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

Summary

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.

Abstract

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.

Introduction

The human pathogen, Aeromonas, has been shown clinically to cause gastroenteritis, wound infections, septicemia, and urinary tract infections^1,2. 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....

Protocol

1. Preparation of the Culture Medium

NOTE: See Table 1 for solution preparation.

1. 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 °C for 20 min. Wait until cooled to room temperature and then add 0.5 mL of 1 M MgSO₄ before first use.
2. To prepare nematode growth medium (NGM)¹², dissolve 3 g NaCl, 2.5 g bacterial peptone, and 20 g agar in 1 L of deionized water. Autoclave at 121 °C for 20 min. Wait until cooled to 55 °C in....

Results

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 .......

Discussion

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 intestine^9,13. The epidermis and bands of muscle of C. elegans resemble the soft-tissue structures in mammals and humans⁶. Because of these characteristics, C. ele.......

Materials

List of materials used in this article

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