Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Summary

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Abstract

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Introduction

C. difficile is a gram-positive, spore-forming, anaerobic, and toxin-producing bacillus that is the leading cause of severe infections in the gastrointestinal tract¹. Typical symptoms of CDI include diarrhea, abdominal pain, and fatal pseudomembranous colitis, and it can sometimes lead to death^1,2. Evidence has shown that host immune responses play a critical role in both the progression and outcome of this disease³. In addition to the immune response, the indigenous gut microbiota is crucial for the onset and pathogenesis of CDI⁴. In the past decade, both the number of cases and the mortality rate of CDI have increased significantly due to the emergence of a hypervirulent strain of C. difficile (BI/NAP1/027)^5,6. A better understanding of underlying immune mechanisms and the role of microbiota during CDI will help lead to new therapeutic developments and advances, enabling better control of this epidemic.

Several animal models, such as the hamster and mouse, have been developed to provide insight into the immune defense against C. difficile^7,8. However, the role of innate immune cells is still poorly understood, particularly since innate immune cell behavior is mainly derived from histological analysis or cultured cells in vitro. Therefore, establishing a transparent zebrafish model to reveal the innate immune response to C. difficile inside of a living vertebrate organism will facilitate such studies. Zebrafish larvae have a functional innate immune system, but they lack the adaptive immune system until 4–6 weeks after fertilization⁹. This unique feature makes zebrafish larvae an excellent model to study the isolated response and function of innate immune cells in CDI.

This report describes new methods using zebrafish larvae to study the interactions between C. difficile and innate immune cells, such as macrophages and neutrophils. First, a localized microinjection protocol that includes C. difficile inoculum and staining is presented. Using in vivo confocal time-lapse imaging, the response of neutrophils and macrophages towards the infection site is recorded, and the phagocytosis of bacteria by neutrophils and macrophages is observed. However, it has been reported that the injection itself causes tissue damage and leads to the recruitment of leukocytes independent of the bacteria¹⁰. Therefore, a noninvasive microgavage protocol to deliver C. difficile into the intestine of zebrafish larvae is subsequently described. Previous studies have demonstrated that indigenous gastrointestinal microbiota protect a host against the colonization of C. difficile¹¹. Therefore, gnotobiotic zebrafish larvae are also used to predispose the zebrafish that are infected ¹². Afterwards, intestinal dissection is performed to recover viable C. difficile and validate the duration of their presence in zebrafish intestinal tracts.

Protocol

All animal work described here was performed in accordance with legal regulations (EU-Directive 2010/63, license AZ 325.1.53/56.1-TUBS and license AZ 33.9-42502-04-14/1418).

1. Preparation of Low Melting Agarose, Gel Plate, and Microinjection/Microgavage Needles

1. Dissolve 0.08 g of low melting agarose (Table of Materials, agarose A2576) in 10 mL of 30% Danieau's medium (0.12 mM MgSO₄, 0.18 mM Ca [NO₃]₂), 0.21 mM KCl, 1.5 mM HEPES (pH = 7.2), and 17.4 mM NaCl, stored at room temperature (RT) to obtain a 0.8% solution.
   NOTE: Higher or lower concentrations of agarose can be used. However, the required time to solidify varies for different brands of agarose, even at the same concentration.
2. Prepare microinjection and microgavage needles from glass capillaries (Table of Materials).
   1. Use a micropipette puller with the following settings (note that units are specific to the puller used here; see Table of Materials): microinjection needles (air pressure = 500; heat = 400; pull = 125; velocity = 75; time = 150); and microgavage needles (air pressure = 500; heat = 400; pull = 100; velocity = 75; time = 150). Use a microloader tip to load 3 µL of nuclease-free H₂O into the pulled needle.
   2. Introduce the needle into the injector and fasten it properly. Adjust the needle to a suitable angle for injection. Set the injection pressure between 600–900 hPa for microinjection needle and 200–300 hPa for gavage needle.
   3. Place a drop of mineral oil onto the black circle of the calibration slide. Use fine forceps to clip the tip of the needle. Inject one drop into the mineral oil to measure the size of the droplet.
   4. For microinjection, adjust the injection time to obtain a droplet with a diameter of 0.10–0.12 mm, which equals a volume of 0.5–1.0 nL. For microgavage, obtain a droplet with a diameter of 0.18–0.20 mm, which equals a volume of 3–5 nL.
3. Prepare a 1.5 % agarose plate with agarose (Table of Materials, 8050) in a 10 cm Petri dish in 30% Danieau's medium using a plastic mold as the microgavage mold. Store at 4 °C to prevent desiccation until needed. Warm to RT or 28 °C prior to the experiment.

2. Preparation and Labeling of C. difficile and Spores with Fluorescent Dye

1. Prepare a 1 mM stock solution of a fluorescent dye (Table of Materials). Because the dye is sold in 50 µg aliquots of powder, add 69 µL of DMSO to the vial to obtain a 1 mM stock concentration.
2. Prepare a 100 µM working solution of the fluorescent dye by adding 2 µL of 1 mM stock solution to 18 µL of DMSO in a centrifuge tube, and mix well.
3. Culture C. difficile (R20291, a ribotype 027 strain) by inoculating 10 mL of BHIS liquid medium with two to three colonies from a plate in an anaerobic hood without shaking overnight. BHIS is BHI supplemented with 0.5% (w/v) yeast extract and 0.1% (w/v) L-cysteine. Dissolve 1 g of L-cysteine in 10 mL of ddH₂O and sterilize by filtration, then add to the autoclaved medium to obtain a final concentration of 1 g/L. Plates are prepared by adding 15 g/L agar-agar to the medium before autoclaving. Selective culturing of C. difficile is done by using chromID C. difficile plates (Table of Materials).
4. Staining C. difficile with the fluorescent dye
   1. Harvest C. difficile at an OD₆₀₀ of 1.0–1.2 and wash 1x with 1 mL of 1x PBS (5,000 x g for 3 min at RT). Resuspend in 1 mL of 1 x PBS.
   2. Add 3 µL of working solution of the fluorescent dye into 1 mL of bacteria suspension. Incubate the sample for 15 min at RT in the dark. Wash the stained C. difficile once with 1 mL PBS to remove residual dye and resuspend in 1x PBS to an OD₆₀₀ of 1.0 (1.0 OD₆₀₀ is approximately equivalent to 10⁸ cfu/mL).

3. Injection of Stained C. difficile into Zebrafish Larvae

1. Anesthetize 20–30 zebrafish larvae at 5 days post-fertilization (referred to here as 5 dpf) with 0.02%–0.04% tricaine (tricaine powder is dissolved in double-distilled water and adjusted to pH = 7 with 1 M Tris-HCL solution) in 30% Danieau's's medium ~10 min before injection. Transfer the anesthetized larvae to a fresh 10 cm Petri dish and remove any excess 30% Danieau's's medium.
2. Place a drop of 0.8% low melting agarose onto the zebrafish larvae to cover. Gently adjust the larvae to a lateral position. Place the Petri dish on ice for 30–60 s to allow the low melting agarose to solidify. Add 30% Danieau's medium containing 0.02%–0.04% tricaine to cover the agarose.
3. Prepare the injection solution. Add 1 µL of 0.5% phenol red in PBS solution into 9 µL of the dye-stained C. difficile inoculum to visualize the injection process.
4. Load a calibrated microinjection needle with the injection solution using a microloader. Mount the loaded needle onto a micromanipulator and position it under a stereomicroscope.
5. Adjust the injection pressure between 600–900 hPa. Set the injection time to 0.1–0.3 s to obtain 0.5–1.0 nL. Set the needle in the micromanipulator at a ~45° angle pointing toward the embedded larvae.
6. Place the needle tip above the gastrointestinal tract close to the urogenital pore. Pierce through agarose then the muscle with the needle tip, then insert it into the intestinal lumen and inject 0.5–1.0 nL of C. difficile. Use a fluorescence microscope to monitor the injected larvae and pick up the properly injected larvae for confocal imaging.

4. Generation of Gnotobiotic Zebrafish Larvae

1. Use the well-established natural breeding method to generate gnotobiotic zebrafish embryos, including: in vitro fertilization, washing with antibiotic-containing medium (1 µg/mL amphotericin B, 10 µg/mL kanamycin, and 20 µg/mL ampicillin), washing with 0.1% wt/vol polyvinyl pyrrolidone-iodine (PVP-I) solution, and incubation of the embryos in a cell culture hood¹².
2. Maintain all gnotobiotic zebrafish larvae under gnotobiotic conditions until 5 dpf or just before the gavage. After the gavage, zebrafish larvae will be transferred into a standard incubator but with sterile 30% Danieau's medium.

5. Gavage of Zebrafish Larvae

1. Calibrate the microgavage needle as described in step 1.2.
2. Measure the diameter of the tip of the needle by placing the needle on a calibration slide with one drop of mineral oil. Ensure that the tip is 30–40 µm in diameter, blunt, and smooth. Discard the sharp or rough needles.
   NOTE: Sharp edges of needles can be blunted by quick flaming.
3. Prepare the gavage solution as described in step 3.3.
4. Load and mount the needle onto a micromanipulator as described in step 3.4. Adjust the micromanipulator to position the needle at a 45° angle.
5. Anesthetize the zebrafish larvae referred to in step 3.1. When the larvae stop moving, transfer them to the groove of a microgavage mold using a Pasteur pipette.
6. Place a drop of 0.8% low melting agarose onto the zebrafish larvae to cover. Gently adjust the larvae with heads facing upright at 45° angles in the groove and tails against the wall of the groove. Ensure that the angles of the heads are approximately the same so that they are aligned with the angle of the gavage needles. Place the microgavage mold on ice for 30–60 s, allowing the low melting agarose to solidify in order to stabilize the positions of the larvae.
7. Adjust the injection pressure between 200–300 hPa. Set the injection time to 0.1–0.3 s to obtain an injection volume of 3–5 nL of C. difficile.
8. Gently operate the needle through the agarose then into the mouth of zebrafish larvae, through the esophagus. Once the tip of the needle is inside the anterior intestinal bulb, press the injection pedal to release the bacteria. Fill the lumen of the intestine with the delivered volume. Do not let it overflow from the esophagus or cloaca. Gently withdraw the needle from the mouth of the zebrafish.
9. Following gavage, rescue the infected zebrafish larvae from the agarose with a flexible microloader tip by first cutting the agarose away then by lifting the larvae. Transfer these larvae into sterile 30% Danieau's medium. Rinse the larvae in sterile medium twice. Transfer the larvae to a fresh 10 cm Petri dish. The larvae will be maintained for up to 11 dpf.

6. Confocal Microscopy Analysis of Injected Zebrafish Larvae

1. Anesthetize zebrafish larvae referred to step 3.1. Make a hole in the bottom of a 35 mm Petri dish with a glass slide attached to the hole, referred as the imaging chamber. Transfer embryos to the bottom of the imaging chamber with an adequate amount of 30% Danieau's medium.
2. Add 200–300 μL of 1% low melting agarose to cover the anesthetized larvae. Since an inverted confocal microscope is used, place the infected region of the larvae against the glass slide as closely as possible.
3. Let the agarose solidify on ice for 30–60 s. Submerge the agarose with 30% Danieau's containing 0.02%–0.04% tricaine.
4. Image the larvae with a confocal laser scanning microscope (Table of Materials).

7. Dissection of Larval Zebrafish Intestine to Recover Viable C. difficile

1. Isolate gastrointestinal tracts from larvae to analyze bacterial load. Start by euthanizing zebrafish larvae with 0.4 % tricaine.
2. Rinse the zebrafish briefly with sterile 1x PBS and transfer them to a fresh agarose plate.
3. Dissection of zebrafish
   1. Insert a needle into the dorsal trunk of zebrafish larvae close to the head to immobilize the zebrafish. Remove the head behind the gills with a lancet.
   2. Insert the second needle into the middle of the dorsal trunk. Insert the third needle into the abdomen of the zebrafish and pull the intestine out of the body cavity.
      NOTE: Extreme care is needed to isolate the intact intestine. If it is difficult to do so, perform additional pulls to separate the rest of the intestine from the remaining internal organs.
   3. Use a microinjection needle to transfer 10–15 intestines into a 1.5 mL tube containing 200 µL sterile of 1x PBS.
   4. Homogenize the intestines with a pestle to disrupt the tissue and prepare homogenates. Ensure the pestle reaches the bottom of the tube to disrupt all intestines completely.
4. Incubate the homogenates in C. difficile rearing medium containing D-cycloserine and cefoxitin, with or without taurocholate (TCA, a germinant of C. difficile spores) in an anaerobic chamber.
5. Incubate the plate anaerobically for 48 h at 37 °C.
6. Use bacterial culture for 16S rRNA-PCR.
   1. Resuspend a colony in 50 µL of H₂O and boil it at 95 °C for 15 min. Pellet the lysed debris by centrifugation (14,000 rpm for 2 min at RT) and use 2 µL of the supernatant as a template in 25 µL of PCR-reaction using C. difficile-specific primers (Cdiff16Sfw: 5' GTG AGC CAG TAC AGG 3'; Cdiff16Srev: 5' TTA AGG AGA TGT CAT TGG 3').
   2. When bacteria from liquid culture are used, harvest 1 mL of culture and wash once with 1 mL of PBS (14,000 rpm for 2 min at RT). Resuspend the pellet in 100 µL of H₂O_dd and treat as done above. To further characterize bacterial colonies, streak on a BHIS- or chromID-plate (Table of Materials).

   

Results

C. difficile is strictly anaerobic, but the chromophore of fluorescent proteins usually requires oxygen to mature. To overcome this problem, a fluorescent dye was used to stain live C. difficile cells that were actively growing (R20291, a ribotype 027 strain; Figure 1A). Using the Gal4/UAS system, stable transgenic zebrafish lines were generated for live imaging, in which the mpeg1.1 or lyZ promoters drove the expression of EGFP fluorescen...

Discussion

The presented methods modify and extend an existing approach to infect zebrafish larvae by performing both injection and microgavage^10,14. It also demonstrates an approach to study anaerobic pathogens with zebrafish larvae²². In addition, the protocol facilitates the analysis of innate immune cell responses in vivo upon CDI and upon colonization of C. difficile in zebrafish. The method is reproducible and easy to conduct in a rout...

Materials

Name	Company	Catalog Number	Comments
Agarose	Sigma-Aldrich	A2576	Ultra-low gelling agarose
Agarose low-melting (LM)	Pronadisa	8050	It is used in agarose plates
BacLight Red Bacterial Stain	Thermo Fisher Scientific	B35001	Fluorescent dye
Brain-Heart-Infusion Broth	Carl Roth GmbH	X916.1
Brass (wild-type)			deficient in melanin synthesis, used to generate stable transgenic lines
Calcium nitrate (Ca(NO3)2)	Sigma-Aldrich	C1396
Capillary Glass	Harvard Apparatus	30-0019	Injection needles
Clostridioides difficile			R20291,, a ribotype 027 strain, TcdA+/TcdB+/CDT+ production
DMSO	Carl Roth GmbH	A994
FIJI	open-source platform		Image processing
HEPES	Carl Roth GmbH	6763
Horizontal needle puller	Sutter instrument Inc	P-87
L-cysteine	Sigma-Aldrich	168149
Leica Application Suite X (LAS X)	Leica		Image processing
Magnesium sulfate (MgSO4)	Carl Roth GmbH	P026
Micro injector	eppendorf	5253000017
Microinjection molds	Adaptive Science Tools	TU1
Leica SP8 confocal microscope	Leica
Phenol Red	Sigma-Aldrich	P0290
Potassium chloride (KCl)	Carl Roth GmbH	5346
Sodium chloride (NaCl)	Carl Roth GmbH	9265
Taurocholate	Carl Roth GmbH	8149
Tg(lyZ: KalTA4)bz17/Tg(4xUAS-E1b:EGFP)hzm3			stable transgenic line in which in which the lyZ promoters drive the expression of EGFP fluorescent protein in neutrophils
Tg(mpeg1.1: KalTA4)bz16/Tg(4xUAS-E1b:EGFP)hzm3			stable transgenic line in which in which the mpeg1.1 drive the expression of EGFP fluorescent protein in macrophages
Tricaine	Sigma-Aldrich	E10521
Yeast extract	BD Bacto	212750