless, even without accompanying metadata, the methods outlined in this paper are amenable to identifying bacterial isolates with inhibitory activity toward specific pathogens or target microbes of interest.
An additional limitation is that these assays are not commercially available, and the bioassay plates must be hand-prepared, which can limit the assay's efficiency. Plate preparation is critical to the experiment, and care should be taken while preparing the plates. If the wells are uneven, then the inoculation stamp may not be able to inoculate all wells. However, missed wells can simply be inoculated by directly pipetting the target organism culture onto the right side of each uninoculated well. Indeed, even if a few wells on each plate are missed by the stamp, the procedure is still more efficient than directly pipetting the target organism into every single well. Alternatively, users can forgo the inoculation stamp and pipette the target organism into each well, but this process is more time-consuming, especially compared to stamping 12 wells simultaneously.
Finally, the test organism may consume the available nutrients in the well during monoculture before the target organism is inoculated. Though nutrient depletion may affect the observed inhibition patterns, it appears to be uncommon among the pairwise combinations that have been tested thus far. Notably, depletion of iron in the wells by C. propinquum allowed for the discovery of siderophore-mediated competition from members of the human nasal microbiota16.
These co-culture interaction assays are customizable by modifying the media composition, timing, or even including multiple organisms or microbial consortia as the test organism. Furthermore, different scoring systems can be used depending on the desired level of detail required to describe the interaction phenotype. Examples scoring scales include those from 0-2 used to describe competitive interactions among bacteria isolated from the human nasal cavity16 (Figure 2) and from 0-3 used to assess the antimicrobial potential of Streptomyces isolated from insect microbiomes18. However, with more nuanced scales, scoring becomes increasingly difficult. Thus, inhibition is most easily recognized using a binary scoring system (e.g., 0 is defined as no inhibition and 1 as inhibition), which can eliminate any confusion and standardize scoring across multiple individuals. Moreover, in addition to scoring for inhibition phenotypes, these assays can also be scored for other phenotypes, including pigment production, sporulation, or any other phenotype that can be assessed visually. As these assays are highly scalable with analysis based on visual inspection of the target organism, training sets for machine learning algorithms can be generated to facilitate phenotype scoring and further increase assay throughput.
A major strength of these co-culture assays is their ability to facilitate screening many combinations of pairwise interactions inexpensively and rapidly to uncover activities or inhibition patterns of interest. Subsequently, more complex and intensive methods (i.e., genomic characterization, MALDI-TOF-IMS, or natural product isolation and characterization) may be used for deeper characterization of the microbes and interactions of interest identified by co-culture assays. As a recent example, these co-culture inhibition assays were used to show that insect-derived Streptomyces can inhibit Gram-negative bacteria and fungi better than their soil-derived counterparts. The inhibition assays allowed for quick and efficient visualization of inhibition patterns among 2,003 Streptomyces isolates and led to the discovery of a new antifungal called cyphomycin, which is active against drug-resistant fungal pathogens18. Thus, these co-culture interaction assays are a powerful tool for microbiome research, antimicrobial discovery, and gaining deeper insights into patterns of microbial interactions.
The authors have nothing to disclose.
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.
1 μL disposable polystyrene inoculating loops, blue | VWR | 12000-806 | |
10 μL disposable polystyrene inoculating loops, yellow | VWR | 12000-810 | |
12-well cell culture plate, sterile with lid | Greiner bio-one | 665 180 | |
14 mL polystyrene round bottom tube, 17 x 100mm style, nonpyrogenic, sterile | Falcon | 352057 | |
2.0 self standing screw cap tubes with caps, sterile | USA scientific | 1420-9710 | |
25 mL serological pipet | Cell Treat | 229225B | |
Agar, bacteriological | VWR | J637 | |
Brain Heart Infusion Broth | Dot Scientific | DSB11000-5000 | |
Polycarbonate filament, white, 3mm diameter | Keene Village Plastics | 12.1-3MM-WH-581.2-1KG-R | |
School Glue | Elmer's | EPIE304 | |
Taz 6 3D printer | Lulzbot |