Abstract
BackgroundLow-abundance microorganisms of the gut microbiome are often referred to as a reservoir for antibiotic resistance genes. Unfortunately, these less-abundant bacteria can be overlooked by deep shotgun sequencing. In addition, it is a challenge to associate the presence of resistance genes with their risk of acquisition by pathogens. In this study, we used liquid culture enrichment of stools to assemble the genome of lower-abundance bacteria from fecal samples. We then investigated the gene content recovered from these culture-enriched and culture-independent metagenomes in relation with their taxonomic origin, specifically antibiotic resistance genes. We finally used a pangenome approach to associate resistance genes with the core or accessory genome of Enterobacteriaceae and inferred their propensity to horizontal gene transfer.ResultsUsing culture-enrichment approaches with stools allowed assembly of 187 bacterial species with an assembly size greater than 1 million nucleotides. Of these, 67 were found only in culture-enriched conditions, and 22 only in culture-independent microbiomes. These assembled metagenomes allowed the evaluation of the gene content of specific subcommunities of the gut microbiome. We observed that differentially distributed metabolic enzymes were associated with specific culture conditions and, for the most part, with specific taxa. Gene content differences between microbiomes, for example, antibiotic resistance, were for the most part not associated with metabolic enzymes, but with other functions. We used a pangenome approach to determine if the resistance genes found in Enterobacteriaceae, specifically E. cloacae or E. coli, were part of the core genome or of the accessory genome of this species. In our healthy volunteer cohort, we found that E. cloacae contigs harbored resistance genes that were part of the core genome of the species, while E. coli had a large accessory resistome proximal to mobile elements.ConclusionLiquid culture of stools contributed to an improved functional and comparative genomics study of less-abundant gut bacteria, specifically those associated with antibiotic resistance. Defining whether a gene is part of the core genome of a species helped in interpreting the genomes recovered from culture-independent or culture-enriched microbiomes.
Highlights
Low-abundance microorganisms of the gut microbiome are often referred to as a reservoir for antibiotic resistance genes
Culture of fecal samples allows the sequencing of lessabundant bacteria Stool samples were grown for 7 days under four culture conditions using the same base media (MCDA broth) and were sequenced using shotgun metagenomics to generate culture-enriched microbiome (CEM)
This was to be expected as E. coli, not dominant in the gut microbiome, is well adapted for Notable gut microbiome bacteria like Akkermansia or Prevotella were not observed in CEM while they were prevalent in culture-independent microbiome (CIM)
Summary
Low-abundance microorganisms of the gut microbiome are often referred to as a reservoir for antibiotic resistance genes. These less-abundant bacteria can be overlooked by deep shotgun sequencing. Culture on different media in Petri dishes, followed by colony picking, allowed the discovery and sequencing of the genome of hundreds of new species [16, 17]. This culture-based approach can potentially allow the characterization of bacteria that are less abundant in the microbiota but still play a significant role in maintaining health [18]. Others have grown bacteria on solid or in liquid culture media to complement microbiome sequencing and to find culture conditions that allow the obtention of an accurate community representation of the complete microbiota [20, 21]
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