Mycobacterium avium subspecies paratuberculosis (Mpt) is the cause of Johne's disease, a gastrointestinal disease that mainly affects ruminants. Despite being an obligate intracellular pathogen, Mpt can survive in the environment for months. How Mpt survives in the environment has yet to be determined, but one tactic Mpt may use is the formation of biofilms. Biofilms may provide sufficient protection and allow Mpt endure in the environment until a new host is encountered. The conditions affecting Mpt biofilm formation have not been investigated. Both in the host and in the environment, Mpt experiences physiological stresses that may induce biofilm formation including suboptimal pH, oxidative stress and hypoxic stress. Mpt was subjected to various levels of pH, oxidative stress or hypoxia and cultured for 20 or 30 days. Adherent biofilms were characterized for biomass, viability, cellularity and metabolic activity. Biofilm biomass was measured by staining biofilms with crystal violet, destaining with ethanol and measuring the crystal violet-ethanol solution for optical density. Viability and cellularity were measured using the fluorescent DNA dyes SYTO 9 and propidium iodide to differentially stain cells based on membrane integrity. Metabolic activity was assessed using a bioluminescence reaction to measure ATP. Under standard conditions for culturing Mpt, biofilms were the most robust. Alkaline conditions inhibited biofilm formation. Oxidative stress limited biofilm formation. Hypoxic shock may have forced Mpt into early stationary phase or dormancy. In conclusion, Mpt will form single species biofilms and do so under conditions optimal got planktonic growth in vitro.


Timothy Secott

Committee Member

Dorothy Wrigley

Committee Member

Michael Bently

Date of Degree




Document Type



Master of Science (MS)


Biological Sciences


Science, Engineering and Technology

Creative Commons License

Creative Commons Attribution-NonCommercial 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License

Included in

Microbiology Commons



Rights Statement

In Copyright