Abstract
Epidemiological data show that the composition of gut microbiota influences host health, disease status, and even behaviour. However, to confirm these epidemiological observations in controlled experiments, pure cultures of gut anaerobes must be obtained. Since the culture of gut anaerobes is not a simple task due to the large number of bacterial species colonising the intestinal tract, in this study we inoculated 174 different culture media with caecal content from adult hens, and compared the microbiota composition in the original caecal samples and in bacterial masses growing in vitro by 16S rRNA sequencing. In total, 42% of gut microbiota members could be grown in vitro and since there were some species which were not cultured but for which the culture conditions are known, it is likely that more than half of chicken gut microbiota can be grown in vitro. However, there were two lineages of Clostridiales and a single lineage of Bacteroidetes which were common in chicken caecal microbiota but resistant to culture. Of the most selective culture conditions, nutrient broths supplemented with mono- or di-saccharides, including those present in fruits, positively selected for Lactobacillaceae. The addition of bile salts selected for Veillonellaceae and YCFA (yeast casitone fatty acid agar) enriched for Desulfovibrionaceae. In addition, Erysipelotrichaceae were positively selected by colistin, trimethoprim, streptomycin and nalidixic acid. Culture conditions tested in this study can be used for the selective enrichment of desired bacterial species but also point towards the specific functions of individual gut microbiota members.
Highlights
Assessing gut microbiota composition is currently relatively simple due to recent developments in DNA sequencing techniques
These studies concluded that between 50 to 90% of total gut microbiota could be cultured in vitro if the appropriate culture conditions are provided [13,14]. These studies did not perform phylogenetic analyses of the OTUs capable or incapable of in vitro growth. It is not known whether the bacterial species resistant to in vitro growth are randomly distributed among known bacterial phyla, classes, orders, families, genera or species, or whether the microbiota members resistant to in vitro growth are clustered in particular lineages, which is exactly what we investigated in this study
Of the most common chicken gut microbiota members belonging to phyla Bacteroidetes, Firmicutes and Proteobacteria, 20 OTUs belonging to Proteobacteria did not grow in vitro, and this phylum comprised the highest proportion of in vitro non-growing OTUs (62.5%)
Summary
Assessing gut microbiota composition is currently relatively simple due to recent developments in DNA sequencing techniques. Various studies have associated the composition of gut microbiota with age, disease or behaviour [1,2,3,4]. The vast majority of published data are based on epidemiological studies and correlation analyses, which may not necessarily prove the causative link between the tested intervention, microbiota composition and host response. The correlation analysis cannot distinguish between the cause and the consequence—is the change in gut microbiota composition the cause of the behavioural change or its consequence?. Studies administering defined bacteria, followed by verification of their presence in the intestinal tract and their association with tested characteristics, are much less frequent and most frequently involve the administration of Lactobacillaceae family members [5,6,7,8,9]. We and others have initiated systematic culturing of gut anaerobes [11,12,13,14] to have them available for whole genome sequencing and subsequent experiments
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