The overall goal of this experimental system is to examine the negative side effects to a host organism after antibiotic treatment. So this method can help answer key questions in the microbiome field, such as enbac associated side effects on the host's health that is mediated by a disrupted microbiome. So the main advantages of this technique are that this system is straightforward, inexpensive in examining vertebrate hosts'physiological phenotypes, and allows for noninvasive disruption to the mucosal microbiome.
This technique's implications characterize how a disruption in the microbiome of an animal can lead to major side effects. To begin, streak a previously obtained and frozen infectious strain of Edwardsiella ictaluri onto E.Ictaluri selective and differential medium agar. Incubate the culture at 27 degrees Celsius for two days.
Transfer a pure isolated colony from the culture and innoculate a 150 milliliter flask containing 40 milliliters of nutrient broth. Place the flask in a shaking incubator at 27 degrees Celsius and 150 RPM for two days. Following the incubation, to calibrate the E.Ictaluri culture for desired infectious dose volumes, use PBST to dilute a sample of the culture to an OD 650 of 0.2.
After collecting Gambusia affinis, and treating the fish with rifampicin in the water column for three days, transfer both antibiotic treated and untreated control fish groups into individual plastic cups containing 130 milliliters of fresh artificial pond water, or APW, for approximately 10 hours for drug clearance from fish tissues. Then, separate the fish again into individual eight ounce plastic cups containing 130 milliliters of clean APW. Administer a lethal dose of E.Ictaluri culture to each fish and incubate the fish at 27 degrees Celsius for 24 hours.
Following the E.Ictaluri bath, transfer each fish into a new cup with 130 milliliters of APW. Without feeding the fish, record fish mortality over the duration of a week. Gather collected fecal matter into a sterile plastic bag before transferring it into a sterile 15 milliliter conical tube, then use PBST to create a stock concentration of the fecal matter at 500 to 800 milligrams per milliliter, using a one milliliter or larger plastic pipette with the tip cut off, prepare 130 milliliters per cup of 15 milligram per milliliter or 10 milligram per milliliter of fecal matter in APW.
Transfer antibiotic treated or untreated fish into individual cups of the solution, and then incubate the fish at 25 degrees Celsius for two days, closely observing and recording mortality during this time. To treat fish with soil, collect one to kilograms of rich organic topsoil, approximately seven for 15 centimeters deep, and transfer it into a clean plastic container. Prepare individual plastic cups containing 18.2 grams of soil and 130 milliliters of APW, mixing the soil well by hand, then transfer the antibiotic treated or untreated fish into the cups and leave at 25 degrees Celsius for three days while recording any mortality.
After a 10 hour rest period in APW following antibiotic treatment, transfer fish into individual cups containing 17.5 milligram per milliliter of sodium chloride and APW, observe the fish over 36 hours for mortality, to carry out a nitrate toxicity challenge, after a 10 hour rest period as before, separate fish into individual cups containing either 10 or 17.5 milligram per milliliter of sodium nitrate and 130 milliliters of APW. Observe fish over the course of one day for mortality at the high dose of sodium nitrate, and four days for the fish at the low dose. After completing three day antibiotic or control exposure in groups, for individual fish, fill eight ounce styrofoam cups with 150 milliliters of APW, and after weighing the filled cups, transfer the fish into the cups, then record the total body weight for initial assessment.
Feed the fish daily with two pellets of food per fish, monitor the consumption by visually recording uneaten pellets. At the end of each week over the course of four weeks, determine the fish weight by first weighing a cup of fresh APW and record the weight, then pour the fish into a dip net, and transfer it into the new cup, before weighing the cup again. For groups of fish, place 150 milliliters of APW into a cup and tare the scale.
Keep the fish together in both groups when transferring them into new containers of APW, then record the total group weight. Repeat the weighing at the end of each week over the course of a month. To analyze gut transit time, add 360 microliters of sterile deionized water and 52 milligrams of gelatin into a sterile 1.7 milliliter tube, and place it on a 60 degrees Celsius heat block until the gelatin melts and is fully dissolved.
Using a mortal and pestle, grind the goldfish food flakes into a fine powder, and add 40 milligrams of the powder to the tube along with 20 microliters of fish oil. After mixing, add 1.6 milligrams of FitC dextran. Dispense 20 microliter drops of the hot liquid onto parafilm on the lab bench, and let them quickly cool and solidify.
Place the parafilm onto half of a Petri dish and float it on sterile water inside a sealed plastic container. Store the solidified drops in the refrigerator for up to four days before they become too hard for the fish to eat. Just before feeding, use a razor blade to cut drops into quarter sections.
Acclimate the fish to individual styrofoam cups for two days without feeding, place four quarters of the food into the fish cup, and observe the fish for 30 minutes for how many pieces of the food they each eat. To begin monitoring, transfer individual fish into cups with 80 milliliters of APW, and every two hours, take a 1 milliliter sample. Store it in a labeled 1.7 milliliter tube at four degrees Celsius.
After completion of the assay, place an entire one milliliter sample into a cuvette, and use a fluorescence spectrophotometer to record fluorescence at an excitation of 495 nanometers, and an emission of 520 nanometers for all the samples at the same time. as shown here, fish with an antibiotic-altered microbiome were more susceptible to an E.Ictaluri infection than control fish. The difference in mean time to death for the treated versus control fish was not significant.
This is likely due to the small group size. This graph illustrates that fish exposed to rifampicin were more susceptible to osmotic stress than control fish, with this assay, salinity levels above 18 milligrams per milliliter resulted in rapid fish death. As demonstrated in this table, when exposed to the toxin nitrate in the water column, pre-exposure to antibiotic treatment did not affect survival.
At 10 milligrams per milliliter, 50%lethality was observed for both the antibiotic treated and control groups at 90 hours. In this experiment using control fish in an aquarium, either two sections of FitC dextran labeled food, or one section were fed to fish to examine food transit time by measuring fluorescence in the surrounding water over time. Following this procedure, other methods like measuring total body fat stores and quantifying skin mucous production can be performed in order to understand mechanisms, and narrow down confounding factors that might contribute to negative host effects.
