Application of culturomics to explore the cultivable microbiota and enable targeted bacterial isolation from the ceca of broiler chickens

Metagenomic analyses have significantly advanced our understanding of microbial composition in the poultry gut. However, many microbes identified through metagenomic studies remain uncultured, largely due to the lack of understanding of their cultivation conditions, which hinders efforts to explore their functional roles in gut health and metabolism. In this study, we performed culturomics, a culture-dependent approach that combines diverse culture conditions with high-throughput 16S rRNA gene sequencing, to comprehensively assess the cultivability of chicken cecal microbiota and provide guidance for isolating target species of interest. Microbial profiling was performed using both culture-dependent (CD) and culture-independent (CI) approaches. For CI, genomic DNA (gDNA) was directly extracted from six broiler chicken cecal samples and subjected to full-length 16S rRNA gene sequencing. For CD, the same samples were cultured under 28 conditions, yielding 161 colony mixtures for sequencing. Based on diversity profiles of the colony mixtures, 10 conditions were selected for single-colony isolation and analysis. Results showed that CD and CI approaches identified 350 and 502 bacterial species, respectively, with 160 species detected by both methods. The dominant species recovered by the CD approach,including Escherichia coli, Proteus mirabilis, Limosilactobacillus reuteri, Enterococcus faecalis, and Ligilactobacillus salivarius, were detected at much lower abundances in the CI analysis, highlighting the capacity of culturomics to enrich and recover minority taxa that are often poorly detected by CI apparoach. Cultivation profiling showed that MRS selectively enriched Limosilactobacillus and Ligilactobacillus as well as Lactobacillus, whereas CNAB and MSA enriched Enterococcus and Bacillus, respectively. Community diversity and structure were significantly influenced by culture conditions (P < 0.01), with medium as the primary factor and air condition as a secondary factor. Subsequent single-colony analysis from 10 selected culture conditions identified 150 single-species isolates belonging to 14 distinct bacterial species. This study provides foundational insight into the cultivability of chicken cecal microbiota, facilitating future research to isolate specific strains and characterize their roles in poultry health and nutrition.