This project was partially funded by a FAST (Faculty and Student Team) Award to TPP and JMC from EURECA (Center for Enhancing Undergraduate Research Experiences and Creative Activities) at Sam Houston State University.

All animal experiments were conducted under approval of IACUC protocols, numbered 14-05-05-1018-3-01, 13-04-29-1018-3-01, and 14-04-17-1018-3-01.
1. Animal Collection, Handling, and Ethical Care
<ol>
	<li>Collect Gambusia affinis from the field site (identification guide at http://www.sms.si.edu/irlspec/Gambusia_affinis.htm) using a small dip net and place into 19 L buckets. Use visual inspection to identify species.</li>
	<li>Rest fish for 1 - 2 d&#160;in a bucket with pond water. Afterwards, transfer fish into 76 or 189 L aquarium tanks filled with half pond water/half tap water at 25 &#176;C, aerated with a bubbling airstone. Use a small dip net when handling fish as to minimally disturb their skin microbiome. 
	NOTE: Fish density should be no higher than 20 fish/38 L of aquarium water. Fish are acclimated to the aquarium for at least 5 d before experimentation.</li>
	<li>Feed fish on a daily regimen with 5 mg of flake food per fish. Hold fish with a 12 h light/ 12 h dark cycle.</li>
</ol>
2. Initial Antibiotic Exposure for All Experiments
<ol>
	<li>Drawing all fish from each experiment from the same aquarium, place into two separate groups (treated: exposed to antibiotic &#38; control: unexposed) in 19 L buckets. Previous data collected shows that fish in the same aquarium have homogenized skin microbiomes that have little variation in community composition.12</li>
	<li>During experiments, keep fish in 2 L of artificial pond water (APW; 0.33 g/L CaCl2, 0.33 g/L MgSO4, 0.19 g/L NaHCO3) that is sterilized by autoclaving before usage.</li>
	<li>Prepare the rifampicin antibiotic stock solution by adding 50 mg/mL into dimethyl sulfoxide (DMSO). Rifampicin is a representative broad-spectrum antibiotic that is non-toxic to the fish. In humans and mice, it distributes well in the tissues.</li>
	<li>From the rifampicin stock administer a final concentration of 25 &#181;g/mL in 2 L of APW for antibiotic exposure of the treated fish group for 3 days. Note that after 3 d, culturable skin bacterial numbers return similar to that of pretreated fish skin.</li>
	<li>In parallel, keep a group of untreated fish in APW and give an equivalent amount of DMSO (0.5 mL per L of APW) into the water column to act as a control group.</li>
	<li>As experiments are intended to measure effects of an antibiotic-altered microbiome on fish phenotypes, in order to avoid effects directly from the antibiotic itself, following the three-day antibiotic exposure (or control) period, transfer fish into a bucket with 2 L of fresh APW.
	<ol>
		<li>Use a 10 h rest period so the antibiotic is removed by the bodies of the fish. Base this elimination time on experiments where fish were exposed to 25 &#956;g/mL of antibiotic for three days. Euthanize the fish by snipping the spinal cord followed by pithing, then homogenize the entire body using a tissue grinder.</li>
		<li>Determine antibiotic presence by placing 50 &#956;L of this suspension after 0.2 &#181;m filtering into the center of an agar petri dish. Make a hole for this suspension by pressing an upside down sterile 200 &#956;L pipette tip into the agar, which removes a small plug.</li>
		<li>Use agar plates with 20 mL measured volume of nutrient agar, and spread evenly using a cotton swab with an indicator organism, Bacillus subtilis, which is sensitive to rifampicin. Obtain the B. subtilis from a nutrient agar incubated at 25 &#176;C O/N and using the cotton swab to obtain a colony. Observing a zone of inhibition after overnight incubation at 25 &#176;C indicates presence of antibiotic in the fish tissues.</li>
	</ol>
	</li>
</ol>
3. Microbiome Extraction
<ol>
	<li>Extract a skin microbiome sample from the outer mucosal epidermis by placing the fish into a sterile 15 mL conical tube filled with 2 mL sterile PBST (137 mM NaCl, 10 mM phosphate, 0.1% Tween-20, pH 7.4) solution and vortexing at a setting of 10 for 1 min with 10 s pausing increments. Detergent and salt in the buffer promotes even dispersion of bacteria in solution. Comparable results were found with 0.85% saline but lowered bacterial counts with pure water.</li>
	<li>Transfer the fish from the conical tube to a recovery bucket. Lethality of skin extraction is typically lower than 10%. Either, analyze the bacterial suspension in the tube immediately upon extraction or pellet the bacteria by a 2 min spin in a centrifuge at room temperature at 15,000 x g and store at -80 &#176;C for future analysis. 
	NOTE: All culture and biochemical analyses are immediate; DNA or protein extraction can be from frozen samples.</li>
	<li>To obtain a gut microbiome sample, first place the fish in a sterile petri dish. Euthanize the fish by severing the cervical spinal cord directly behind the head with surgical scissors, followed by pithing, and then externally sanitize the body with 70% ethanol wipes.</li>
	<li>After dissection, remove the entire gut by cutting after the esophagus and before the anus.</li>
	<li>Cut the gut into small sections 1 - 2 mm in length and place all sections into two mL PBST solution in a conical tube. After vortexing for 1 min, remove supernatant into another clean tube (take caution to not draw up the gut sections). 
	NOTE: The supernatant contains extracted gut bacteria that can either be used for direct analyses or pelleted by the previous method mentioned for skin samples.</li>
</ol>
4. Infection Model Preparation and Bath of a Specific Pathogen
<ol>
	<li>Obtain an infectious strain of Edwardsiella ictaluri (E. ictaluri) and keep the culture stored at -80 &#176;C. Strain 93 - 146 used in this study, isolated from an infected catfish, was obtained from Mark Lawrence, College of Veterinary Medicine, Mississippi State University.</li>
	<li>Streak E. ictaluri stock onto a selective &#38; differential media called E. ictaluri media (EIM) agar19 to culture the bacteria and incubate for 2 d at 27 &#176;C.</li>
	<li>Transfer a pure isolated colony from the culture and inoculate a 150 mL flask containing 40 mL of nutrient broth (NB; 5 g/L peptone, 4 g/L beef extract). Place flask in a shaker set at 150 rpm for 2 d&#160;at 27 &#176;C.</li>
	<li>After incubation, dilute a sample of the culture in PBST to a spectrometer reading of 0.2 OD at 650 nm to calibrate E. ictaluri culture for desired infectious dose volumes.</li>
	<li>Next transfer both treated (after 3 d&#160;of antibiotic exposure) and untreated fish groups into individual plastic cups containing 130 mL of fresh artificial pond water, or APW for approximately 10 h&#160;for drug clearance from fish tissues.</li>
	<li>Then from both groups, transfer again each fish into 8-ounce plastic cups containing 130 mL of clean APW.</li>
	<li>Give each fish a lethal dose containing 2.8 x 10 6 CFU/mL of E. ictaluri culture into the APW (bath infection model) and keep in a 27 &#176;C incubator for 24 h.</li>
	<li>After the E. ictaluri bath, transfer fish individually into new cups with 130 mL of APW.</li>
	<li>Following water replacement, record fish mortality over the duration of a week. Fish are not fed over this period. In contrast to catfish, Gambusia show no external signs of infection, therefore it is essential to use lethality as an experimental endpoint.</li>
</ol>
5. Polymicrobial Challenge with Feces &#38; Soil
<ol>
	<li>Feces Treatment
	<ol>
		<li>Obtain fresh human feces from a healthy volunteer subject who has not received antibiotics in the previous two weeks, using sterile gloves and a sterile 50 mL conical tube.</li>
		<li>Upon collection, gather fecal matter in a sterile plastic bag and then transfer into a sterile 15 mL conical tube. Create a stock concentration by suspending feces in PBST to give a stock solution of 500 - 800 mg/mL. This thick mixture is best to transfer using a 1 mL or larger plastic pipette tip that has been cut at the bottom with sterile scissors so that the opening is larger.</li>
		<li>After initial antibiotic exposure of treated and control untreated fish groups, separate into individual plastic cups containing fecal suspension at final concentrations of either 15 mg/mL or 10 mg/mL into APW with a total volume of 130 mL. Hold cups in an incubator at 25 &#176;C.</li>
		<li>Record fish mortality during fecal exposure by close observation over 2 d.</li>
	</ol>
	</li>
	<li>Soil Treatment
	<ol>
		<li>Collect 1 - 2 kg of rich organic topsoil (should contain the highest density and diversity of microbes) approximately 7 - 15 cm down in depth and place into a clean plastic container. Note that the soil should be used within two days following collection.</li>
		<li>Directly after initial exposure period, separate both groups of antibiotic treated and untreated fish into individual plastic cups containing 18.2 g of soil in 130 mL of APW. Mix the soil in the water well by hand before adding the fish. Most of the particulates do not suspend and stay at the bottom of the cup.</li>
		<li>Expose fish for a duration of 3 d at 25 &#176;C and record any mortality.</li>
	</ol>
	</li>
</ol>
6. Osmotic Stress Challenge
<ol>
	<li>After treatment followed by a 10 h rest period as described above, individualize fish into separate cups containing NaCl at 17.5 mg/mL (300 mM) in 150 mL of APW. This is half of the typical salinity of seawater, which is not fully lethal (&#60;50%) to control fish but is strongly stressful, requiring effective osmoregulation.</li>
	<li>Observe for fish mortality over a duration of 36 h.</li>
</ol>
7. Nitrate Toxicity Challenge
<ol>
	<li>Rest fish for a 10 h period after antibiotic exposure, and then separate fish into individual cups containing a sodium nitrate concentration of either 10 mg/mL (118 mM) or 17.5 mg/mL (206 mM) in 130 mL of APW.</li>
	<li>Record for mortality over 4 d&#160;for low dose and 1 d&#160;for high dose.</li>
</ol>
8. Growth Analysis of Individualized or Grouped Fish
<ol>
	<li>Complete 3 d antibiotic exposure or control period as groups in buckets.</li>
	<li>For Individual, fill 8-oz Styrofoam cups with 150 mL APW each, transfer an individual fish into the cup and record total body weight for initial assessment.
	<ol>
		<li>Repeat for all fish in both treated and untreated groups. Use white Styrofoam cups instead of clear plastic cups because fish will not eat when in the clear cups.</li>
	</ol>
	</li>
	<li>Feed fish daily with the standard diet of two pellets per fish. Pellets were used rather than flakes because pellets have low variance in individual mass (3.53 &#177;0.42 mg each, or 8% variation), resulting in fish receiving similar amounts of food. Monitor consumption by visually recording uneaten pellets. Consumption rates were typically above 80%.</li>
	<li>At the end of each week over 4 weeks, determine fish weight. Prepare a new cup with fresh APW, place on a balance, and then record the weight. Then, pour a fish into a dip net from the original cup and transfer into the new cup, which is then weighed again. Fish are not handled to avoid stress.</li>
	<li>For Groups, place 2 L of APW into a 19 L bucket and tare the scale. Keep fish together in both groups when transferring into new APW buckets then record total group weight. Record fish group weight each week over a month time span by transferring to a new bucket.</li>
</ol>
9. Gut Transit Time
<ol>
	<li>Use fluorescein-labeled 70 kDa anionic dextran. Dextran is not significantly digested, and is too large to be absorbed across the gut layer, thus passage will measure transit time through the gut. Labeled dextran was incorporated into fish food.</li>
	<li>Into a sterile 1.7 mL tube, add 360 &#956;L of sterile deionized water and 52 mg of gelatin (13% solution), and place the tube on a 60 &#176;C heat block until the gelatin melts and is fully dissolved.
	<ol>
		<li>Grind goldfish food flakes to a fine powder using a mortar and pestle, and add 40 mg of this powder to the tube, along with 20 &#956;L of fish oil. After mixing, add 1.6 mg of FITC-dextran.</li>
	</ol>
	</li>
	<li>Dispense the hot liquid mixture into 20 &#956;L drops onto Parafilm on the lab bench, and let them quickly cool and solidify. Drops can be stored for up to 4 d&#160;before they become too hard for fish to eat. Store drops in the refrigerator by placing the Parafilm onto half of a petri dish, which is then floated on sterile water inside a sealed plastic container, which keeps the drops humidified.</li>
	<li>Just before feeding, quarter drops into sections using a razor blade. Acclimate the fish to the Styrofoam cups individually for 2 days without feeding (Note: Only unexposed control fish were used). Place four quarters of the food into the cup with the fish, and observe the fish for 30 minutes for how many pieces of the food they each eat.</li>
	<li>To begin the monitoring period, transfer fish individually into cups with 80 mL of APW using a dip net. Every 2 h, gently swirl the APW by hand, and then take a 1 mL sample and store in a labeled 1.7 mL tube at 4 &#176;C. Take samples for 36 h.</li>
	<li>Record fluorescence, with a fluorescence spectrophotometer, of all samples at the same time after completion of the assay. Place the entire 1 mL sample into a cuvette, and record the fluorescence measured using excitation at 495 nm and emission at 520 nm.</li>
</ol>

<ol>
	<li>Panda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-Term+Effect+of+Antibiotics+on+Human+Gut+Microbiota.">Short-Term Effect of Antibiotics on Human Gut Microbiota.</a> PloS ONE. 9 (4), 95476 (2014).</li><li>Perez-Cobas, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbiota+disturbance+during+antibiotic+therapy:+a+multi-omic+approach.">Gut microbiota disturbance during antibiotic therapy: a multi-omic approach.</a> Gut. 62 (11), 1591-1601 (2013).</li><li>Theriot, C. M., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+and+metabolic+interactions+between+the+gastrointestinal+tract+and+Clostridium+difficile+infection.">Microbial and metabolic interactions between the gastrointestinal tract and Clostridium difficile infection.</a> Gut Microbes. 5 (1), 86-95 (2014).</li><li>Buffie, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+microbiome+reconstitution+restores+bile+acid+mediated+resistance+to+Clostridium+difficile.">Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile.</a> Nature. 517, 205-208 (2015).</li><li>Sekirov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic-Induced+Perturbations+of+the+Intestinal+Microbiota+Alter+Host+Susceptibility+to+Enteric+Infection.">Antibiotic-Induced Perturbations of the Intestinal Microbiota Alter Host Susceptibility to Enteric Infection.</a> Infect Immun. 76 (10), 4726-4736 (2008).</li><li>Looft, T., Allen, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+effects+of+antibiotics+on+mammalian+gut+microbiomes.">Collateral effects of antibiotics on mammalian gut microbiomes.</a> Gut Microbes. 3 (5), 463-467 (2012).</li><li>Magnadottir, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+of+fish.">Innate immunity of fish.</a> Fish Shellfish Immunol. 20 (2), 137-151 (2006).</li><li>Gomez, D., Sunyer, J., Salinas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mucosal+immune+system+of+fish:+the+evolution+of+tolerating+commensals+while+fighting+pathogens.">The mucosal immune system of fish: the evolution of tolerating commensals while fighting pathogens.</a> Fish Shellfish Immunol. 35 (6), 1729-1739 (2013).</li><li>Nunes, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Effects+of+Tetracycline+Exposure+in+the+Freshwater+Fish+Gambusia+holbrooki:+Antioxidant+Effects,+Neurotoxicity+and+Histological+Alterations.">Acute Effects of Tetracycline Exposure in the Freshwater Fish Gambusia holbrooki: Antioxidant Effects, Neurotoxicity and Histological Alterations.</a> Arch Environ Contam Toxicol. 68 (2), 331-381 (2014).</li><li>Fryxell, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+ratio+variation+shapes+the+ecological+effects+of+a+globally+introduced+freshwater+fish.">Sex ratio variation shapes the ecological effects of a globally introduced freshwater fish.</a> Proc Biol Sci. , 22 (2015).</li><li>Nunes, B., Miranda, M. T., Correia, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+effects+of+different+types+of+detergents+on+the+cholinesterase+activity+and+histological+markers+of+mosquitofish+(Gambusia+holbrooki)+after+a+sub-lethal+chronic+exposure.">Absence of effects of different types of detergents on the cholinesterase activity and histological markers of mosquitofish (Gambusia holbrooki) after a sub-lethal chronic exposure.</a> Environ Sci Pollu Res Int. , 1-8 (2016).</li><li>Leonard, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Skin+Microbiome+of+Gambusia+affinis+Is+Defined+and+Selective.">The Skin Microbiome of Gambusia affinis Is Defined and Selective.</a> Adv Microbiol. 4, 335-343 (2014).</li><li>Carlson, J. M., Hyde, E. R., Petrosino, J. F., Manage, A. B. W., Primm, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+effects+of+Gambusia+affinis+with+an+antibiotic-disrupted+microbiome.">The host effects of Gambusia affinis with an antibiotic-disrupted microbiome.</a> Comp Biochem Physiol C Toxicol Pharmacol. 178, 163-168 (2015).</li><li>Karsi, A., Gulsoy, N., Corb, E., Dumpala, P. R., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bioluminescence-based+mutant+screening+strategy+for+identification+of+bacterial+virulence+genes.">High-throughput bioluminescence-based mutant screening strategy for identification of bacterial virulence genes.</a> Appl Environ Microbiol. 75 (7), 2166-2175 (2009).</li><li>Hawke, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Edwardsiellosis+caused+by+Edwardsiella+ictaluri+in+Laboratory+Populations+of+Zebrafish+Danio+rerio.">Edwardsiellosis caused by Edwardsiella ictaluri in Laboratory Populations of Zebrafish Danio rerio.</a> J Aquat Anim Health. 25 (3), 171-183 (2013).</li><li>Petrie-Hanson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Zebrafish+Danio+rerio+as+a+Model+for+Enteric+Septicemia+of+Catfish+(ESC).">Evaluation of Zebrafish Danio rerio as a Model for Enteric Septicemia of Catfish (ESC).</a> J Aquat Anim Health. 19 (3), 151-158 (2007).</li><li>Fultz, R. S., Primm, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Laboratory+Module+for+Host-Pathogen+Interactions:+America's+Next+Top+Model.">A Laboratory Module for Host-Pathogen Interactions: America's Next Top Model.</a> J. Microbiol. Biol. Educ. 11, (2010).</li><li>Uliano, E., Cataldi, M., Carella, F., Migliaccio, O., Iaccarino, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+changes+in+salinity+and+temperature+on+routine+metabolism+and+nitrogen+excretion+in+gambusia+(Gambusia+affinis)+and+zebrafish+(Danio+rerio).">Effects of acute changes in salinity and temperature on routine metabolism and nitrogen excretion in gambusia (Gambusia affinis) and zebrafish (Danio rerio).</a> Comp Biochem Physiol A. 157, 283-290 (2010).</li><li>Shotts, E. B., Waltman, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+medium+for+the+selective+isolation+of+Edwardsiella+ictaluri.">A medium for the selective isolation of Edwardsiella ictaluri.</a> J Wildl Dis. 26, 214-218 (1990).</li><li>Under animal toxicity studies, sodium chloride entry. TOXNET - Hazardous Substances Data Bank Available from: <a target="_blank" href="https://toxnet.nlm.nih.gov/">https://toxnet.nlm.nih.gov/</a> (2016)</li><li>Under animal toxicity studies, sodium nitrate entry. TOXNET - Hazardous Substances Data Bank Available from: <a target="_blank" href="https://toxnet.nlm.nih.gov/">https://toxnet.nlm.nih.gov/</a> (2016)</li><li>Under animal toxicity studies, sodium nitrite entry. TOXNET - Hazardous Substances Data Bank Available from: <a target="_blank" href="https://toxnet.nlm.nih.gov/">https://toxnet.nlm.nih.gov/</a> (2016)</li><li>Vilz, T. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Assessment+of+Intestinal+Motility+and+Gut+Wall+Inflammation+in+Rodents:+Analyses+in+a+Standardized+Model+of+Intestinal+Manipulation.">Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation.</a> J Vis Exp. (67), e4086 (2012).</li><li>Katoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Harmonization+of+Laboratory+Animals.">International Harmonization of Laboratory Animals.</a> National Research Council (US) International Committee of the Institute for Laboratory Animal Research. Microbial Status and Genetic Evaluation of Mice and Rats: Proceedings of the 1999 US/Japan Conference. , (2000).</li></ol>

The authors have nothing to disclose.

Some challenges require a rest period in clean APW after antibiotic treatment for the drug to be depleted in fish tissues. If the rest period is skipped then antibiotic presence can confound the results, especially when the assay involves exposure to bacteria. In order to examine effects from an altered microbiome composition without large changes in the total number of microbes on the host, preliminary experiments monitoring microbiome composition (16S profiling or whole genome sequencing) and population density (16S quantitation via qPCR) during antibiotic exposure would be required. While 3 d&#160;is optimal in this system, changing the host and/or antibiotic would require recalibration.
Typical of biomedical research, the mouse model is commonly utilized for microbiome studies. The most common fish model is zebrafish. In order to get a better understanding of the universal properties of the structure and function of mucosal microbiomes and host interactions, new and atypical models are a necessity. Our WT fish model serves as an authentic source for studying host-microbiome interactions by including natural host genetic variability that other models have lost due to generations of lab-raised animal inbreeding24. A major experimental advantage of Gambusia is their hardy nature, tolerating a wide range of conditions. High survival rates have been observed in water that varies in temperature (4 - 38 &#176;C), salinity (0 - 17 mg/mL), dissolved oxygen (110&#160;- 25%), and pH (4 - 8). This allows not only examining many environmental conditions, but also for multi-step experimental procedures that are often too stressful for other fish species.
The procedures presented here are suited for rapid screening to discover host effects from a particular treatment. Follow-up studies are required to determine direct causality and mechanism. Example extension studies for the fish microbiome host effects include: measuring intestinal inflammation, examination of fish health by quantifying fat stores, and measurement of mucus levels on the skin and in the gut. A gnotobiotic system is being developed to study in detail links of specific microbes to particular host phenotypes.

It has been established that antibiotics can disrupt the human microbiome leading to dysbiosis, meaning a microbial community imbalance. The microbiota&#39;s compositional alteration after antibiotic treatments has been shown to lower the community&#39;s diversity, reduce key members, and alter community metabolism, especially in the gut1,2. Antibiotic disturbance of the gut microbiome can reduce colonization resistance to Clostridium difficile3,4 and Salmonella5.
Additionally, the disruption of the microbiota has been linked to the development of many syndromes and diseases in humans (e.g., antibiotic-associated enterocolitis, inflammatory bowel disease, metabolic disorders, etc.). Antibiotics are also widely implemented in agriculture as growth promoters in livestock and poultry production6. The usage of these powerful tools is not without collateral effects, which is evident in the rapid rise of antibiotic resistance, as well as the effects of a disrupted microbiome has with its inhabited host. Many studies have shown that broad-spectrum antibiotic usage has long lasting consequences to the structure and function of the microbiota, yet the side effects from an antibiotic-disrupted microbiome impacting host physiology are only speculations which have yet to be supported.
The interplay between host, microbiota, and antibiotics is far from being understood in a concise manner. Therefore, a simple and more tractable model is advantageous to shedding light on the highly complex mammalian system. Mucosal surfaces in humans, including the gut, harbor the highest density and diversity of microbes, and also the most intimate microbe-host interactions. The mucosal skin microbiome of fish offers several advantages as a model system. The Teleostei (bony fish) is one of the earliest lineages to diverge within the Vertebrata meaning that teleosts have both innate and acquired immune systems that have co-evolved a relationship with commensal bacterial communities7. Fish skin shares many characteristics with type 1 mucosal surfaces of mammals, such as physiological functions, immunity components, and arrangement of mucus-producing cells8. The external location of the fish skin mucosal surface offers a microbiome easy to experimentally manipulate and sample.
The Western mosquitofish, Gambusia affinis (G. affinis), is a model fish that has been used in the past for studying mating and toxicology9,10,11. Given the small size, population abundance in the wild as an invasive species, minimal care cost, and hardy nature, we have developed G. affinis as a mucosal microbiome model. Further, Gambusia share the physiology of giving birth to live young with viviparous mammals, which is uncommon in fish species. We completed the most extensive study at the time of fish skin normal microbiota using 16S profiling with Gambusia12. Further work demonstrated three negative effects on the host following disruption of the skin and gut microbiota using a broad-spectrum antibiotic13.
Five different effects were examined in the fish following antibiotic exposure. The most well established host benefit of the microbiome is competitive exclusion of pathogens. The fish pathogen Edwardsiella ictaluri is known to cause outbreaks of enteric septicemia in commercial catfish farms14. E. ictaluri has also been shown to lethally infect zebrafish15,16 and Gambusia17. A challenge with this pathogen from the water column can serve as a measure of exclusion. As a comparison to susceptibility to an individual pathogen, survival during exposure to a high density of mixed organisms was also carried out. Feces and organic-rich soil were used as commonly-encountered sources of microbial communities.
Another established role the bacterial gut community performs is nutrient processing and energy harvest, thus affecting the overall nutritional uptake for the host. As a gross measurement of nutrition, fish body weight was compared before and after one month of being fed a standard diet. Antibiotic-treated fish as an average lost weight while control fish on average gained weight over the month. The mechanism for this lack of weight gain is unclear. One possible contributing factor is transit time of food in the gut. A GI motility method was adapted from zebrafish (Adam Rich, SUNY Brockport, personal communication) to determine transit time. It has not yet been determined if antibiotic-treated fish have an altered transit time.
A common challenge experienced in the natural environment by all organisms, especially fish, is osmotic stress. Gambusia have been shown to quickly adapt when acutely stressed in high concentrations of salinity18. Surprisingly, fish with an antibiotic-altered microbiome exhibited lowered survival to a high salt stress. The mechanism for this novel phenotype is under investigation. Another common stress on aquatic animals, especially in aquaria, is toxic forms of nitrogen (ammonia, nitrate, and nitrite). Survival against nitrate was not significantly different between antibiotic-treated and control fish. The methods presented in this manuscript can be used with Gambusia or similar fish model organisms, such as zebrafish and medaka, to measure phenotypes in the fish following experimental manipulation.

<table><tbody><tr><td>Rifampicin</td><td>Calbiochem</td><td>557303-1GM</td><td></td></tr><tr><td>Sodium Nitrate</td><td>Sigma Aldrich</td><td>S5506</td><td></td></tr><tr><td>Fluorescein-labeled 70 kDa anionic dextran</td><td>ThermoFisher Scientific</td><td>D1823</td><td></td></tr><tr><td>Phosphate-buffered Saline (PBS) tablets</td><td>Calbiochem</td><td>6500-OP</td><td>tablets dissolve in water to make PBS</td></tr></tbody></table>

examination of host phenotypes in gambusia affinis following antibiotic treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

An overall schematic diagram of the experimental system used to study fish host effects from antibiotic exposure13 is represented in Figure 1A and includes the technique for extracting the skin (Figure 1B) and gut (Figure 1C) microbiomes from the fish. Three days was selected as the antibiotic period of exposure because previous data reveals that while the total skin culturable number drops early in treatment, it has returned to pre-treatment levels after 3 d. Meanwhile, the community composition, as determined by 16S profiling, has been strongly altered. Therefore, the 3-day period should be optimal for analyzing the effects from a changed community of approximately the same density. Note that culture analysis, using colony numbers on agar plates, may leave out a number of species. Efficiency of the skin microbiome dispersion method (Figure 1B) was analyzed by comparing colony number on plates from suspension buffer (typically in range of 104&#160;- 105 CFU/g fish weight) to colony number from several fish subjected to the same suspension procedure a second time (to quantify any remaining bacteria). Counts from this second suspension were lower than 100 CFU/g, suggesting the suspension method is effective (unpublished).
Fish with an antibiotic-altered microbiome appeared to be more susceptible to an E. ictaluri infection than control fish (Figure 2). The difference in mean time to death of 56.1 &#177;15 h for the treated fish and 98.5 &#177;48 h for control fish was not statistically significant (two-tailed Student&#39;s t-test, p = 0.12). This is likely due to the small group size (treated n=6, control n=5). Group sizes of at least a dozen are therefore recommended. An advantage of this infection model is the bath protocol, which does not require needles for infection or tracking of food intake. E. ictaluri naturally invades catfish from the water column. Safety is high because E. ictaluri is temperature sensitive, and thus poorly infectious to humans. Other studies have revealed that the time to death in G. affinis correlates with the initial dose of bacteria. The incubation temperature of 27 &#176;C was selected as optimal for both bacteria and fish.
Treated or control fish did not have significant differences in survival in water contaminated with high counts of mixed microbes. No mortality was observed over 4 days for control (n=8) or treated (n=9) fish when soil was used as the microbial source. While some mortality did occur with human feces as the source of microbes (Table 1), antibiotic treatment made no difference. When the concentration of feces in APW was at 20 mg/mL, 40 - 50% of the fish died. However, the dissolved oxygen concentration was only 10% (compared to &#62;80% in buckets or aquaria), thus the hypoxic conditions confounded the interpretation. When lower levels of 16 or 10 mg/mL were used, the survival rates were matched (two-sided Fisher&#39;s exact test, p=0.95 or greater for a difference between groups). Dissolved oxygen levels in these two trials were 65% or above. Gambusia are a very hardy invasive species that can live in low-quality water. It would be interesting to use these assays of natural microbial exposure to measure survival of other species against a challenge of contaminated water. Samples of water from specific environmental sites, especially those subjected to eutrophication, could be used in a similar assay, although water quality (dissolved O2, nitrate, salinity, etc.) would need to be determined, as a potentially confounding factor.
Fish exposed to rifampicin were more susceptible to osmotic stress than control fish (Figure 3). The log-rank test observed a significant difference (p = 0.049) in survival rate (43% death in control group and 88% death in treated group, n = 9 for both groups) when challenged with elevated salinity. Results from this assay agreed with a determination of 18.1 mg/mL as the LC50 for NaCl with G. affinis at 24 h in freshwater20. Signs of saline stress that fish exhibit include reddening around the gills and decreased swimming movement. With this assay, salinity levels above 18 mg/mL resulted in rapid fish death.
When exposed to the toxin nitrate in the water column, pre-exposure to antibiotic did not affect survival (Table 2). For each trial, death was recorded at both a short and long time point, representing acute and more chronic effects. The concentration of 10 mg/mL was selected based on it being the LD50 for G. affinis in freshwater at 48 h21. Results from this assay were consistent with this LD50 value, with a 50% lethality in both treated and control groups after 90 h. A higher challenge of nitrate (17.5 mg/mL) was also examined, leading to more rapid death. Again there was not difference in treatment groups. Nitrite is dramatically more toxic than nitrate to G. affinis, with an LD50 of 0.0015 mg/mL at 48 h22. Community biochemical analysis using the analytical profile index system (unpublished) shows that the fish skin microbiota has the potential to reduce nitrate. However, nitrite levels in the APW during both trials remained below the detection limit (0.001 mg/mL). This challenge method could be used with any small soluble chemicals.
When individualized in cups and fed the same amount to each fish for a month, a trend was observed for the antibiotic-treated fish to not gain weight as well as control fish, without a statistically significant difference (data not shown). To avoid the stress of individualization, fish were housed together as a group for treated and untreated fish in buckets for one month and given matched amounts of food. The limitation of this setup is that fish may not be receiving the same food on an individual basis. However, in the group model, treated fish on average lost weight and control fish on average gained weight (Table 3). The amount of food the fish were consuming did not seem to be affected by prior antibiotic exposure, so appetite suppression is an unlikely candidate explanation for lack of weight gain. Numerous other factors could contribute, including changes in inflammation in the gut, levels of mucus production, gut permeability, and/or gut motility. A major advantage of this assay to measure nutritional effects is simplicity. It is inexpensive, and only requires a laboratory balance as instrumentation. It is suitable to screen for an effect, leading to other involved experimentation to determine mechanism.
One example factor to examine related to fish weight gain is food transit time, which is related to gut motility. FITC-labeled dextran can be incorporated into gelatinized food and is nonlethal to the fish. Measuring fluorescence in the surrounding water over time gives a measurement of how fast the FITC-dextran is passing through the gut. Fluorescence above background can be detected as soon as 2 hours, with a maximum reached after 16 hours post-feeding (Figure 4). This result is with control fish from an aquarium. Reliable results from antibiotic-treated fish have not yet been obtained. One limitation of this procedure is that a 2-d&#160;starvation period is required for fish to eat the food. An advantage of the protocol is high sensitivity (low background fluorescence), as fish eating only one food section can give results, although data is more consistent when two sections are eaten. This protocol is less complicated than a similar and lethal method for mice23.
<img alt="Figure 1" src="/files/ftp_upload/55170/55170fig1.jpg" /> 
Figure 1: Schematic Overview of the Common Experimental Protocol. A is a flow chart illustrating, left to right, fish transferred from aquarium tank, separated into antibiotic-treated (represented by red water) or control (blue) groups, and then placed into individual cups to track phenotypes. B and C depict the process of extracting the fish skin and gut microbiomes. After vortexing, bacteria are suspended into the solution for analysis of the microbial community. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55170/55170fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55170/55170fig2.jpg" /> 
Figure 2: Pathogen Susceptibility. A survival curve during exposure to E. ictaluri for fish pre-treated with rifampicin (red line) or an untreated control group (black line) fish. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55170/55170fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55170/55170fig3.jpg" /> 
Figure 3: Susceptibility to Osmotic Stress. A survival curve during exposure to high salinity for fish in both antibiotic-treated and untreated groups. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55170/55170fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55170/55170fig4.jpg" /> 
Figure 4: Food Transit Time. Fluorescence over time in the water from two fish fed FITC-dextran. Fish A ate two gelatin food sections and fish B ate one. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55170/55170fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/55170/55170table1.jpg" /> 
Table 1: Susceptibility to High Microbial Environments. Exposure to human fecal matter was assessed at 3 different concentrations. Death ratio of x:y shows the total number of fish dead at the indicated time point x compared to the total number of fish in the experimental group y.
<img alt="Table 2" src="/files/ftp_upload/55170/55170table2.jpg" /> 
Table 2: Nitrate Toxicity Challenge. Recorded time of fish mortality in treated and control groups throughout exposure to a toxic nitrate concentration. Death ratio of x:y shows the total number of fish dead at the indicated time point x compared to the total number of fish in the experimental group y.
<img alt="Table 3" src="/files/ftp_upload/55170/55170table3.jpg" /> 
Table 3: Growth Analysis. Changes in total body weight after one month following antibiotic or control treatment. The percent difference in initial mean body weight (for the fish in that trial group) compared to final mean body weight is the column &#916;weight. N is the number of fish in that group. The mean weight per fish at the end of the trial is underweight/fish.

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Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

examination-host-phenotypes-gambusia-affinis-following-antibiotic

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.6K Views.  Sam Houston State University.  Deze studie omvat werkwijzen effecten op een modelvis gastheer na verandering van de huid en darmen microbiome gemeenschappen compositie van een antibioticum onthullen. 

Video: Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

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Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

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Optimizing GF Zebrafish Models: Generation, Maintenance, and Microbial Studies

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

Author Spotlight: Establishing Germ-Free Zebrafish for Microbiota-Host Relationship Studies

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

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Developmental Biology

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.

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Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms.

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Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

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Single&#45;Copy Gene Expression for Characterizing Multidrug Efflux Systems

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

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Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

Biology

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

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