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Comparison of Ultrastructure, Extracellular Matrix, and Drug Susceptibility in M. avium subs. hominissuis Biofilms
William R McManus1, Jeffrey S Schorey1
1Department of Biological Sciences, Galvin Life Science Center, University of Notre Dame, Notre Dame, IN 46556, USA.
Abstract:
Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate similar structures and cell phenotypes. To make a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and the drug susceptibility of biofilm resident bacteria, we used two published methods to generate M. avium biofilms: four-week incubation in M63 medium or 24 h exposure to dithiothreitol (DTT). Scanning electron microscopy revealed differences in the biofilm ultrastructure between the two methods, including variation in the appearance of ECM materials and morphology of resident cells, while light microscopy and staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Measuring the susceptibility of biofilms to degradation by enzymes suggested differences in structurally important ECM molecules, with DTT biofilms having important protein and, to a lesser extent, cellulose components, and M63 biofilms having moderate protein, cellulose, and DNA components. Both biofilms conferred resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at greater than 10-fold lower rates than planktonic cells at almost all concentrations. These comparisons indicate differences in biofilm responses by M. avium under differing conditions, but also suggest common features of biofilm formation, including cellulose production and antimicrobial resistance.
Insights
Different methods for creating Mycobacterium avium biofilms yield distinct structures and compositions. However, both methods result in cellulose production and significant antimicrobial resistance in biofilm-grown bacteria.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Biofilm Formation
Background:
- Mycobacterium avium causes pulmonary infections in susceptible individuals.
- M. avium persists in environmental biofilms, posing a public health challenge.
- Understanding M. avium biofilm variability is crucial for effective treatment.
Purpose of the Study:
- To compare the in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and drug susceptibility of M. avium generated by two distinct methods.
- To investigate how different biofilm formation conditions influence M. avium characteristics.
Main Methods:
- Two established methods were used to generate M. avium biofilms: four-week incubation in M63 medium and 24-hour exposure to dithiothreitol (DTT).
- Scanning electron microscopy (SEM) and light microscopy were employed to analyze biofilm ultrastructure and ECM components.
- Enzymatic degradation assays and antimicrobial susceptibility testing (amikacin, clarithromycin) were performed.
Main Results:
- SEM revealed significant differences in biofilm ultrastructure, ECM appearance, and cell morphology between the M63 and DTT methods.
- Both methods produced biofilms containing cellulose, a key polysaccharide component.
- Enzymatic assays indicated variations in ECM composition, with DTT biofilms rich in protein and cellulose, and M63 biofilms containing protein, cellulose, and DNA.
- Both biofilm types demonstrated substantial resistance to amikacin and clarithromycin, with >10-fold lower killing rates compared to planktonic cells.
Conclusions:
- Different in vitro conditions induce distinct M. avium biofilm phenotypes and ECM compositions.
- Common biofilm features, including cellulose production and significant antimicrobial resistance, are observed across different formation methods.
- These findings highlight the complexity of M. avium biofilms and their implications for therapeutic strategies.
