Modeling Aeromonas Pathogenesis in C. elegans Using a Survival Assay

Abstract

Source: Chen, Y., et al. Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model. J. Vis. Exp. (2018)

This video demonstrates a survival assay using C. elegans to model Aeromonas pathogenesis, where oral ingestion of the bacteria leads to intestinal infection and mortality, enabling quantitative assessment of bacterial virulence in a live host system.

Protocol

1. C. elegans Survival Assay with Aeromonas

1. On the day of seeding L1 worms on the ENGM (enriched nematode growth medium) 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 representative clinical isolates from National Cheng Kung University Hospital.
2. Measure the OD₆₀₀ (optical density at 600 nm) 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 on each 6 cm NGM (nematode growth medium) plate. Culture the plates at room temperature overnight.
   NOTE: To prepare NGM, dissolve 3 g NaCl (sodium chloride), 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 (calcium chloride), 1 mL of 1 M MgSO₄ (magnesium sulfate), 1 mL of 5% cholesterol in ethanol, and 25 mL of phosphate buffer. Swirl to mix well.
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 number 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Shaker incubator	YIH DER	LM-570R	Bacteria incubation
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
6 cm Petri plate	ALPHA PLUS	46	Agar plate preparation
Bacterial peptone	Affymetrix/USB	AAJ20048P2	Culture medium preparation
FudR	SIGMA	1271008	Culture medium preparation
Microscope system	Nikon	Eclipase Ti inverted	Microscope imaging