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.

Protocollo

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 1.5 h. Screen around 6,000 individualized clones from the 96-well plate Tn mutants stock.
      NOTE: The MOI (multiplicity of infection) is unknown at this step of the protocol; however, all the 96-well plates containing Tn mutants were cultivated in exactly the same way. This first screen aims to quickly identify intracellular deficient mutants, without considering the variations in inoculum density.
   3. After 1.5 h of infection, invert the 96-well plate on sterile gauze placed in a sterilized metal tray to remove the AC medium under a fume hood. Refill with 200 µL of AC medium. Repeat this wash twice more.
   4. Add 200 µL of fresh medium supplemented with 100 µg/mL amikacin to eliminate remaining extracellular mycobacteria.
   5. Incubate for 2 h before proceeding with 3 additional washings. After washing, maintain the cultures with 50 µg/mL amikacin in 200 µL of AC buffer.
   6. Add 5 x 10⁷ heat-inactivated Escherichia coli bacteria at the end of the infection and every 24 h to prevent encystment of amoebae.
   7. After 48 h of co-culture, lyse the cells by adding 10 µL of 10% SDS (Sodium dodecyl sulfate) to each well and incubate for 30 min at 32 °C. Proceed with the serial dilution and add on Columbia agar plates containing 5% sheep blood (COS plates) to evaluate the number of intracellular mycobacteria. Seal the plate with the parafilm and incubate at 37 °C.
   8. When colonies appear on the agar plates after 3-4 days, photograph the plate and identify the mutants impaired for intracellular growth by observing the deposits with the lowest number of bacterial colonies.
      NOTE: Do not mind about the shape, height or density of the colony (Figure 1A). 136/6000 mutants were identified.

Risultati

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...

Materiali

Elenco dei materiali utilizzati in questo articolo

Nome	Azienda	Numero di catalogo	Commenti
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