In vitro fermentation test bed for evaluation of engineered probiotics in polymicrobial communities

Abstract

In vitro fermentation systems offer significant opportunity for deconvoluting complex metabolic dynamics within polymicrobial communities, particularly those associated with the human gut microbiome. In vitro gut models have broad experimental capacity allowing rapid evaluation of multiple parameters, generating knowledge to inform design of subsequent in vivo studies. Here, our method describes an in vitro fermentation test bed to provide a physiologically-relevant assessment of engineered probiotics circuit design functions. Typically, engineered probiotics are evaluated under pristine, monoor co-culture conditions and transitioned directly into animal or human studies, commonly resulting in a loss of desired function when introduced to complex gut communities. Our method encompasses a systematic workflow entailing fermentation, molecular and functional characterization, and statistical analyses to validate an engineered probiotic’s persistence, plasmid stability and reporter response. To demonstrate the workflow, simplified polymicrobial communities of human gut microbial commensals were utilized to investigate the probiotic Escherichia coli Nissle 1917 engineered to produce a fluorescent reporter protein. Commensals were assembled with increasing complexity to produce a mock community based on nutrient utilization. The method assesses engineered probiotic persistence in a competitive growth environment, reporter production and function, effect of engineering on organism growth and influence on commensal composition. The in vitro test bed represents a new element within the Design-Build-Test-Learn paradigm, providing physiologically-relevant feedback for circuit re-design and experimental validation for transition of engineered probiotics to higher fidelity animal or human studies.

In Vitro Fermentation Evaluation of Engineered Sense and Respond Probiotics in Polymicrobial Communities

“…In Vitro Fermentation. Polymicrobial community fermentation was performed as described in Arcidiacono et al 40 Briefly, organisms were grown to mid log in various growth media (Table S1), with CFU/mL determined, and processed in reduced phosphate buffered glycerol (0.1 M phosphate buffer pH 7.2 with 15% w/v glycerol and 0.08% L cysteine) for storage at −80 °C. The engineered organism (5 mL) was combined in equal volumes under anaerobic conditions with commensal organisms and/or phosphate buffer with 15% glycerol to create 40 mL inocula for fermentation vessels.…”

“…Fluorescent reporter molecules were extracted as described in Arcidacono et al for Lp_GFP and using ethyl acetate extraction as described in Sangwan et al for EcN_TDC. 40,47 Fluorescence was measured using an HJY Fluorolog 3 fluorimeter (Horiba Scientific, NJ, USA). Aliquots (700 uL) were placed in a quartz cuvette (1 mL) and analyzed in right angle mode for liquid-phase samples.…”

“…They are generally less expensive to operate, allow precise control and manipulation of environmental parameters (e.g., temperature, pH, agitation rate, and growth medium), and are useful for mechanistic studies − Additionally, the technique has been employed for understanding functional metabolic changes within the human gut microbiome during acute stress states and exposure of regionally specific microbiota composition and microbial activity in luminal and mucosal domains. , At relevant use of these systems is as an in vitro -based test bed for engineered organisms that employs a systematic approach to increasing metabolic competition using simplified, defined communities to assess engineered probiotic persistence, functional output, and impact on commensal organisms …”

“…38,39 At relevant use of these systems is as an in vitro-based test bed for engineered organisms that employs a systematic approach to increasing metabolic competition using simplified, defined communities to assess engineered probiotic persistence, functional output, and impact on commensal organisms. 40 Defined synthetic communities offer less complexity than fecal inoculum but have been used to mimic complex environments to conduct studies not feasible using in vivo methods. Furthermore, evidence is emerging that simplified microbial communities can facilitate understanding that translates to complex microbial community dynamics realized within the human gut microbiome.…”

Current Perspectives on Gastrointestinal Models to Assess Probiotic-Pathogen Interactions

“…A more recent batch fermentation method simulating the distal colon could potentially be used for studying probiotic pathogen interactions, however so far only E. coli Nissle have been tested in this model with simplified communities of gut bacteria ( Arcidiacono et al, 2021 ).…”