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Page 2 of 16 Li et al. Microbiome Res Rep 2024;3:26 https://dx.doi.org/10.20517/mrr.2023.57

methods using RapidAIM 2.0 showed the minimal effect of sample processing on live microbiota functional
responses to kestose.

Conclusions: Depth and reproducibility of RapidAIM 2.0 are comparable to previous manual label-free
metaproteomic analyses. In the meantime, the protocol realizes culturing and sample preparation of 320 samples
in six days, opening the door to extensively understanding the effects of xenobiotic and biotic factors on our
internal ecology.

Keywords: Gut microbiome, metaproteomics, high-throughput in vitro assay, biobanking, functional responses

INTRODUCTION
Numerous studies have shown that xenobiotic compounds can functionally affect the gut microbiota. These
include pharmaceutical compounds, not only those developed for combating microbial infections, but also
those non-antimicrobial drugs developed to target the host functions . Biotic factors from external sources,
[1]
such as probiotics, pathogens, phages, etc., also influence the gut microbiome functionality in various ways.
To deconvolute the complexity of microbiome responses to these factors, in vitro approaches in the absence
of the host component have been used. Studies have shown that commensal bacterial species in the gut can
be directly affected by 24% of commonly used host-targeted drugs , and therapeutic drugs can accumulate
[1]
in gut bacteria without altering their abundances . However, studying gut microbes in isolation has its
[2]
limitations, because microbial species function differently in complex community settings in comparison to
pure cultures. Cooperative and antagonistic interactions collectively contribute to microbiome diversity and
[3]
resilience . Several synthetic communities have also been used to evaluate the effect of drugs on
community composition and cross-feeding interactions . Nevertheless, the natural human gut microbiome
[2]
harbors hundreds of microbial species, with a considerable variation in taxonomic functions and
compositions among different individuals . The complexity and variability are much greater than those of
[4-6]
synthetic gut microbial communities. Studies have shown long-term stability of the individual gut
microbiome across the life span , and this stability can be irreversibly perturbed by xenobiotic
[7]
[8,9]
stimulation . Therefore, when it comes to the context of individual gut microbiomes, it is necessary to
adopt an assay that maintains the individuality of the community composition and function in vitro. In
addition, since evaluating xenobiotic compounds or biotic components against different individual
microbiomes requires large matrices of samples, high-throughput-compatible models and assay readouts
are necessary to perform such studies. There have been various models to evaluate microbiome responses.
Early in vitro gut microbiome models were based on large-scale bioreactors that are low-throughput and,
due to the large volume of culturing, very costly owing to the considerable amounts of compounds added.
More recent advances in modeling the gut ecosystem include realizing the culturing of complex human gut
microbiome in anaerobic intestine-on-a-chip models, enabling the observation of host-microbiome
interactions . However, for the purpose of high-throughput compound screening, these models are not
[10]
easily adaptable. The use of deep-well plates has the advantage of easy setup,cost-effectiveness, and time
efficiency when scaling up; additionally, it is compatible with automated downstream analysis.

In terms of assessing the functional response of cultured microbiomes, liquid chromatography - tandem
mass spectrometry (LC-MS/MS) is capable of metaproteomic analysis of microbial communities . Briefly,
[11]
LC-MS/MS separates peptides by mass-to-charge ratio, and fragments separated peptides to generate MS/
MS spectra, which are subsequently matched to peptide sequences through database search approaches.
With its fast-growing measurement depth and capacity, metaproteomics techniques have been used to study
microbiome-associated health and diseases such as inflammatory bowel disease [12,13] , colorectal cancer ,
[14]
diabetes , mental illnesses , and COVID-19 . It has also been used to evaluate in vitro responses of gut
[17]
[15]
[16]