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 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 Aeromonas^6,7,8. 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 Aeromonas^6,7,9 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.

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 a water bath and then add 1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄, 1 mL of 5% cholesterol in ethanol, and 25 mL of phosphate buffer. Swirl to mix well.
3. Pour about 5 mL NGM into each 6 cm Petri dish. Avoid bubble production on the surface of plate. Leave plates at room temperature for 1 night and package to save at 4 °C. Bring back to room temperature before use.
4. To prepare enriched NGM (ENGM), dissolve 3 g NaCl, 5 g bacterial peptone, 1 g yeast extract, and 30 g agar in 1 L of deionized water. Autoclave at 121 °C for 20 min. Wait until cooled to 55 °C in a water bath, and add 1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄, 1 mL of 5% cholesterol in ethanol, and 25 mL of phosphate buffer. Swirl to mix well.
5. Pour about 10 mL ENGM into each 9 cm Petri dish. Avoid bubble production on the surface of plate. Leave plates at room temperature for 1 night and package to save at 4 °C. Bring to room temperature before use.
6. To prepare S medium¹², mix 40 mL S Basal, 0.4 mL 1 M potassium citrate, 0.4 mL trace metals solution, 0.12 mL of 1 M CaCl₂, 0.12 mL of 1 M MgSO₄, 40 µL of 5% cholesterol in ethanol and 1 mL of 8 mM FudR before use.

2. Synchronization of C. elegans^6,9,12

1. Wash about 2,000 worms in the gravid-adult stage with 10 mL of sterile deionized water in a 15 mL tube. Wash out the bacteria 3x, keeping the worms at the bottom of the tube.
2. Centrifuge the sample for 1 min at 500 x g to pull down the worms. Remove the supernatant, and keep the worms in 3.5 mL deionized water.
3. Add 1 mL NaOCl (10–15%) and 0.5 mL 5 M KOH into the tube. Mix evenly by shaking for <6 min to lyse the worm bodies.
4. After the eggs are released from the worms, add 10 mL deionized water to stop the lysis. Centrifuge for 1 min at 1,200 x g to pull down the eggs and remove the supernatant as much as possible. Wash with 15 mL M9 medium at least 3x.
   NOTE: See step 1.1 for the composition of M9 medium.
5. Keep eggs in 1 mL M9 medium. Transport eggs to a 3.5 cm dish for incubation at 20 °C for one night (about 12–18 h).
   NOTE: After the incubation, the eggs will hatch to worm larva which is called as first larva (L1) stage.
6. Pipette out 10 µL of L1 worms in M9 medium. Count the worm number and calculate the concentration of L1 worms in M9 medium.
7. Seed at most 10,000 incubated L1 stage worms with a pipet on a 9 cm ENGM plate spread with Escherichia coli OP50.
   1. In this step, spread 0.5 mL overnight cultured E. coli OP50 with Luria-Bertani (LB) broth on the 9 cm ENGM plate. Incubate the plate at 37 °C for 16–18 h
      NOTE: To avoid contamination, the spreading step should be performed in a laminar flow hood.
   2. Cool to room temperature before seeding L1 worms. Seed at most 10,000 incubated L1 stage worms on the ENGM plate with E. coli OP50.
8. Incubate the worms at 20 °C for 44 h or until they grow to the 4^th larva (L4) stage.
   NOTE: See step 1.3 for the composition of ENGM medium. A worm has a white dot in half-moon shape at the middle of body side at the L4 stage.
9. Use the synchronized L4 stage worms in the following assays. Use wild type N2 worms in the C. elegans survival assay with Aeromonas dhakensis and the C. elegans liquid toxicity assay with Aeromonas dhakensis. Use RW1596 worms (myo-3(st386);stEx30[myo-3p::GFP::myo-3 + rol-6(su1006)]) in the C. elegans muscle necrosis assay with Aeromonas dhakensis.

3. C. elegans Survival Assay with Aeromonas^6,9

1. On the day of seeding L1 worms on the ENGM spreading with E. coli OP50, pick a single colony of each of the four Aeromonas strains or E. coli OP50 and culture with 2 mL LB broth respectively at 37 °C for 16 h.
   NOTE: To avoid contamination, this step should be performed in a laminar flow hood. Here, the following bacterial strains were used: E. coli OP50, the normal food source of C. elegans. A. dhakensis AAK1, the first fully-sequenced pathogenic clinical A. dhakensis isolate. A. hydrophila A2-066, A. veronii A2-007, and A. caviae A2-9307121 are representatively clinical isolates from National Cheng Kung University Hospital.
2. Measure the OD₆₀₀ absorbance of bacterial broth and adjust the bacterial broth to OD₆₀₀ = 2.0 with LB broth.
3. Spot and spread 30 µL of bacterial broth each 6 cm NGM plate. Culture the plates at room temperature overnight.
   NOTE: See step 1.2 for the composition of NGM medium.
4. On the day the L1 worms grow to the L4 stage, randomly pick and transfer 50 worms to an NGM plate with each of the four Aeromonas strains or E. coli OP50.
5. Incubate the NGM plates at 20 °C until the assay is finished.
6. Transfer all the living worms to a newly prepared NGM plate with bacteria and count the numbers of live, dead, and sensor worms every day until the last worm is dead.
   NOTE: Transfer worms to a fresh-prepared NGM plate every day for the maintenance of infection, even if using sterile worms (e.g., glp-4, or with FudR).
7. Plot the survival curves based on the daily data calculation.

4. C. elegans Liquid Toxicity Assay with Aeromonas⁷

NOTE: To avoid contamination, steps should be performed in a laminar flow hood.

1. The day after seeding the L1 worms on the ENGM plate spread with E. coli OP50, pick a single colony of each of the four Aeromonas strains and E. coli OP50, and culture with 5 mL LB broth each at 37 °C for 16 h.
   NOTE: In the protocol, the following bacteria strains were used: E. coli OP50, the normal food source of C. elegans. A. dhakensis AAK1, the first fully-sequenced pathogenic clinical A. dhakensis isolate. A. hydrophila A2-066, A. veronii A2-007, and A. caviae A2-9307121 are representatively clinical isolates from National Cheng Kung University Hospital.
2. Measure the OD₆₀₀ absorbance of the bacteria broth.
3. Centrifuge the bacterial broth at 3,500 x g for 15 min to remove the LB broth. Resuspend the bacteria and adjust the bacterial broth to OD₆₀₀ = 3.0 with S medium.Add 195 µL of bacterial broth in S medium to at least 8 wells of a 96-well plate.
   NOTE: See step 1.6 for the composition of S medium.
4. Wash off the L4 worms on the ENGM plate with E. coli OP5 using M9 medium. Adjust the worm solution to a concentration of 5 worms per µL and add 5 µL of the worm solution to each well of the 96-well plate. Ensure that there are approximately 25 worms per well.
5. Incubate the 96-well plate on a shaker at 200 rpm at 25 °C.
6. Count the number of live worms and dead worms after 24, 48, and 72 h. Calculate the survival rates of each well with the following formula: (Live worms/Total worms) × 100%.
7. Draw a scatter plot graph with the daily data calculation.

5. C. elegans Muscle Necrosis Assay with Aeromonas⁶

NOTE: To avoid contamination, these steps should be performed in a laminar flow hood.

1. On the day the L1 worms are seeded on the ENGM spread with E. coli OP50, pick a single colony of each of the four Aeromonas strains and E. coli OP50 and culture with 2 mL LB broth each at 37 °C for 16 h.
   ​NOTE: Here, the following bacteria strains were used: E. coli OP50, the normal food source of C. elegans. A. dhakensis AAK1, the first fully-sequenced pathogenic clinical A. dhakensis isolate. A. hydrophila A2-066, A. veronii A2-007, and A. caviae A2-9307121 are representatively clinical isolates from National Cheng Kung University Hospital.
2. Measure the OD₆₀₀ absorbance of the bacteria broth and adjust the bacteria broth to OD₆₀₀ = 2.0 with LB broth. Spot and spread 30 µL of bacterial broth on 6 cm NGM plates. Culture the plates at room temperature overnight.
3. On the day the L1 worms grow to the L4 stage, transfer 50 worms to each NGM plate with each of the four Aeromonas strains or E. coli OP50. Incubate the NGM plates at 20 °C.Transfer worms every 24 h to newly prepared NGM plates with bacteria.
4. Take muscle images using a fluorescence microscope.
   1. Randomly pick 10 worms onto a 2% agarose gel in M9 medium on a slide. Paralyze the worms using 2 µL of 1% sodium azide in M9 medium on agarose for less than 5 min before taking images.
   2. Place a cover slip and capture the muscle images using a green fluorescent protein (GFP) filter on a fluorescent microscope with charge-coupled device camera. Capture the muscle images at 24, 48, and 72 h post the L4 stage.
5. Conduct image analysis and figure preparation with an image processing software.
   NOTE: The definitions of the scores and the muscle damage levels are shown in Figure 3, for which the criteria are listed as follows: 3 points for missing muscle fibers; 2 points for ruptured or broken muscle fibers; 1 point for bent muscle fibers; 0 points for healthy muscle fibers.

6. Statistical Analyses

1. Perform all experiments a minimum of three times independently.
2. Use the Kaplan-Meier method to assess the C. elegans survival assays, and use the log-rank test in analyzing survival differences. Set statistical significance at 0.05.
3. Use Student's t-test to analyze the statistical results of the C. elegans liquid toxicity assays for the two different groups. Use one-way ANOVA test in analyzing differences among three or more values for one independent variable. Set statistical significance at 0.05.

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

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