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Method Article
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.
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.
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' advantages and applications. The model organism, Caenorhabditis elegans, is a bacterivorous nematode which intakes bacteria as foodnaturally. C. eleganshas developed a complicated innate immune system against bacterial infection over the course of its evolution. Under the stress of bacterial infection, 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.
1. Preparation of the Culture Medium
NOTE: See Table 1 for solution preparation.
2. Synchronization of C. elegans6,9,12
3. C. elegans Survival Assay with Aeromonas6,9
4. C. elegans Liquid Toxicity Assay with Aeromonas7
NOTE: To avoid contamination, steps should be performed in a laminar flow hood.
5. C. elegans Muscle Necrosis Assay with Aeromonas6
NOTE: To avoid contamination, these steps should be performed in a laminar flow hood.
6. Statistical Analyses
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 ...
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 authors have nothing to disclose.
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.
Name | Company | Catalog Number | Comments |
Shaker incubator | YIH DER | LM-570R | Bacteria incubation |
K2HPO4 | J.T.Baker | MP021519455 | Culture medium preparation |
KH2PO4 | J.T.Baker | 3246-05 | Culture medium preparation |
Na2HPO4 | J.T.Baker | MP021914405 | Culture medium preparation |
NaCl | SIGMA | 31434 | Culture medium preparation |
MgSO4 | SIGMA | M7506 | Culture medium preparation |
agar | Difco | 214530 | Culture medium preparation |
CaCl2 | SIGMA | C1016 | Culture medium preparation |
cholesterol | SIGMA | C8503 | Culture medium preparation |
ethanol | SIGMA | 32205 | Culture medium preparation |
KOH | SIGMA | P5958 | Culture medium preparation |
6 cm Petri plate | ALPHA PLUS | 46 | agar plate preparation |
96-well plate | FALCON | 353072 | liquid assay |
bacterial peptone | Affymetrix/USB | AAJ20048P2 | Culture medium preparation |
yeast extract | SIGMA | 92144 | Culture medium preparation |
citric acid•H2O | SIGMA | C1909 | Culture medium preparation |
tri-potassium citrate•H2O | SIGMA | 104956 | Culture medium preparation |
FudR | SIGMA | 1271008 | Culture medium preparation |
disodium EDTA | SIGMA | E1644 | Culture medium preparation |
FeSO4•7 H2O | SIGMA | 215422 | Culture medium preparation |
MnCl2•4 H2O | SIGMA | 221279 | Culture medium preparation |
ZnSO4•7 H2O | SIGMA | 204986 | Culture medium preparation |
CuSO4•5 H2O | SIGMA | C8027 | Culture medium preparation |
tryptone | SIGMA | 16922 | Culture medium preparation |
Microscope system | Nikon | Eclipase Ti inverted | microscope imaging |
Scientific CCD Camera | QImaging | Retiga-2000R Fast 1394 | microscope imaging |
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