A multidimensional gut microbiome analysis platform for interrogating substrate utilization by pathogenic and probiotic microbes under aerobic and anaerobic environments

A multidimensional gut microbiome analysis platform for interrogating substrate utilization by pathogenic and probiotic microbes under aerobic and anaerobic environments

Even though E. coli is perhaps the most extensively studied gut microbe, more work is still needed to understand how prebiotic substrates modulate its influence on the gut microbiome and host. Novel solutions seeking to reduce the prevalence and impact of pathogenic E. coli go beyond antibiotics and leverage probiotic E. coli strains like E. coli Nissle 1917 (ECN), which is capable of impeding the growth of pathogenic bacteria, including ECO. 

Studying probiotic bacteria and their interactions with prebiotic substrates is pivotal to understanding gut health and engineering a healthy microbiome balance to protect against pathogens. 

While omics techniques like NGS can identify genes related to prebiotic substrate utilization, there are numerous steps separating genes from functional utilization of prebiotic substrates. As such, genomics often fails at predicting prebiotics’ functional impact on the gut microbiome. Other omics techniques that directly monitor these interactions, like metabolomics, are cost-prohibitive, highly biased on sample preparation and the analysis technique used, and require sophisticated laboratory infrastructure, which is unattainable in most research settings. Biolog’s phenomics technology can elucidate prebiotics’ direct influence on the gut microbiome with minimal bias, at high throughput, and relative accessibility. 

Biolog’s line of PreBioM microplates offers a multidimensional metabolic phenotype profiling platform tailored to interrogate the intricate interplay between prebiotics and the microbiome. The three-plate system contains 90 unique prebiotic substrates ranging from simple sugars to complex food extracts (Figure 1). 

Combined with the Odin™ family of instruments, these plates enable multidimensional kinetic characterization under either aerobic or anaerobic conditions by monitoring how strains or even communities of organisms, can consume prebiotic substrates, providing an accurate profile of microbial function. While this study focuses on facultative anaerobes, we offer an Anaerobic MediaMatcher plate to assist in selecting the optimal media for culturing obligate anaerobes. For more details, please visit biolog.com.

This application note seeks to develop a mechanistic understanding of prebiotic substrate utilization by both probiotic and pathogenic E. coli strains, with the potential for developing prebiotic and synbiotic interventions to mitigate the impacts of pathogenic microbes. Herein, we outline how PreBioM plates and Odin can be leveraged to analyze two genetically similar (Figure 2) E.coli strains with opposing health impacts, elucidating the functional dynamics
of prebiotics and their broader impact on the
gut microbiome. 

Methods

Aerobic experiments
The standard Biolog “PreBioM Protocol for Aerobic Organisms” was used to aerobically grow and phenotype ECN and ECO strains. Briefly, cells were grown on Biolog Universal Growth Agar with 5% Sheep’s Blood (BUG+B) at 36 ºC for 24 hours. Tubes containing inoculating fluid were prepared for cell growth and metabolism experiments. The cell growth kinetics tube was prepared by mixing 10 mL of IF-0 (1.2x), 0.04 mL of 20% yeast extract, and 1.96 mL of sterile water. The cell metabolism tube was prepared by mixing 10 mL of IF-0 (1.2x), 0.12 mL Dye Mix A (100x), and 1.88 mL sterile water. 

Cell suspensions for each strain were then made using the prepared Biolog inoculating fluids at a transmittance of 80% T measured via a turbidimeter. 

Four plates of each PreBioM variant were pre-warmed to room temperature, and each well was inoculated with 100 µL of the ECN or ECO cell suspensions. Each sector of the PreBioM plate was used as a replicate (n=3).

Inoculated PreBioM plates were transferred to Odin L for incubation at 36 ºC for 24 hours with reads every 20 minutes at 590 and 740 nm.

Anaerobic Experiments
The standard Biolog “PreBioM Protocol for Facultative Anaerobes” was used to grow and phenotype ECN and ECO strains anaerobically. 

Briefly, cells were grown in an anaerobic chamber on Biolog Universal Growth Agar with 5% Sheep’s Blood (BUG+B) at 36 ºC for 24 hours. Tubes containing inoculating fluid were prepared in the anaerobic chamber for cell growth and metabolism experiments.  The cell growth kinetics tube was prepared by mixing the following deoxygenated components 15 mL of IF-0a (1.2x), 0.04 mL of 20% yeast extract, and 2.96 mL of sterile water. The cell metabolism tube was prepared by mixing the following deoxygenated components: 15 mL of
IF-0a (1.2x), 0.18 mL Dye Mix A (100x), 0.022 mL of 1M potassium ferricyanide, 0.018 mL menadione 0.5 mM, and 2.78 mL sterile water. 

Cell suspensions for each strain were then made using the prepared Biolog inoculating fluids at a transmittance of 80% T measured via a turbidimeter. 

Four deoxygenated plates of each PreBioM variant were pre-warmed to room temperature, and each well was inoculated with 150 µL of the ECN or ECO cell suspensions. Each sector of the PreBioM plate was used as a replicate (n=3).

Inoculated PreBioM plates were sealed in the anaerobic chamber and transferred to Odin L for incubation at 36 ºC for 24 hours with reads every 20 minutes at 590 and 740 nm.

Two assays were performed under each atmospheric condition using the 24 plates from both protocols in a single instrument run: cell metabolism (with dye) and growth kinetics (no dye). This yielded four study factors per strain: growth kinetics aerobic, growth kinetics anaerobic, cell metabolism aerobic, and cell metabolism anaerobic. 

Kinetic metabolic and growth profiles were compared by plotting OD590 data for each plate using the Curation function in Odin software.

Data analysis
An unpaired t-test, adjusting for substrates using maximum optical density (Max OD) and correcting for multiple comparisons with the Holm-Sidak method, was performed alongside principal component analysis (PCA) using GraphPad Prism version 10.3.0 for Windows (GraphPad Software, Boston, Massachusetts, USA; www.graphpad.com). Log2 fold change (Log2FC) values and relative standard deviations (RSD%) were calculated using Excel (Microsoft, Redmond, WA)

Results

Differential substrate utilization between the two strains under the two growth conditions was examined in the context of cell growth and energy metabolism. Despite genetic similarities, fifteen substrates distributed across the PreBioM plate line PB-M1, PB-M2, and PB-M3 (Table 1) presented statistically significant utilization differences.

Principal component analysis using the substrates as loadings for cell growth and metabolism reveals atmosphere-dependent class separation on PC1 and strain-dependent class separation on PC2 (Figure 3). This is supported by the limited overlap in differential substrate utilization under the two atmospheric conditions. 

ECN’s utilization of allose for growth underscores the importance of Odin’s kinetic measurement capabilities for studying the gut microbiome. Despite reaching equivalent endpoint measurements under aerobic and anaerobic conditions, the growth rate under anaerobic conditions lags behind that of aerobic conditions. Selecting the wrong endpoint could result in incorrect observations (Figure 4).
ECN successfully used substrates like allose for growth and energy metabolism under both aerobic and anaerobic culture conditions. Conversely, allose was inaccessible under all conditions to its pathogenic counterpart, ECO. A Biocyc search provided a possible explanation for this difference, with specific genes present in ECN but absent
for ECO:

GO:0008786 – allose 6-phosphate isomerase activity
GO:0008787 – allose kinase activity
GO:0015593 – allose transmembrane transporter activity. 

Figure 4:  A) Allose utilization for kinetic cell growth. B) Raffinose utilization for kinetic cell growth. Pink represents ECN under aerobic conditions, Orange represents ECN under anaerobic conditions, blue represents ECO under aerobic conditions, and cyan represents ECO under anaerobic conditions. C) Allose utilization growth, D) Allose utilization metabolism, E) Inulin utilization growth, F) Inulin utilization metabolism, G) Sucrose utilization growth, H) Sucrose utilization metabolism, I) Raffinose utilization growth, and J) Rafifinose utilization metabolism. In all bar graphs, ECN is represented by purple bars and ECO is represented by magenta bars.  

Conclusion

This study highlights the significant differences in substrate utilization between pathogenic and probiotic E. coli strains. Our multidimensional approach, utilizing PreBioM plates and the Odin platform, provided a detailed mechanistic insight into how prebiotic substrates influence bacterial growth and metabolism. The observed variations in substrate utilization, especially under different atmospheric conditions, underscore the limitations of relying solely on genetic information to predict metabolic functions. The findings emphasize the importance of phenotypic profiling for accurately understanding and manipulating microbial interactions within the gut microbiome. Such insights are instrumental in developing effective prebiotic and synbiotic interventions to enhance gut health and combat pathogenic microbes.

References
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Apr;69(4):2217-22. doi: 10.1128/AEM.69.4.2217-2222.2003. PMID: 12676703; PMCID: PMC154817.