Back to the bench: the trials and tribulations of creating a public culture collection for the pig gut

The creation of public culture collections requires both time, effort and an array of skills!

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Pigs are important domestic animals, as well as models for biomedical research. With the advent of next generation sequencing, multiple studies have looked into the pig microbiome with both 16S rRNA amplicon and metagenomic sequencing. While these studies provide correlations with host phenotypes and associations with dietary changes, mechanistic understanding requires that we know who is present in the gut and what they can do. 

Over the last decade the impact of strain level variation has been highlighted, hence host specific culture collections are needed for accurate analysis in the context of host-microbe interactions. In 2020, just before publication of the PiBAC collection, only 31 bacterial isolates from the pig gut were deposited at the German Collection of Microorganisms and Cell Culture (DSMZ). By making the PiBAC isolates publicly available, novel strains are able to be named and the strains are maintained for future scientific research or applications.

Although all bacteria can be cultured in theory, a major challenge is identifying the nutrients necessary for growth in vitro. Additionally, you must prevent fast growing bacteria from outcompeting slow growing bacteria. Therefore, multiple media recipes must be used, identified from the literature research and from our own imagination. As an example, the unconventional inclusion of fat from pan cooked beef mixed with rumen fluid and porcine faecal water led to the isolation of many of the novel taxa within the collection.  

Figure 1: Working in the anaerobic chamber to isolate strict and facultative anaerobes using a range of agar media.

Beside their initial isolation, maintaining bacteria in culture can pose their own unique challenges. In case of Bullifex porci, an isolate representing a novel genus that grows as unusual spherical cells, several months were required for visible growth to occur, highlighting why it may have been missed by previous isolation efforts. Supplementing the media with rumen fluid (10%) reduced the time needed by 600%, meaning only 2 weeks were required.

Through intensive cultivation, a final collection of 117 strains, representing 110 species across 9 phyla and 40 families was generated. However, when compared to a collection of metagenomic assembled genomes (MAGs) from the pig gut, very few of the isolates matched MAGs, suggesting different communities of bacteria from the pig gut are captured by cultivation-dependant and -independent methods. 

With recent advances in high-throughput isolation methods, the speed at which novel taxa are isolated will increase, facilitating in vitro study and valid naming of these as yet uncultured species. Describing novel taxa for valid naming, as done within the presented work, requires a lot of time and could be automated, something we are currently working towards.

While this collection provides an open resource of pig isolates for the community to use (www.dsmz.de/pibac), they must be studied further to be understood! We provide initial insights by exploring cell morphology, biosynthetic gene clusters encoding potentially new anti-microbial substances, novel CAZymes, and the generation of a minimal consortium for pigs, but the real benefit will be what can be done with these isolates next!

To read the full story the PiBAC paper is available here 

The poster image was created by Patrick Buchta, AVMZ team, UK Aachen.

Thomas

Post-doc, University Hospital Aachen

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