Capturing the diversity of the human gut microbiota through culture-enriched molecular profiling.

Jennifer T Lau,Michael G Surette,Premysl Bercik,Stephen M Collins,Isiri Herath,Christine H Lee,Fiona J Whelan

doi:10.1186/s13073-016-0327-7

Abstract

BackgroundThe human gut microbiota has been implicated in most aspects of health and disease; however, most of the bacteria in this community are considered unculturable, so studies have relied on molecular-based methods. These methods generally do not permit the isolation of organisms, which is required to fully explore the functional roles of bacteria for definitive association with host phenotypes. Using a combination of culture and 16S rRNA gene sequencing, referred to as culture-enriched molecular profiling, we show that the majority of the bacteria identified by 16S sequencing of the human gut microbiota can be cultured.MethodsFive fresh, anaerobic fecal samples were cultured using 33 media and incubation of plates anaerobically and aerobically resulted in 66 culture conditions for culture-enriched molecular profiling. The cultivable portion of the fecal microbiota was determined by comparing the operational taxonomic units (OTUs) recovered by 16S sequencing of the culture plates to OTUs from culture-independent sequencing of the fecal sample. Targeted isolation of Lachnospiraceae strains using conditions defined by culture-enriched molecular profiling was carried out on two fresh stool samples.ResultsWe show that culture-enriched molecular profiling, utilizing 66 culture conditions combined with 16S rRNA gene sequencing, allowed for the culturing of an average of 95 % of the OTUs present at greater than 0.1 % abundance in fecal samples. Uncultured OTUs were low abundance in stool. Importantly, comparing culture-enrichment to culture-independent sequencing revealed that the majority of OTUs were detected only by culture, highlighting the advantage of culture for studying the diversity of the gut microbiota. Applying culture-enriched molecular profiling to target Lachnospiraceae strains resulted in the recovery of 79 isolates, 12 of which are on the Human Microbiome Project’s “Most Wanted” list.ConclusionsWe show that, through culture-enriched molecular profiling, the majority of the bacteria in the human gut microbiota can be cultured and this method revealed greater bacterial diversity compared to culture-independent sequencing. Additionally, this method could be applied for the targeted recovery of a specific bacterial group. This approach allows for the isolation of bacteria of interest from the gut microbiota, providing new opportunities to explore mechanisms of microbiota–host interactions and the diversity of the human microbiota.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0327-7) contains supplementary material, which is available to authorized users.

Highlights

The human gut microbiota has been implicated in most aspects of health and disease; most of the bacteria in this community are considered unculturable, so studies have relied on molecular-based methods
Using 66 culture conditions and applying the same 16S rRNA gene sequencing method to both the cultured community and the fecal samples, we demonstrate that the majority of operational taxonomic unit (OTU) could be detected through culture-enriched molecular profiling and culture detected greater diversity than culture-independent methods
In this study, the cultivable proportion of the human gut microbiota was determined from the fecal samples of Culture-enriched molecular profiling detects the majority of OTUs in fecal samples An average of 76 % of all OTUs observed in cultureindependent sequencing of the fecal samples was detected by culture-enrichment (Fig. 1b)

Summary

Introduction

The human gut microbiota has been implicated in most aspects of health and disease; most of the bacteria in this community are considered unculturable, so studies have relied on molecular-based methods. These methods generally do not permit the isolation of organisms, which is required to fully explore the functional roles of bacteria for definitive association with host phenotypes. Most studies characterizing the human gut microbiome have relied on culture-independent sequencing methods These studies have provided insights into the community composition of the gut microbiota in healthy individuals [3], how it changes with environmental perturbation [4, 5], and its potential role in a variety of diseases [6, 7]. Culture using selective media allows for the growth and detection of less abundant bacteria that may be missed by insufficient sequencing depth in culture-independent studies [8]

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