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>

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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 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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		<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 ...

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

Research

JoVE Journal

Immunology and Infection

7.6K Views.  Sam Houston State University. This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities composition by an antibiotic.

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

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 ...

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 ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

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 ...