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. 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.
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’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.
1. Prepare Strains for Coincubation
2. Coincubate Bacterial Strains
3. Visualizing Coincubations Using Fluorescence Microscopy
4. Data Analysis
5. Determining Whether Interaction is Contact-dependent
NOTE: If you find that one strain kills or inhibits the reference strain, the interaction may be diffusible or contact-dependent. To determine whether the interaction is dependent on cell-cell contact, perform a coincubation assay as described above for steps 1-2 with the following modifications.
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 identified. As an example, the following experiments were performed using V. fischeri isolates. Coincubated strains harbored one of two stable plasmids: pVSV102 expressing kanamycin resistance and GFP+, or pVSV208 expressing chloramphenicol resistance and DsRed+. In the sample data, strains were mixed in a 1:1 ratio and incubated on LBS agar plates for 5 h. As a control treatment, differentially tagged versions of the reference strain were coincubated with each other. The experimental treatments were performed with the reference strain (harboring pVSV102) and either competitor strain 1 or competitor strain 2 (harboring pVSV208). Cultures of each strain were prepared and coincubated as described above and as shown in Figure 1 and Figure 2.
In Figure 3, data analyzed to determine whether competitor strain 1 or 2 outcompeted the reference strain are presented in two ways. In Figure 3A, the proportion of each strain type for each time point during the experiment was calculated according to step 4.2.1. In the experimental treatments, the sample data show the reference strain and competitor strain 1 are present at a proportion of 0.5 at the beginning (0 h) and end (5 h) of the experiment, which is consistent with what is observed in the control treatment. These data show the proportion of the strains did not change after a 5 h coincubation, and therefore no competition was observed. By contrast, when the reference strain was incubated with competitor strain 2, the reference strain was present at a proportion of 0.5 at the beginning (0 h), and a proportion <0.01 at the end (5 h) of the experiment, which was significantly lower than the control treatment (Student’s t-test: P < 0.001). These data indicate that the proportion of the reference strain decreased in the presence of competitor strain 2, and therefore suggests competition between competitor strain 2 and the reference strain occurred. This type of analysis should be applied when determining how the proportion of strains within a community changes over time but cannot be used to determine the mechanism of the competitive interaction, and therefore should be combined with additional analysis. For example, the proportion of the reference strain decreasing in the presence of competitor strain 2 could be attributed to several types of interactions: (i) strain 2 grew more quickly than the reference strain, (ii) strain 2 inhibited the growth of the reference strain, or (iii) strain 2 eliminated the reference strain through killing.
In Figure 3B, the log relative competitive index (RCI) was calculated for each treatment according to step 4.2.2. When the reference strain was incubated with competitor strain 1, log RCI values were not statistically different from zero or from the control treatment (P > 0.05), suggesting competition between strains was not observed. When the reference strain was incubated with competitor strain 2, however, log RCI values were significantly greater than zero and the control treatment (Student’s t-test: P < 0.001). These data suggest strain 2 outcompeted the reference strain. Analyzing log RCI values provides a simple method to determine whether one strain outcompeted the other during the incubation period. Because this analysis incorporates the ratio of strains at the end (5 h) and the beginning (0 h) of the experiment, the starting ratio can dramatically impact the result. Therefore, the starting ratio should be examined and considered when deriving conclusions from log RCI data. Furthermore, this analysis does not provide information about the competitive mechanism and simply reports how the ratio of strains change during the incubation.
Figure 4 displays two methods of data analysis to determine the mechanism of competition for a given interaction. In Figure 4A, total CFUs for each strain at each time point of the experiment are displayed. When the reference strain was incubated with competitor strain 1, CFUs of both strains increase over the course of 5 h and CFUs for the reference strain were not significantly different from strain 1 or the control at 5 h (P > 0.05). These data indicate that the reference strain grew in the presence of strain 1, and suggest no competition occurred. However, when the reference strain was incubated with competitor strain 2, strain 2 CFUs increased after 5 h but CFUs for the reference strain decreased. Reference strain CFUs were significantly lower than strain 2 CFUs and the control at 5 h (Student’s t-test: P < 0.002). These data indicate that the reference strain CFUs decrease in the presence of strain 2, and suggest strain 2 kills reference strain cells. If the reference strain did not show a decrease in CFUs, but rather no change (no statistical difference between reference strain CFUs at 0 h and 5 h), these data would suggest strain 2 outcompeted the reference strain by inhibiting the growth of the reference strain. Analyzing untransformed total CFU data is particularly informative, as CFUs for both strains at each time point are displayed independently and can be used to identify the mechanism of competition.
Figure 4B shows the percent recovery of the reference strain in order to determine how the presence of a competitor strain affects the reference strain. When the reference strain was incubated with competitor strain 1, a ~3,200 percent recovery was observed, which was not statistically different from the control and indicates strain 1 did not affect the percent recovery of the reference strain. When the reference strain was incubated with competitor strain 2, a ~4 percent recovery was observed, which was significantly lower than the control (Student’s t-test: P < 0.002). The percent recovery was also significantly less than 100 (Student’s t-test: P < 0.002), indicating strain 2 outcompeted the reference strain by killing reference strain cells. If the percent recovery was not statistically different from 100, those data would suggest strain 2 inhibited the growth of the reference strain. Percent recovery data provides a simplified way to characterize the mechanism of competition by examining how the reference strain population responds to the presence of a competitor strain. However, displaying the data in this way excludes information about the starting ratio as well as how the abundance of the competitor strain changed throughout the incubation.
Figure 1: Flowchart illustrating the coincubation assay. (A) Bacterial strains harboring either pVSV102 (reference strain indicated Ref. Strain or R.S.) or pVSV208 (competitor strain indicated Comp. Strain or C.S.) are grown separately on media selective for either the reference strain (LBS Kan) or competitor strain (LBS Cam). Strains are then resuspended in LBS broth and normalized to an OD = 1.0. (B) The reference strain and competitor strain are mixed at a 1:1 ratio by volume. A serial dilution is performed with this mixture to determine CFUs for both strains at 0 h. (C) The strain mixture is then spotted onto 24-well plates containing LBS agar. Each replicate is spotted into its own well. Spots are allowed to dry and then incubated at 24 °C for 5 h. After 5 h, a serial dilution is performed to quantify CFUs for each strain. (D) The strain mixture from panel B is also spotted onto LBS agar Petri plates allowed to dry and incubated at 24 °C for 24 h. At 24 h, the coincubation spot is imaged using a fluorescence dissecting microscope that is adapted to detect green (reference strain) and red (competitor strain) fluorescence. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 2: Representative images of plates required for a serial dilution. (A) 96-well plate used to perform serial dilution. The plate is rotated such that there are 12 rows and 8 columns. Descriptors of each treatment include the strains used and the plasmids they harbor (e.g., Reference strain with pVSV102 and Competitor strain with pVSV208) and the replicate number for each row (R1, R2, R3, or R4). The first column is the undiluted sample and each column to the right represents a 10-fold dilution from the previous column (dilution factor listed above). (B) LBS agar plate used to determine CFUs for the reference strain on LBS Kan plates (top) and competitor strain on LBS Cam plates (bottom) from the experimental treatment. Each row is one replicate in a treatment (e.g., R1) and the dilution factor of each spot is listed at the top of the plate. The number of CFUs counted for each replicate is listed to the right. Please click here to view a larger version of this figure.
Figure 3: Sample data for assessing whether competitor strains outcompete the reference strain. (A) The proportion of coincubating strains harboring either pVSV102 (dark gray) or pVSV208 (light gray). R.S. indicates the reference strain and C.S. indicates the competitor strain. Dashed horizontal line indicates a proportion of 0.5. Asterisk indicates the reference strain made up a statistically smaller proportion of the population than the competitor strain or reference strain in the control at 5 h (Student’s t-test: P < 0.001); ns indicates not significant (P > 0.05). (B) Log relative competitive index (RCI) for coincubation assays. Dashed vertical line indicates log RCI = zero. Asterisk indicates the log RCI value is statistically greater than zero and the control (Student’s t-test: P < 0.001). Error bars indicate SEM. Please click here to view a larger version of this figure.
Figure 4: Sample data for determining the mechanism of competition. (A) Total CFU counts for coincubation assays performed with V. fischeri isolates that were differentially tagged with either pVSV102 or pVSV208. CFUs were collected at the beginning of the experiment (0 h) and after 5 h incubation. R.S. indicates reference strain and C.S. indicates competitor strain. Dashed horizontal line indicates the average 0 h CFUs for both strains; asterisk indicates reference strain CFUs were statistically lower in the experimental treatment relative to the control treatment at 5 h (Student’s t-test: P < 0.002). (B) Percent recovery of the reference strain. Horizontal dashed line indicates 100% recovery (no increase or decrease in CFUs); asterisk indicates percent recovery was statistically lower than 100% and the control treatment (Student’s t-test: P < 0.002). Error bars indicate SEM. Please click here to view a larger version of this figure.
Figure 5: Sample data for incubation time and imaging optimization. (A) Total CFUs for coincubation assays where CFUs were collected at the beginning of the experiment (0 h), after 5, 12, and 24 h incubation. R.S. indicates reference strain and C.S.2 indicates competitor strain 2. Dashed horizontal line indicates the average 0 h CFUs for both strains; asterisks indicate reference strain CFUs were statistically lower in the experimental treatment relative to the control treatment at the given time point (Student’s t-test: P < 0.002). Error bars indicate SEM. (B) Fluorescent microscopy images corresponding with CFU data in panel A. Scale bar =1 mm. Please click here to view a larger version of this figure.
Figure 6. Sample data for coincubation ratio optimization. Coincubation experiments were performed between the reference strain (R.S.) harboring pVSV102 (green, top row) and competitor strain (C.S.) harboring pVSV208 (red, bottom row) and fluorescent microscopy images were taken at 24 h. (A) Strains were mixed in a 1:1 ratio or (B) a 1:5 ratio where the reference strain, containing pVSV102, was outnumbered by the competitor strain containing pVSV208. Scale bar = 1 mm. Please click here to view a larger version of this figure.
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 competitive mechanisms are often conditionally regulated5,6,23,24,25,26,27,28, thus small differences in growth conditions (e.g., shaking vs standing culture, temperature, etc.) and media type (e.g., salt content) can dramatically affect the results. Therefore, optimizing coincubation conditions is likely necessary for different bacterial species as well as different competitive mechanisms. It is best to choose culture conditions that closely reflect the natural environment of the isolates. For example, coincubation assays between V. fischeri strains were performed with LBS media at 24 °C, to reflect the salinity and temperature of the marine environment. However, some bacteria are naturally competent in their environment27,28 and therefore could take up genetic material released by lysed cells during antagonistic interactions29.To prevent such DNA transfer from impacting coincubation results, it is important to use conditions that do not promote competence or strains that are not competent, either naturally or through inactivation of DNA uptake machinery. Moreover, experimental parameters such as cell growth phase, culture density, incubation time, or starting strain ratio may also require optimization for different bacterial species or competitive mechanisms. For example, initial culture density will dictate the amount of cell-cell contact between strains, which can affect the ability of bacteria to deploy contact-dependent mechanisms of competition.
Figure 5A displays the process of optimization of coincubation assays with V. fischeri isolates. Here, a range of incubation times for CFU collection and fluorescent microscopy imaging were evaluated to determine the optimal time for each metric to be collected. CFUs were collected immediately after the mixture of the reference strain and competitor strain 2 was spotted onto LBS agar plates (0 h), and CFU measurements and fluorescent microscopy images were taken immediately and after 5, 12, and 24 h. These sample data highlight the importance of thorough optimization prior to drawing any conclusions about the interaction between two strains. For example, two different conclusions about the mechanism of interaction can be deduced based on when CFUs are collected: CFUs from 5 or 12 h indicate strain 2 killed the reference strain, while CFUs collected at 24 h suggest strain 2 inhibits the growth of the reference strain.
The optimal time for visualization of coincubation spots through fluorescent microscopy may be different than the optimal time for CFU collection. Figure 5B displays fluorescent microscopy images of coincubation spots at 0, 5, 12, and 24 h. At 0 and 5 h, the coincubation spots are not visible with fluorescent microscopy. For images taken at 12 h, both strains in the control treatment are visible, yet the RFP (reference strain harboring pVSV208) is notably dimmer. In the experimental treatment at 12 h, competitor strain 2 is visible (yet dim) and the reference strain is not detectable. Strain-specific differences between bacterial isolates can affect the expression of fluorescent proteins, and thus brightness of the cells in the mixed spot. Because RFP is notably dimmer than GFP in the control, the coincubation spots should continue to be incubated and be imaged again at a later time. In images taken at 24 h, both strains are visibly detectable and at a similar brightness in the control experiment. In the experimental treatment strain 2 is visible while the reference strain is not observed within the coincubation spot. 15 – 24 h incubation time is sufficient to visualize GFP and RFP for V. fischeri using stable plasmids pVSV102 and pVSV208, respectively, but the incubation time may need to be adjusted for different plasmids or bacterial species. Although the optimal time for visualization of coincubation spots and collecting CFU data are different, imaging at 24 h is a good way to quickly screen interactions for V. fischeri, because the result obtained from imaging at 24 h (target is visible or not) reflects the more time-intensive quantitative data obtained from plating CFUs at 5 or 12 h.
The starting ratio can significantly impact results, particularly when incubating two inhibitory strains, and may need to be adjusted to account for strain specific differences in killing efficiency or growth rate. For example, Figure 6A displays fluorescent microscopy images of experiments where the reference strain was coincubated with itself (control) and three other V. fischeri isolates starting at a 1:1 ratio. In these sample data, the reference strain is visibly detected when incubated with itself, competitor strain 1, and competitor strain 3 after 24 h. However, when the starting ratio was adjusted to 1:5 (i.e., 50 μL of reference strain mixed with 250 μL of competitor strain) the reference strain is only visibly detected when coincubated with itself and strain 1, indicating that both strain 2 and strain 3 outcompete the reference strain. This adjustment prevents the faster growth rate of the reference strain from obscuring the effect of any interference competition mechanisms exhibited by the competitor strains. Based on the results in Figure 6A, a ratio of 1:5 (reference strain : competitor strain) should be used to screen additional V. fischeri strains for the ability to kill the reference strain.
This protocol discriminates between coincubating strains by differentially labeling strains with plasmids containing either kanamycin or chloramphenicol resistance genes. However, different antibiotics or other selection methods may be better suited for different bacterial species. Other methods for differential selection could include: 1) exploiting a strain/species-specific auxotrophy for specific growth factors (e.g., DAP or thymidine), 2) conditional growth requirements (e.g., one strain grows at 37 °C while the other does not), or 3) counterselection markers that eliminate or inhibit the growth of the tagged strain when grown under appropriate conditions to express a “kill” gene (e.g., ccdB or sacB).
Selecting the appropriate reference strain is critical for obtaining and interpreting reproducible results from the coincubation assay. A reference strain should be well-studied (i.e., have a broad body of scientific literature), have no apparent killing or inhibitory ability, and ideally have a sequenced genome. For example, certain strains of Escherichia coli are common reference strains for many bacterial coincubation experiments30,31. However, E. coli may not be ecologically relevant for a given competitive mechanism or competitor, which can affect results. For example, some bacteria may have evolved mechanisms specifically targeting closely-related species or competitors for the same ecological niche and their competitive mechanism would not be effective against an E. coli reference strain.
In summary, the method described here aims to provide an easily modified and robust approach to evaluate interbacterial interactions and competition. This method can be applied to bacterial isolates relevant to environmental or clinical research, and can be used to explore diverse mechanisms of microbial interaction that have been previously unknown or difficult to investigate.
The authors have nothing to disclose.
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.
1.5 mL Microcentrifuge Tubes | Fisher | 05-408-129 | |
10 μL multichannel pipette | |||
100 μL multichannel pipette | |||
300 μL multichannel pipette | |||
10 μL single channel pipette | |||
20 μL single channel pipette | |||
200 μL single channel pipette | |||
1000 μL single channel pipette | |||
24-well plates | Fisher | 07-200-84 | sterile with lid |
96-well plates | VWR | 10062-900 | sterile with lid |
Calculator | |||
Chloramphenicol | Sigma | C0378 | stock (20 mg/mL in Ethanol); final concentration in media (2 μg /mL LBS) |
Fluorescence dissecting microscope with camera and imaging software | |||
forceps | Fisher | 08-880 | |
Kanamycin Sulfate | Fisher | BP906-5 | stock (100 mg/mL in water, filter sterilize); final concentration in media (1 μg/mL LBS) |
Nitrocellulose membrane (FS MCE, 25MM, NS) | Fisher | SA1J788H5 | 0.22 μm nitrocellulose membrane (pk of 100) |
petri plates | Fisher | FB0875713 | sterile with lid |
Spectrophotometer | |||
Semi-micro cuvettes | VWR | 97000-586 | |
TipOne 0.1-10 μL starter system | USA Scientific | 1111-3500 | 10 racks |
TipOne 200 μL starter system | USA Scientific | 1111-500 | 10 racks |
TipOne 1000 μL starter system | USA Scientific | 1111-2520 | 10 racks |
Vortex | |||
Name | Company | Catalog Number | Comments |
LBS media | |||
1M Tris Buffer (pH ~7.5) | 50 mL 1 M stock buffer (62 mL HCl, 938 mL DI water, 121 g Trizma Base) | ||
Agar Technical | Fisher | DF0812-17-9 | 15 g (Add only for plates) |
DI water | 950 mL | ||
Sodium Chloride | Fisher | S640-3 | 20 g |
Tryptone | Fisher | BP97265 | 10 g |
Yeast Extract | Fisher | BP9727-2 | 5 g |