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Updated: Dec 11, 2025
A Rapid Image-based Bacterial Virulence Assay Using Amoeba
Published on: June 27, 2018
Increased Sensitivity of Amoeba-Grown Francisella Species to Disinfectants
Maša Knežević1, Valentina Marečić1, Mateja Ožanič1
1Department of Microbiology and Parasitology, University of Rijeka, Faculty of Medicine, 51000 Rijeka, Croatia.
Abstract:
Francisella tularensis is a highly infectious, intracellular bacterium and it is the causative agent of tularemia. The bacterium has been isolated from more than 250 species, including protozoa. Previous studies have shown that the growth of Legionella pneumophila within the amoeba results in a dramatic increase in the resistance to disinfectants. Since Francisella persists in the environment for years, this study investigates whether Acanthamoeba castellanii-grown F. novicida exhibits an alteration in the resistance to disinfectants. The disinfectants used are didecyldimethylammonium chloride (DDAC) combined with isopropyl alcohol (D1), benzalkonium chloride combined with DDAC and formic acid (D2), and polyhexamethylene biguanide (PHMB, D3). The effect of disinfectants on the bacterial viability is determined by a colony-forming unit (CFU), by transmission electron microscopy (TEM), by fluorescence microscopy, and the damage of the bacterial membrane. Our data has shown that only a one-log10 loss in bacterial viability is exhibited upon treatment of agar-grown Francisella, while in amoeba-grown Francisella there was a three-log10 difference with D3. The D1 disinfectant sterilized the bacteria within 10 s. The treatment of agar-grown F. novicida with D2 reduces bacterial viability by seven-log10 within 10 s and 15 min, respectively. Surprisingly, the treatment of amoeba-grown F. novicida with D2 results in a total loss of bacterial viability. In conclusion, A. castellanii-grown F. novicida is more susceptible to many disinfectants.
Insights
Francisella novicida grown within Acanthamoeba castellanii showed increased susceptibility to certain disinfectants, contrary to expectations for environmental persistence. This finding impacts understanding of bacterial resistance and disinfection strategies.
Area of Science:
- Microbiology
- Environmental Science
- Infectious Diseases
Background:
- Francisella tularensis is a highly infectious bacterium causing tularemia.
- Francisella species are found in over 250 species, including protozoa.
- Intracellular bacterial growth, like Legionella pneumophila in amoebae, can increase disinfectant resistance.
Purpose of the Study:
- To investigate if Acanthamoeba castellanii-grown F. novicida exhibits altered resistance to disinfectants.
- To determine the impact of amoebal cultivation on F. novicida's susceptibility to common disinfectants.
Main Methods:
- Cultivation of F. novicida within Acanthamoeba castellanii.
- Treatment of both agar-grown and amoeba-grown F. novicida with disinfectants: D1 (DDAC/IPA), D2 (Benzalkonium chloride/DDAC/formic acid), and D3 (PHMB).
- Assessment of bacterial viability using colony-forming unit (CFU) counts, transmission electron microscopy (TEM), and fluorescence microscopy.
Main Results:
- Amoeba-grown F. novicida showed a three-log10 difference in viability with D3 compared to agar-grown bacteria.
- Disinfectant D1 rapidly sterilized both agar-grown and amoeba-grown F. novicida within 10 seconds.
- Agar-grown F. novicida viability was reduced by seven-log10 with D2, while amoeba-grown F. novicida showed a complete loss of viability.
Conclusions:
- Contrary to hypotheses, Acanthamoeba castellanii-grown F. novicida demonstrated increased susceptibility to disinfectants D2 and D3.
- The intracellular environment within A. castellanii may not confer enhanced disinfectant resistance to F. novicida.
- Findings suggest that F. novicida's interaction with protozoa could alter its susceptibility profiles to disinfectants.
