This method can answer the key question about the role of innate immune system in the Clostridium infection, like if and how macrophages recognize or phagocytose this pathogen. The main advantage of this method is that it can be applied to many different anaerobic bacteria, that the infection time can be manipulated, and that oral administration represents the normal human infection much closer. After culturing, transfer one milliliter of the culture into a spectrophotometer cuvette, and measure the OD600.
Transfer the required amount into a fresh 1.5-milliliter tube in order to reach a final OD of one to 1.2 in one milliliter of final volume. Wash one time with one milliliter of 1X PBS, and centrifuge at 5, 000 times g for three minutes at room temperature. Resuspend in one milliliter of 1X PBS.
Add three microliters of working solution of the prepared fluorescent dye into the one-milliliter bacterial suspension. Incubate the sample for 15 minutes at room temperature in the dark. After that, wash the stained Clostridioides difficile once with 1X PBS to remove residual dye, and resuspend in one milliliter of 1X PBS to achieve an OD600 of 1.0.
First, place a drop of 0.8%low-melting agarose onto the zebrafish larvae in a Petri dish to cover. Gently adjust the larvae to a lateral position. Place the Petri dish on ice for 30 to 60 seconds to allow the low-melting agarose to solidify.
Add 30%Danieau's medium containing 0.02%to 0.04%tricaine to cover the agarose. To prepare injection solution, add one microliter of 0.5%phenol red in PBS solution into nine microliters of the dye-stained Clostridioides difficile inoculum. Load a calibrated microinjection needle with the injection solution using a Microloader.
Mount the loaded needle into a micromanipulator, and position it under a stereo microscope. Adjust the injection pressure between 600 and 900 hectopascals. Set the injection time to 0.1 to 0.3 seconds to obtain 0.5 to 1.0 nanoliters.
Set the needle in the micromanipulator at an approximately 45-degree angle, pointing toward the embedded larvae. Place the needle tip above the gastrointestinal tract, close to the urogenital pore. Pierce through the agarose, then the muscle with the needle tip.
Then insert it into the intestinal lumen, and inject 0.5 to 1.0 nanoliters of Clostridioides difficile. Use a fluorescence microscope to monitor the injected larvae, and use a flexible Microloader tip to pick up the properly injected larvae for confocal imaging. In a 1.5%agarose plate with grooves, 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-degree angles in the groove and tails against the wall of the groove. 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 0.5 to one nanoliters of bacteria culture.
Fill the lumen of the intestine. Do not let it overflow from the esophagus or cloaca. Gently withdraw the needle from the mouth of the zebrafish.
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, and rinse twice. After anesthetizing the zebrafish larvae, add 200 to 300 microliters of 1%low-melting agarose to cover the anesthetized larvae.
Place the infected region of the larvae against a glass slide as closely as possible. Let the agarose solidify on ice for 30 to 60 seconds. Submerge the agarose into 30%Danieau's medium containing 0.02%to 0.04%tricaine.
Proceed to image the larvae with a confocal laser scanning microscope. After euthanizing the infected zebrafish larvae, 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.
Insert a second needle into the middle of the dorsal trunk. Insert a third needle into the abdomen of the zebrafish, and pull the intestine out of the body cavity. Use a microinjection needle to transfer 10 to 15 intestines into a 1.5-milliliter tube containing 200 microliters of sterile 1X PBS.
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. Into the homogenates, add Clostridioides difficile rearing medium containing D-cycloserine and cefoxitin, with or without taurocholate, and incubate in an anaerobic chamber.
In this study, both neutrophils and macrophages reach the infection sites. The example of an activated macrophage phagocytizing two bacteria shows the clearing by phagocytosis and digestion of the labeled Clostridioides difficile. The microgavage to deliver fluorescence-labeled Clostridioides difficile into the intestinal lumen of macrophage and neutrophil reporter lines at five days post-fertilization mimics the natural path of Clostridioides difficile infection.
However, neutrophils and macrophages did not show obvious migration to the gastrointestinal tract for up to 12 hours after microgavage. In the meantime, fluorescence of the labeled Clostridioides difficile disappeared after around five hours post-microgavage. Intestinal samples 24 hours post-infection were dissected and showed bacterial growth.
No bacteria grew in the control group. At later time points, incubated samples only grew in the medium containing TCA, suggesting that total Clostridioides difficile in the gut had formed spores. 16S rDNA PCR identified the grown bacterium as Clostridioides difficile, which produce specific PCR amplicons of predictable size around 800 base pairs.
Bacteria cultures grown onto a CHROMID plate appeared as typical black colonies, which further indicated that bacteria from the zebrafish intestines were Clostridioides difficile. Working with anaerobic bacteria requires that the aerobic steps are kept short because that influences the biology of the bacteria of course. Innate immune cell in zebrafish can be manipulated.
For example, they can decipher the mechanism of the innate immune cell responses to the infection. Our method indicated that zebrafish can be a tool to study anaerobic pathogen from human gut. Working with multi-resistant pathogens requires that appropriate safety measures are taken.
