Unraveling the Impact of Xenobiotics on Gut Microbiota: A High-Throughput Approach under Anaerobic Conditions

Unraveling the Impact of Xenobiotics on Gut Microbiota: A High-Throughput Approach under Anaerobic Conditions
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The human gut microbiome has been the subject of extensive research for decades, much of it based on sequencing techniques. This is because, until recently, a large proportion of the bacterial species in the gut microbiome were considered 'unculturable', as we lacked the methods and knowledge to grow them in vitro. As microbiome research has sought to move from association to causation, culture-based approaches have become increasingly important and the protocols required to study gut bacteria have improved over the years. This has made it possible to investigate the role of small molecule xenobiotics, such as drugs, in modulating the human gut microbiome [1]. While such compounds have previously been associated with changes in the composition of the microbiome, it has remained unclear whether they have direct antibacterial effects on gut bacteria. This is a critical question for understanding and mitigating potential collateral damage to the gut microbiome and for exploring opportunities for drug repurposing.

About a decade ago, the Zeller, Patil, Bork and Typas labs at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, initiated a comprehensive study of the direct inhibitory effects of marketed drugs on gut bacterial species in monoculture. They started by installing a simple microplate reader in an anaerobic chamber to quantify growth effects of about 1,200 drugs on gut bacteria by optical density (OD) measurements in 96-well plates. It soon became clear that a plate reader holding only one plate at a time would not allow the throughput required to study the effects of drugs on a wider range of different bacteria of the human gut microbiome. To optimize the protocol, Lisa Maier, a postdoc at EMBL at the time, integrated the microplate reader with an automated stacking system that allowed the simultaneous measurement of multiple plates. This development laid the foundation for our current protocol. In 2019, Lisa established her own lab at the University of Tübingen, Germany, where we jointly developed the protocol to its current state (Figure 1). Sarela Garcia-Santamarina, another former postdoc from the Typas lab, further extended the protocol to study the effects of drugs on gut microbial communities, again in high numbers.

The high-throughput screening is conducted in an anaerobic chamber. A variety of compounds can be screened, including drugs, natural compounds, environmental pollutants, endogenous metabolites, etc. Preparation of drug master plates. Compounds of interest are dissolved in a solvent (e.g. DMSO) and arrayed in a 96-well plate at 100x the desired concertation. Preparation of screening plates. Sufficient drug master plates allowing to test all strains in replicates. Strain or community inoculation. This protocol is suitable for bacterial monocultures and synthetic- and stool-derived communities. Compound Screening. The screening plates are inoculated with 50 µl of bacterial culture and growth curves are acquired by hourly measurement of optical density (OD) with the help of a microwell plate reader and a matching stacking device. Growth Analysis and Hit Identification. Resulting growth measurements are then analyzed with R package 'neckaR'. Created with BioRender.com

Figure 1: Overview of the workflow. The high-throughput screening is conducted in an anaerobic chamber. A variety of compounds can be screened, including drugs, natural compounds, environmental pollutants, endogenous metabolites, etc. Preparation of drug master plates. Compounds of interest are dissolved in a solvent (e.g. DMSO) and arrayed in a 96-well plate at 100x the desired concertation. Preparation of screening plates. Sufficient drug master plates allowing to test all strains in replicates. Strain or community inoculation. This protocol is suitable for bacterial monocultures and synthetic- and stool-derived communities. Compound Screening. The screening plates are inoculated with 50 µl of bacterial culture and growth curves are acquired by hourly measurement of optical density (OD) with the help of a microwell plate reader and a matching stacking device. Growth Analysis and Hit Identification. Resulting growth measurements are then analyzed with R package 'neckaR'. Created with BioRender.com

Our automated high-throughput, cultivation-based approach allows the simultaneous evaluation of the direct inhibitory effect of thousands of compounds on hundreds of bacterial isolates under anaerobic conditions. It offers cost-effective screening of approximately 5,000 compounds against a single microbial species in just five days [2]. Moreover, it is adaptable to study microbial communities assembled from pure isolates or complex stool samples [3]. Importantly, our protocol is versatile and extends beyond drugs, allowing the exploration of the influence of diverse small molecules, such as natural compounds and metabolites, or other environmental factors like pH and temperature. To manage and analyze the large datasets generated by our protocol, we (main contributor Jacobo de la Cuesta-Zuluaga) developed the 'neckaR' R package, which is available for download from our GitHub page (https://github.com/Lisa-Maier-Lab/neckaR). This software tool streamlines the large-scale analysis of the inhibitory effects of small molecules on bacterial growth.

One of the unique challenges we faced was working within the confines of an anaerobic chamber, which is more time-consuming and spatially limited compared to conventional laboratory settings. At the heart of our protocol is the automated high-throughput anaerobic screening platform, comprising a microplate stacker coupled to a microplate spectrophotometer inside the anaerobic chamber. This setup allows for swift loading, unloading, restacking, and continuous feeding of microplates to the microplate reader, all within a confined workspace. To ensure a constant incubation temperature of all plates at 37°C during the entire screening process, the stacker-reader combination had to be surrounded by an incubator. Bulky conventional incubators that could accommodate our platform would not fit into the chamber. We therefore collaborated with the local mechanical workshop at EMBL in Heidelberg to develop a modular incubator system. This system, made out of an aluminum profile frame and PVC plates, can be assembled within the chamber around the reader. (Figure 2). Our customized incubation solution underscores the benefits of bringing biologists together with dedicated onsite engineers who are willing to go the extra mile for scientific progress.

A) Illustration of the custom-made heatable incubator (closed) with microplate spectrophotometer. Frontal and lateral view. B) Illustration of the arrangement of the stacker and plate reader in the incubator in frontal view. C) Schematic drawings of the incubator housing (bottom, walls, roof and 2 magnetic doors). D) Schematic drawings of the aluminum profile frame of the incubator. The housing consists of black PVC plates that are screwed to the aluminum profile frame. E) Images of the incubator in the anaerobic chamber with the BioStack 4 and Epoch2.

Figure 2: Custom modular incubator system. A) Illustration of the custom-made heatable incubator (closed) with microplate spectrophotometer. Frontal and lateral view. B) Illustration of the arrangement of the stacker and plate reader in the incubator in frontal view. C) Schematic drawings of the incubator housing (bottom, walls, roof and 2 magnetic doors). D) Schematic drawings of the aluminum profile frame of the incubator. The housing consists of black PVC plates that are screwed to the aluminum profile frame. E) Images of the incubator in the anaerobic chamber with the BioStack 4 and Epoch2.

 Cultivation-based approaches are experiencing a resurgence due to their potential to explore the mechanisms that govern the complex gut microbiome ecosystem. Our automated, high-throughput protocol provides a platform for studying the impact of small molecules on gut microbial species in monoculture and communities under anaerobic conditions. We hope that this protocol will help accelerate the exploration of small molecule interactions with gut microbes, and anticipate that it will be used across a wide range of research questions and disciplines, enabling a deeper understanding of the universe within us.

 

Written by Patrick Müller on behalf of all the authors of the protocol:

High-throughput anaerobic screening for identifying compounds acting against gut bacteria in monocultures or communities

References

[1]           G. Falony et al., “Population-level analysis of gut microbiome variation,” Science, vol. 352, no. 6285, pp. 560–564, Apr. 2016.

[2]           L. Maier et al., “Extensive impact of non-antibiotic drugs on human gut bacteria,” Nature, vol. 555, no. 7698, Art. no. 7698, Mar. 2018, doi: 10.1038/nature25979.

[3]           S. Garcia-Santamarina et al., “Emergence of community behaviors in the gut microbiota upon drug treatment.” bioRxiv, p. 2023.06.13.544832, Jun. 14, 2023. doi: 10.1101/2023.06.13.544832.

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Biological Techniques
Life Sciences > Biological Sciences > Biological Techniques
High-throughput Cell Screening
Life Sciences > Biological Sciences > Biological Techniques > Computational and Systems Biology > High-throughput Cell Screening
Microbiology Techniques
Life Sciences > Biological Sciences > Biological Techniques > Microbiology Techniques
Medical Microbiology
Life Sciences > Health Sciences > Biomedical Research > Medical Microbiology