We are grateful for the assistance from the C. elegans core facility in Taiwan and to the Diagnostic Microbiology and Antimicrobial Resistance Laboratory of National Cheng Kung University Hospital for providing the Aeromonas isolates. We also acknowledge the Caenorhabditis Genetics Center (CGC), and the WormBase. We also thank Savana Moore for editing the manuscript.
This study was partially supported by grants from the Ministry of Science and Technology of Taiwan (MOST 105-2628-B-006-017-MY3) and the National Cheng Kung University Hospital (NCKUH-10705001) to P.L. Chen.

1. Preparation of the Culture Medium
NOTE: See Table 1 for solution preparation.
<ol>
	<li>To prepare M9 medium12, dissolve 1.5 g of KH2PO4, 5.66 g of Na2HPO4, and 2.5 g of NaCl in 500 mL of deionized water. Autoclave at 121 &#176;C for 20 min. Wait until cooled to room temperature and then add 0.5 mL of 1 M MgSO4 before first use.</li>
	<li>To prepare nematode growth medium (NGM)12, dissol...

<ol>
	<li>Parker, J. L., Shaw, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aeromonas+spp.+clinical+microbiology+and+disease.">Aeromonas spp. clinical microbiology and disease.</a> Journal of Infection. 62 (2), 109-118 (2011).</li><li>Chao, C. M., Lai, C. C., Tang, H. J., Ko, W. C., Hsueh, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+and+soft-tissue+infections+caused+by+Aeromonas+species.">Skin and soft-tissue infections caused by Aeromonas species.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 32 (4), 543-547 (2013).</li><li>Chao, C. M., Lai, C. C., Tang, H. J., Ko, W. C., Hsueh, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+tract+infections+caused+by+Aeromonas+species.">Biliary tract infections caused by Aeromonas species.</a> European Journal of Clinical Microbiology &amp; Infectious Diseases. 32 (2), 245-251 (2013).</li><li>Chuang, H. 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=Different+clinical+characteristics+among+Aeromonas+hydrophila,+Aeromonas+veronii+biovar+sobria+and+Aeromonas+caviae+monomicrobial+bacteremia.">Different clinical characteristics among Aeromonas hydrophila, Aeromonas veronii biovar sobria and Aeromonas caviae monomicrobial bacteremia.</a> Journal of Korean Medical Science. 26 (11), 1415-1420 (2011).</li><li>Chao, C. M., Lai, C. C., Gau, S. J., Hsueh, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+and+soft+tissue+infection+caused+by+Aeromonas+species+in+cancer+patients.">Skin and soft tissue infection caused by Aeromonas species in cancer patients.</a> Journal of Microbiology, Immunology and Infection. 46 (2), 144-146 (2013).</li><li>Chen, P. 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=A+Disease+Model+of+Muscle+necrosis+caused+by+Aeromonas+dhakensis+infection+in+Caenorhabditis+elegans.">A Disease Model of Muscle necrosis caused by Aeromonas dhakensis infection in Caenorhabditis elegans.</a> Frontiers in Microbiology. 7, 2058 (2016).</li><li>Chen, P. 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=Virulence+diversity+among+bacteremic+Aeromonas+isolates:+ex+vivo,+animal,+and+clinical+evidences.">Virulence diversity among bacteremic Aeromonas isolates: ex vivo, animal, and clinical evidences.</a> PLoS One. 9 (11), 111213 (2014).</li><li>Saraceni, P. R., Romero, A., Figueras, A., Novoa, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+infection+models+in+zebrafish+larvae+(Danio+rerio)+to+study+the+pathogenesis+of+Aeromonas+hydrophila.">Establishment of infection models in zebrafish larvae (Danio rerio) to study the pathogenesis of Aeromonas hydrophila.</a> Frontiers in Microbiology. 7, 1219 (2016).</li><li>Chen, Y. W., Ko, W. C., Chen, C. S., Chen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIOK-1+Is+a+Suppressor+of+the+p38+MAPK+Innate+Immune+Pathway+in+Caenorhabditis+elegans.">RIOK-1 Is a Suppressor of the p38 MAPK Innate Immune Pathway in Caenorhabditis elegans.</a> Frontiers in Immunology. 9, 774 (2018).</li><li>Powell, J. R., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Caenorhabditis+elegans+infection+by+bacterial+and+fungal+pathogens.">Models of Caenorhabditis elegans infection by bacterial and fungal pathogens.</a> Methods in Molecular Biology. 415, 403-427 (2008).</li><li>Chou, T. 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=Enterohaemorrhagic+Escherichia+coli+O157:H7+Shiga-like+toxin+1+is+required+for+full+pathogenicity+and+activation+of+the+p38+mitogen-activated+protein+kinase+pathway+in+Caenorhabditis+elegans.">Enterohaemorrhagic Escherichia coli O157:H7 Shiga-like toxin 1 is required for full pathogenicity and activation of the p38 mitogen-activated protein kinase pathway in Caenorhabditis elegans.</a> Cellular Microbiology. 15 (1), 82-97 (2013).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Engelmann, I., Pujol, N. <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+in+C.+elegans.">Innate immunity in C. elegans.</a> Advances in Experimental Medicine and Biology. 708, 105-121 (2010).</li><li>Feinbaum, R. 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=Genome-wide+identification+of+Pseudomonas+aeruginosa+virulence-related+genes+using+a+Caenorhabditis+elegans+infection+model.">Genome-wide identification of Pseudomonas aeruginosa virulence-related genes using a Caenorhabditis elegans infection model.</a> PLoS Pathogens. 8 (7), 1002813 (2012).</li></ol>

The authors have nothing to disclose.

C. elegans is a bacterivorous nematode that naturally intakes bacteria as food and has developed a complicated innate immunity to bacteria during its evolutionary process. Two of the major organs maintaining and supporting the immunity are the epidermis and intestine9,13. The epidermis and bands of muscle of C. elegans resemble the soft-tissue structures in mammals and humans6. Because of these characteristics, C. ele...

The human pathogen, Aeromonas, has been shown clinically to cause gastroenteritis, wound infections, septicemia, and urinary tract infections1,2. Most associated human diseases have been reported to be associated with four bacterial species: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae 2,3,4,5. Among Aeromonas infectious diseases, soft-tissue infections can cause severe morbidity and mortality in humans. Of note, muscle necrosis is the most severe form of soft tissue infection6. Observation of the survival and muscle necrosis of Caenorhabditis elegans after infection is a convenient method by which to speculate the toxicity of Aeromonas.
Scientists have already developed numerous model organisms to study bacterial infections. In previous studies, mice, zebrafishes, and nematodes were used as animal models to study the pathogenesis and virulence of Aeromonas6,7,8. Every animal model has its&#39; advantages and applications. The model organism, Caenorhabditis elegans, is a bacterivorous nematode which intakes bacteria as foodnaturally.&#160;C. eleganshas developed a complicated innate immune system against bacterial infection over the course of its evolution. Under the stress of bacterial infection,&#160;C. elegans has been proven to be an excellent infection model to study the bacterial pathogenesis of Aeromonas6,7,9 and other pathogens like fungus10 and enterohaemorrhagic Escherichia coli O157:H711. However, there is still no publication that focuses on the methodology in using C. elegans as a model for studying the virulence of Aeromonas.
Here, we introduce three different experiments to study Aeromonas infection using C. elegans as an animal model: assays for survival, liquid toxicity, and muscle necrosis. These methods are a convenient way to evaluate the toxicity among and within Aeromonas species and improve the understanding of the pathogenesis of Aeromonas.

<table border="0" cellpadding="0" cellspacing="0" style="width:660px;" width="661">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaker incubator</td>
			<td style="width:119px;">YIH DER</td>
			<td style="width:119px;">LM-570R</td>
			<td style="width:207px;">Bacteria incubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K2HPO4</td>
			<td style="width:119px;">J.T.Baker</td>
			<td>MP021519455</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KH2PO4</td>
			<td style="width:119px;">J.T.Baker</td>
			<td style="width:119px;">3246-05</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:119px;">J.T.Baker</td>
			<td style="width:119px;">MP021914405</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">31434</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgSO4</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">M7506</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">agar</td>
			<td style="width:119px;">Difco</td>
			<td style="width:119px;">214530</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">C1016</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cholesterol</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">C8503</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ethanol</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">32205</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KOH</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">P5958</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 cm petri plate</td>
			<td style="width:119px;">ALPHA PLUS</td>
			<td style="width:119px;">46</td>
			<td>agar plate preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plate</td>
			<td style="width:119px;">FALCON</td>
			<td style="width:119px;">353072</td>
			<td>liquid assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bacterial peptone</td>
			<td style="width:119px;">Affymetrix/USB</td>
			<td style="width:119px;">AAJ20048P2</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">yeast extract</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">92144</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">citric acid&#8226;H2O</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">C1909</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tri-potassium citrate&#8226;H2O</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">104956</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FudR&#160;</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">1271008</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disodium EDTA</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">E1644</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FeSO4&#8226;7 H2O</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">215422</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MnCl2&#8226;4 H2O</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">221279</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ZnSO4&#8226;7 H2O</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">204986</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CuSO4&#8226;5 H2O</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">C8027</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tryptone</td>
			<td style="width:119px;">SIGMA</td>
			<td style="width:119px;">16922</td>
			<td>Culture medium preparation&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope system</td>
			<td style="width:119px;">Nikon&#160;</td>
			<td style="width:119px;">Eclipase Ti inverted&#160;</td>
			<td>microscope imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scientific CCD Camera</td>
			<td style="width:119px;">QImaging&#160;</td>
			<td style="width:119px;">Retiga-2000R Fast 1394&#160;</td>
			<td>microscope imaging</td>
		</tr>
	</tbody>
</table>

evaluating virulence and pathogenesis of aeromonas infection in a caenorhabditis elegans model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

By following the protocols described above, it is easy to differentiate between the toxicities from the four Aeromonas strains. The survival assay of C. elegans is shown in Figure 1. The survival rates of C. elegans infected with Aeromonas species, shown in order from high to low were: A. caviae, A. veronii, A. hydrophila, and A. dhakensis. Although there is diversity in terms of toxicity among and within ...

Watch this Scientific Journal Video about Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model at JoVE.com

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

This method can help answer key questions in the host-pathogen interaction field such as the pathogenicity and virulence of bacterial infection. The main advantage of this technique is that we can evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection. To begin wash approximately 2, 000 gravid adult worms with sterile deionized water.Repeat the wash three times keeping the worms at the bottom of the tube. After centrifuging the worms remove the supernatant and keep the worms in 3.5 milliliters of deionized water. Add one milliliter of sodium hypochlorite and 0.5 milliliters of potassium hydroxide to the tube.Then shake the tube for six minutes to lyse the worm bodies. After the eggs are released, add 10 milliliters of deionized water to stop the lysis. Then centrifuge the tube to pull down the eggs and remove as much of the supernatant as possible.Wash the eggs with 15 milliliters of M9 medium at least three times. Transfer the eggs to a 3.5-centimeter dish, and incubate them at 20 degrees Celsius for one night. After this remove 10 microliters of L1 worms and M9 medium from the plate.Count the worms and obtain the concentration of L1 worms in the M9 medium. Using a pipette take 0.5 milliliters of E.coli OP50 LB broth and spread it on an ENGM plate. Then incubate the plate at 37 degrees Celsius for 16 to 18 hours.Cool the ENGM to room temperature. Then seed incubated L1 worms on an ENGM plate with E.coli OP50. Incubate the worms at 20 degrees Celsius until they reach the L4 stage.First, select a single colony of each of the four Aeromonas strains and culture them according to the text protocol. Then measure the OD 600 absorbance of the bacterial broth and adjust it until the absorbance is equal to 2.0. Next spot and spread 30 microliters of bacterial broth from each strain into NGM plates.Randomly select and transfer the previously prepared L4 worms to the NGM plates with the Aeromonas strains. Then incubate the plates at 20 degrees Celsius until the assay is complete. Every day transfer all of the living worms to a new NGM plate with bacteria.Count the numbers of living, dead, and sensor worms every day until the last worm is dead. After culturing Aeromonas strains and measuring their absorbance as previously described, centrifuge the bacterial broth at 3, 500 g's for 15 minutes. Adjust the bacterial broth with S Medium.And add 195 microliters of bacterial broth in the S medium to eight wells of a 96-well plate. Wash the worms off of the ENGM plate with them M9 medium. Then adjust the concentration of the worm solution to five worms per microliter.Add five microliters of the worm solution to each of the wells of the 96-well plate. Incubate the plate on a shaker, and count the number of live worms at 24, 48 and 72 hours. After culturing Aeromonas strains and adjusting the absorbance according to the text protocol spot and spread the bacterial broth on NGM plates.After this, transfer 50 L4 worms to each NGM plate. Every 24 hours transfer the worms to fresh NGM plates. Randomly select 10 worms and transfer them to a 2%agarose gel in M9 medium on a slide.Finally position a coverslip on the gel and capture muscle images with a green fluorescent protein filter on a fluorescent microscope equipped with a camera. In this protocol the toxicities of four Aeromonas strains were assessed. The survival rate of C.elegans infected with Aeromonas species was highest in assays using A.caviae, and lowest in assays using A.dhakensis.In a liquid toxicity assay survival of C.elegans decreased significantly when infected with A.dhakensis or A.hydrophilia. In the muscle necrosis assay degrees of muscle damage varied among the Aeromonas species. A.caviae caused the least muscle damage, while A.dhakensis produced the most muscle damage.The methods described in this video offer a convenient way to differentiate virulence diversity among and within Aeromonas species. Additionally, it is also a reliable model by which to study the interaction between a pathogen and host.

evaluating-virulence-pathogenesis-aeromonas-infection-caenorhabditis

Research

JoVE Journal

Immunology and Infection

7.1K Görüntüleme.  National Cheng Kung University. Bu yöntem, bakteriyel enfeksiyonun patojenitesi ve virülansı gibi konak-patojen etkileşim alanındaki anahtar soruların yanıtlatılabiliyor. Bu tekniğin en büyük avantajı, Aeromonas türleri arasındaki toksisiteyi değerlendirebilmek ve Aeromonas enfeksiyonunun patogenezi anlayışımıza katkıda bulunmaktır. Steril deiyonize su ile yaklaşık 2.000 gravid yetişkin solucanlar yıkamabaşlamak için.Solucanları tüpün dibinde tutarak yıkamayı üç kez tekrarlayın. Sol...