Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.
Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.
Bacterial infection is one of the leading causes of mortality in human and animals1,2. The ability to measure virulence of bacteria in cultures or biofilms is important in healthcare and research settings. Here, we describe a versatile, rapid, and relatively simple method to quantify bacterial virulence. The eukaryotic organism Dictyostelium discoideum (amoeba) is used as the model host organism. D. discoideum has been used as a host to identify virulence factors in Pseudomonas aeruginosa (P. aeruginosa)3,4,5 and other bacteria6,7,8 and is susceptible to largely the same virulence factors that kill mammalian cells including type III secretion9,10. Previous virulence assays using D. discoideum have involved prolonged exposure of bacteria with D. discoideum cells over the course of hours3,4,5. The protocol, here, presents a rapid method of determining virulence using this amoeba. This protocol (Figure 1) describes how to: (1) grow the amoebae axenically (in the absence of bacteria), (2) grow bacteria for the assay, (3) prepare bacteria and host cells for microscopy, (4) perform epifluorescence microscopy, and (5) analyze amoeba fluorescence.
Amoebae are initially streaked out from frozen stocks and grown on a lawn of Escherichia coli (E. coli), where the amoebae produce spores. These spores are picked and inoculated into an enriched medium for axenic growth. The amoebae are maintained through axenic growth in nutrient-rich conditions until they are ready to be mixed with bacteria for the assessment of bacterial virulence. The survival or the death of the amoebae is quantified by measuring the fluorescence of calcein-acetoxymethyl (calcein-AM), which is cleaved by intracellular esterases and, thereby, activated for fluorescence11,12. Live amoebae exhibit little or no fluorescence whereas stressed and dying cells fluoresce intensely. This result is due to a little or no incorporation of calcein-AM into healthy amoebae and incorporation and cleavage of the substrate in stressed amoebae13. This behavior is notably distinct from calcein-AM fluorescence in mammalian cells11,14,15,16.
Bacteria that will be assessed for virulence are grown separately. Here, we describe how to measure the virulence of the opportunistic pathogen P. aeruginosa and detail how to quantify the virulence of planktonic (swimming) and surface-attached sub-populations. This protocol may be adapted to test the virulence of other bacteria. In the Representative Results section, we show that virulence is activated in surface-attached cells and is low in planktonic cells, which was reported previously13. Virulence-activated surface-attached P. aeruginosa kills amoebae while non-virulent planktonic cells are consumed by the amoebae. If the virulence of planktonic bacteria is solely being assayed, bacteria can be cultured in ordinary culture tubes rather than using Petri dishes as described in the protocol.
The growth of amoebae and P. aeruginosa cultures must be coordinated such that P. aeruginosa cultures reach the intended growth phase while the amoebae are growing at steady state in nutrient-rich conditions. This condition typically requires amoebae cultures to be diluted at least 1 day prior to when they are mixed with bacteria. Amoebae and bacteria are immobilized using agar pads, are co-incubated for 1 h, and imaged using a low resolution (10X, numerical aperture 0.3) objective, green fluorescence protein (GFP) filters, and an imaging camera. Analysis can be performed using freely-available ImageJ software or customized image analysis software. Our analysis was performed using our own software written using a scientific analysis package13. The software should create a mask using the phase contrast image and extract fluorescence values from the masked areas in the fluorescence image. Fluorescence values are averaged over at least 100 cells, resulting in a numerical host killing index.
All experimental procedures were carried out at the University of California, Irvine.
1. Buffers and Solutions
2. Growth and Maintenance of Amoebae
3. The Growth of P. aeruginosa
4. Microscopy – Preparation of the Agar Pad
5. Microscopy – Preparation of the Bacteria-amoeba Sample for Imaging
6. Microscopy – Image Acquisition
7. Image Analysis and Host Killing Index
We grew wild-type P. aeruginosa strain PA1419 or a ΔlasR strain20 in the same PA14 background in 6 cm-diameter Petri dishes and assayed the virulence of planktonic and surface-attached cells. Cultures were inoculated from single-colonies into PS:DB cultures, grown overnight in culture tubes in a roller drum at 37 °C to saturation, diluted 1:100 into PS:DB, grown for 8 h in 6 cm-diameter Petri dishes shaking at 100 rpm, and planktonic and surface-attached P. aeruginosa sub-populations were isolated as described in section 3.
Surface-attached wild-type P. aeruginosa killed amoebae, which was indicated by a round amoeba cell shape and the observation of calcein fluorescence (Figure 4A, top-left panel). This resulted in a high host killing index (Figure 4B). Planktonic cells were consumed by the amoebae, which was indicated by amorphous amoeba cell shapes and the observation of little or no calcein fluorescence above background (Figure 4A, top-right panel). This resulted in a relatively low host killing index (Figure 4B). Both surface-attached and planktonic cells of the ΔlasR strain were consumed by the amoebae, which was indicated by low host killing indexes (Figure 4B, bottom panel). The results thus show that virulence is upregulated in surface-attached populations and the LasR is required for the surface-activated virulence, which was described previously13. These experiments thus establish robust positive and negative controls for future experiments.
Figure 1: Schematic describing an overview of the virulence assay. (1) Amoeba host cells are grown, (2) bacterial cultures are grown, and (3) planktonic or surface-attached bacterial cells are mixed with amoebae and immobilized onto the same imaging plane using an agar pad. Host cells are quantified for health using calcein-AM, fluorescence microscopy, and image analysis. Scale bars represent 50 µm. Please click here to view a larger version of this figure.
Figure 2: Amoeba spores on a GYP 10 cm-diameter Petri dish plate after 5 days of growth. Spores form above the Petri dish surface. The inset shows a magnified view of a single section of the dish surface. The surface may contain bacteria which cannot be discerned at this resolution. Scale bars in main and inset images represent 1 cm and 2 mm, respectively. Please click here to view a larger version of this figure.
Figure 3: Image analysis of microscopy data using ImageJ. (A) Selection of the peak intensity using the threshold tool. (B) Resulting image after the phase contrast source image from Figure 1 is converted into a binary image. (C) Resulting image after the image is subsequently converted to a mask. (D) Screenshots of the selection of regions of interest using the ROI Manager. Scale bars represent 50 µm. Please click here to view a larger version of this figure.
Figure 4: Representative results of amoeba killing by P. aeruginosa cells. (A) Calcein-AM fluorescence overlaid on phase microscopy images and (B) host killing indexes of surface-attached or planktonic wild-type or ΔlasR P. aeruginosa. Wild-type surface-attached P. aeruginosa kills amoebae, which exhibit significant calcein fluorescence and hence a significant host killing index. Planktonic cells are consumed by amoebae, which exhibit little calcein fluorescence and produce a low host killing index. Both surface-attached and planktonic ΔlasR are consumed by amoebae and result in a low host killing index. Scale bars represent 50 µm. Bars indicate the average of three independent experiments and errors bars indicate standard deviation. Please click here to view a larger version of this figure.
This protocol describes a rapid and quantitative method to assay virulence in P. aeruginosa. This protocol may be tested with other bacteria. However, it is important to keep in mind that the growth medium should be compatible with the amoeba growth conditions. In particular, we have optimized the protocol using PS:DB as the bacterial growth medium. If other media are used, it may be necessary to perform a growth media-only control in which there are no bacterial cells present to verify that the medium is compatible with the amoebae.
This method can be expanded to assay the growth phase dependence of virulence. In particular, bacterial cultures can be grown for a wide range of times instead of a fixed time point. In our experience, it is important to harvest bacterial cultures at specific time points as virulence in P. aeruginosa appears to be dependent on the growth phase. We observed that virulence in P. aeruginosa was induced between 6 – 8 h of growth in Petri dishes.
Many variables associated with the growth environment affect host health and bacterial virulence. Thus, it is important to use proper positive and negative controls for each experiment. We have used wild-type surface-attached P. aeruginosa as a positive control and ΔlasR as a negative control. We suggest performing these controls each time the virulence assay is performed. These controls are important for verifying that host cell deaths are not due to any external factors such as the age of the amoebae, variations in temperature, etc. Furthermore, we suggest performing all experiments in biological replicate to establish the reproducibility of the virulence phenotypes.
We have performed our experiments using amoebae at an optical density of 0.2 to 0.5 after a dilution of at least 1:10 and have regulated the temperature of amoebae culture precisely to 22 °C. Outside of these growth conditions, we have found that the susceptibility of amoebae to virulent bacteria and the reproducibility of the virulence assay are altered. Amoeba cells pellet relatively quickly. Ensure that cultures are resuspended by pipetting up and down immediately before the optical density measurement is made. Do not grow the amoeba cultures higher than an OD600 density of 1 as growth to high densities affects the reproducibility of the experiment. Propagate axenic cultures for no longer than 1 week. In addition, it is important to verify that amoebae are grown axenically (beginning in step 2.7) through 20X or high magnification microscopy such that other microbes are not present in the culture. If cultures are turbid in step 2.10 after 2 days of growth following initial inoculation, this would likely indicate the presence of a microbial contaminant. If bacterial contamination is suspected, we suggest growing 1 mL of the amoebae culture at 37 °C for 4 – 8 h. The observation of a turbid culture under these conditions would suggest bacterial contamination. If repeated bacterial contamination is observed, the antibiotic-antimycotic solution should be replaced.
The host killing index is a reliable indicator of whether a bacterial population is virulent or avirulent. This assay has not been optimized for comparisons between different intermediate levels of virulence (i.e., low virulence compared to medium-low virulence) and over-interpretation of the results should be avoided. Potential methods to address this issue are the repetition of the virulence assay over multiple days using different batches of host cells to establish the confidence in the results, performing additional replicate experiments, and performing appropriate statistical analyses.
The multiplicity of infection (MOI) of planktonic cells can be controlled by normalizing the optical density of the bacterial culture. However, this assay does not control the MOI of surface-attached bacteria cells. We have observed that bacterial surface density increases with time. Thus, adjusting growth time in the Petri dish may result in a corresponding change in surface density. In addition, the surface density depends on the material of the surface. P. aeruginosa cells attach to polystyrene surfaces in the assay described here. However, other surfaces including glass, agar, and polyacrylamide may also be assayed13. The surface density of single-layered bacterial populations can be measured using 100X magnification phase microscopy. If surface densities are multi-layered, confocal microscopy may be appropriate.
Here, we have described a rapid and robust method to measure bacterial virulence. By modifying individual parameters such as incubation times, the fluorescent dye, the bacterial strain, the host cell type, or growth media, this method may be extended to quantify virulence across a wide range of organisms and growth conditions.
The authors have nothing to disclose.
KP and AS wrote and revised the manuscript. KP performed the experiments and the analysis. This work was supported by the National Institutes of Health (NIH) Career Transition Award (K22AI112816) to AS.
Reagents | |||
Bacto agar, dehydrated | BD Difco | 214010 | |
Antibiotic-Antimycotic (100X) | Life Technologies | 15240062 | Aliquot < 1 mL and store at -20 °C |
Calcein-acetoxymethyl ester (calcein-AM) | Life Technologies | C34852 | Calcein Acetoxymethyl (AM) |
Calcium chloride, anhydrous | Sigma-Aldrich | C1016 | |
D-Glucose | Fisher Chemical | D16500 | Dextrose |
Dimethyl sulfoxide | Sigma-Aldrich | D5879 | |
Folic acid | Sigma-Aldrich | F8758 | |
LB-Miller | BD Difco | 244620 | |
Magnesium chloride | Sigma-Aldrich | M8266 | |
Yeast extract | Oxoid | LP0021 | |
Special peptone | Oxoid | LP0072 | |
Potassium hyroxide | Fisher Chemical | P250 | |
Potassium phosphate monobasic | Sigma-Aldrich | P0662 | |
Sodium phosphate dibasic heptahydrate | Fisher Chemical | S373 | |
Vitamin B12 | Sigma-Aldrich | V2876 | |
Strains | |||
Dictyostelium discoideum | Siryaporn lab | Strain AX318 | |
Escherichia coli | Siryaporn lab | Strain B/r17 | |
Pseudomonas aeruginosa | Siryaporn lab | PA14 | PA14 strain19 |
Pseudomonas aeruginosa ΔlasR | Siryaporn lab | AFS20.1 | PA14-derived strain20 |
Supplies | |||
0.22 µm filter | Millipore | SCGPT01RE | For filter sterilization |
Conical tube, 15 mL | Corning | 352097 | |
Glass storage bottles | Pyrex | 13951L | 250 mL, 500 mL, 1000 mL |
Petri dish, 6 cm diameter | Corning | 351007 | 60 x 15 mm polystyrene plates |
Petri dish, 10 cm diameter | Fisher | FB0875712 | 100 x 15 mm polystyrene plates |
Plastic containers with lid | Ziploc | 2.57E+09 | Square 3-cup containers |
Glass plates | Bio-Rad | 1653308 | For preparing agar pads. Other glass plates may be used with similar dimensions. |
Wooden sticks | Fisher | 23-400-102 | |
Equipment | |||
Eclipse Ti-E microscope | Nikon | MEA53100 | Microscope setup |
10X Plan Fluor Ph1 objective 0.3 NA | Nikon | MRH20101 | Microscope setup |
Fluorescence excitation source | Lumencor | Sola light engine | Microscope setup |
Fluorescence filter set | Semrock | LED-DA/FI/TX-3X3M-A-000 | Microscope setup |
Orca Flash 4.0 V2 Camera | Hamamatsu | 77054098 | Microscope setup |
ImageJ | NIH | v. 1.49 | Software for image analysis |
MATLAB | Mathworks | R2013 | Software for image analysis |
Orbital shaker incubator | VWR | 89032-092 | For growth of bacteria at 37 °C |
Platform shaker | Fisher | 13-687-700 | For growth of amoebae at 22 °C |
Spectrophotometer | Biochrom | Ultrospec 10 | |
Undercounter refrigerated incubator | Fisher | 97990E | For growth of amoebae at 22 °C |
Isotemp waterbath | Fisher | 15-462-21Q | For cooling media to 55 °C |