Dissertation

Characteristics of Mycobacterium avium Biofilms and Implications of Biofilm Growth for Pathogenesis

McManus, William R

2024

Overview

Pulmonary infections with Mycobacterium avium subs. Hominissuis occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. M. avium is able to survive and grow in many conditions, including water distribution systems and residential plumbing fixtures, placing the opportunistic pathogen in frequent proximity to humans. Despite the worldwide distribution and increasing incidence of M. avium pulmonary disease, understanding of M. avium biofilms, their development, constituent cell phenotypes, and implications of biofilm growth on subsequent pathogenesis is limited.One source of ambiguity in our understanding of M. avium biofilms is the use of different in vitro models to characterize biofilm development in different studies, leading to uncertainty about whether findings are characteristic of M. avium biofilms in general or whether they arise from conditions specific to a given model. To begin elucidating the similarities and differences between biofilms formed using different methods, we made a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and drug susceptibility of biofilm resident bacteria, using two published methods to generate M. avium biofilms: four week incubation in M63 medium or 24 hour exposure to dithiothreitol (DTT). Differences between the biofilms were observed in ultrastructure appearances visualized by scanning electron microscopy and in the susceptibility of the biofilms to degradation by enzymes targeting ECM components. However, staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Further, bacteria in both biofilms displayed resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at >10-fold lower rates than planktonic cells at almost all concentrations tested.To better understand transcriptional adaptations that occur in M. avium during residence in a biofilm and how those expression patterns change following engulfment by macrophages, we undertook the first study of global differential gene expression in M. avium using RNAseq. This analysis revealed largely divergent transcriptional profiles in M. avium taken from planktonic culture and grown in M63 or DTT biofilms. However, M63 biofilm M. avium and planktonic M. avium used to infect RAW264.7 macrophages for 24 hours had similar transcriptional responses to infection. Comparison of genes that were differentially expressed in both planktonic cells post infection and in M63 biofilm derived cells but were not differentially expressed in M63 cells post infection suggested that gene expression patterns induced by the M63 biofilm environment might prepare M. avium to persist during infection. To examine this possibility further, we carried out aerosol infections of BALB/c mice with M. avium grown either in planktonic culture or in an M63 biofilm. Regardless of the M. avium growth condition, the bacterial loads in the lungs and tracheae of the mice progressively decreased over a 21 day infection period, revealing no apparent difference in survival ability between biofilm and planktonic M. avium under the conditions tested.Taken together, the findings presented in this dissertation indicate that the mechanism of biofilm formation in M. avium, including patterns of differential gene expression, varies under different environmental conditions. However, our analyses of the M63 and DTT biofilm models suggest that some characteristics may be common to M. avium biofilms, including the production and export of cellulose, increased antibiotic resistance, and increased expression of certain genes, such as those involved in stress responses. While growth in a biofilm may cause patterns of expression in some genes that are similar to those observed during infection of macrophages, we did not observe a clear benefit for biofilm derived cells in establishing and persisting in the lungs or tracheae in our mouse aerosol infection model.

• Publisher: ProQuest Dissertations & Theses
• Subject: Bioinformatics / Cellular biology / Microbiology / Pathology
• ISBN: 9798382731353