Name: examination of host phenotypes in gambusia affinis following antibiotic treatment
Uploaded: 2017-02-22T14:00:00
Description: Watch this Scientific Journal Video about Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment at JoVE.com

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 ...

<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>

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 qu...

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.

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	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Rifampicin</td>
			<td style="width:104px;">Calbiochem</td>
			<td style="width:127px;">557303-1GM</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Nitrate</td>
			<td>Sigma Aldrich</td>
			<td>S5506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:184px;">Fluorescein-labeled 70 kDa anionic dextran</td>
			<td style="width:104px;">ThermoFisher Scientific</td>
			<td>D1823</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS tablets</td>
			<td>Calbiochem</td>
			<td>6500-OP</td>
			<td>tablets dissolve in water to make phosphate-buffered saline</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 h...

Watch this Scientific Journal Video about Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment at JoVE.com

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 ...

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.

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Immunology and Infection

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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 ...

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.

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...