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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I7 - Exploring the Effects of Environmental Variables on the Growth of opportunistic Bacteroides members.

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Extracellular matrix (ECM) remodeling is increasingly recognized as a significant factor in inflammatory bowel disease (IBD) onset and progression. [1] Increased proteolytic activity in IBD patients has been attributed to host cell-derived proteases, but recent evidence suggests bacterial-derived contributions. [2] IBD compromises the integrity of the intestinal epithelial barrier, allowing commensal microbes to breach and interact with the ECM, thus altering the ecological microenvironment. Our previous findings have confirmed that multiple Bacteroides species, key members of the core human gut microbiota, have been identified as degraders of ECM components. [3] Notably, some Bacteroides fragilis (B. fragilis) strains contain a pathogenicity island that encodes for multiple proteases. [4] We hypothesize that disease-like conditions can influence microbial growth and the subsequent direct degradation of individual ECM components. To understand and simulate how intestinal environmental changes during IBD impact B. fragilis, this work will alter pH and nutritional sources in bacterial growth media.

References:
1. Shimshoni, Elee, et al. (2015). Gut.
2. Mills, Robert H., et al. (2022). Nature microbiology.
3. Porras, Ana Maria, et al. (2022). MBio.
4. Casterline, Benjamin W., et al. (2017). Gut microbes.

Materials and

Methods:  The Bacteroides species we are working with is Bacteroides fragilis (B. fragilis). We are using Brain Heart Infusion with Supplement (BHIS) media to grow the different B. fragilis strains (ATCC 25285, 43858, 43859, 43860) in an anaerobic chamber. The pH of this media is adjusted by adding sodium hydroxide (NaOH) for more basic conditions and hydrochloric acid (HCl) for more acidic conditions. Each bacterial strain is pre-grown overnight (starter culture) and then transferred to BHIS pH altered media. The growth is monitored using a plate reader (Cerillo) that measures optical density at 600 nm over a 24-hour period, generating an absorbance-based growth curve. This allows us to track and analyze the growth of B. fragilis strains under varying pH conditions. Once the bacterial cultures have reached an OD of 0.9-1.1, they are centrifuged to isolate the supernatant. Then the supernatants are incubated with a fluorescent elastin substrate. Degradation of the substrate is estimated using the increase in relative fluorescence.

Results, Conclusions, and Discussions: In Figure 1A, all B. fragilis strains grown at a pH of 7.4 reached a peak optical density (OD) of 0.6 to 0.8 within the first 6 hours of incubation, following similar growth patterns. Figure 1B shows B. fragilis 25285 as having varying growth rates and patterns when grown in different pH levels, reaching peak OD values ranging from 0.2 to 1.2 when going from lower to higher pH’s, respectively. Figure 1C displays the pH-dependent degradation of elastin through relative fluorescence of a quenched elastin substrate. The cultures grown on the two most acidic levels (pH of 5.5 and 6.0) displayed higher degradation than their basic counterparts (pH of 6.5, 7.0, and 8.0).

The data collected suggest a correlation between acidic environments and increased elastin degradation. To further investigate this trend, we plan to grow additional B. fragilis strains (ATCC 43858, 43859, and 43860) under the same varying pH conditions. These strains will be exposed to the same fluorescent elastin substrate to determine if the observed degradation trend is consistent across different strains. Similarly, we will assess the degradation behavior of these strains using other ECM substrates, such as collagens and glycosaminoglycans. Once we gain a better understanding of pH as an environmental trigger on B. fragilis, we aim to explore whether members of the species exposed to ECM components forage them as nutritional sources. This work highlights the microbe-ECM interactions that may be playing a role in tissue remodeling.

Acknowledgements (Optional):