Cell density alters bacterial community structure in culture-enriched 16S rRNA gene microbiota profiling

OBJECTIVE: Microbial community profiling using 16S rRNA gene has provided invaluable insights into diverse microbial communities. Recently a few studies have attempted to use 16S rRNA gene microbiota profiling in combination with the conventional culture methods to explore bacterial communities. In this “culture-enriched microbiota profiling” approach, microbes in a sample are cultured on solid media, and the resulting colonies are combined and subjected to 16S rRNA gene microbiota profiling. Here we investigated the effect of cell densities as determined by varying levels of sample dilution on the culture-enriched microbiota profiles using De Man, Rogosa and Sharpe (MRS) agar medium as a model system. RESULTS: Cecal samples collected from 10 healthy chickens were serially diluted to 10(2) fold (M-LOW), 10(4) fold (M-MEDIUM), and 10(6) fold (M-HIGH), and the dilutions were plated on MRS agar. 16S rRNA gene profiling showed that the relative abundance of certain genera showed gradual increase (Pediococcus and Enterococcus) or decrease (Lactobacillus and Turicibacter) with higher dilutions, though it was significant only for Pediococcus (p < 0.05). The result indicates that the dilution levels of the samples can alter the resulting microbiota profiles via unknown density-dependent mechanisms and thus should be considered for designing experiments using culture-enriched microbiota profiling.