Detecting Bacterial Virulence Using Amoeba as a Host System

Abstract

Source: Dubois, V., et al. Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes. J. Vis. Exp. (2018)

This video demonstrates the procedure of co-culturing amoebae with mutant bacterial strains to evaluate their intracellular survival and identify virulence markers through differences in colony counts.

Protocol

1. Co-culture of amoebae-Mycobacterium abscessus Tn mutant: first screen
   NOTE: This takes 3–4 months for the screening of 6,000 mutants.
   1. Spread 5 x 10⁴ amoebae(Acanthamoeba castellanii) /well to a 96-well plate in 100 µL of AC (Acanthamoeba castellanii) buffer. Incubate for 1 h at 32 °C without shaking. Allow the floating amoeba trophozoïtes time to adhere to the wells. While incubating, thaw bacteria on ice for 1 h.
   2. Add 5 µL of the thawed bacteria cultures with an electronic multichannel pipette. Infect for 1....

Results

Figure 1: A. First visual screen of M. abscessus Tn mutants in amoeba. (A) large-scale screen of Tn mutants on amoebae is performed in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	Thermofisher	11874235
96-well plates	Thermofisher	10687551
QuBit fluorometer	Thermofisher	Q33226
Acanthamoeba castellanii	ATCC	30010	strain
Amikacin	Mylan	150927-A	powder
CaCl2	Sigma-Aldrich	C1016	>93% granular anhydrous
Columbia agar	Biomerieux	43041	90 mm
D-Glucose	Sigma-Aldrich	G8270	powder
DMEM	Thermofisher	11500596	medium
DNase and RNase free water	Invitrogen	10977-035	solution
Escherichia coli	Clinical isolate	personal stock	bacteria
Fetal Calf Serum	Gibco	10270	serum
Müller-Hinton agar	Biorad	3563901	powder
SDS 20%	Biorad	1610418	solution