Molecular Studies Neglect Apparently Gram-Negative Populations in the Human Gut Microbiota

Studying the relationships between gut microbiota, human health, and diseases is a major challenge that generates contradictory results. Most studies draw conclusions about the gut repertoire using a single biased metagenomics approach.We analyzed 16 different stool samples collected from healthy subjects who were from different areas, hadmetabolic disorders, were immuno-compromised, or were treated with antibiotics at the time of the stool collection. The analyses performed included Gram stain-ing, flow cytometry, transmission electronmicroscopy (TEM), quantitative real-time PCR (qPCR) of the Bacteroidetes and Fir-micutes phyla, and pyrosequencing of the 16S rRNA gene amplicons targeting the V6 region.We quantified 1010 prokaryotes per gram of feces, which is less than was previously described. TheMann-Whitney test revealed that Gram-negative proportions of the prokaryotes obtained by Gram staining, TEM, and pyrosequencing differed according to the analysis used, with Gram-nega-tive prokaryotes yielding median percentages of 70.6%, 31.0%, and 16.4%, respectively. A comparison of TEM and pyrosequenc-ing analyses highlighted a difference of 14.6 % in the identification of Gram-negative prokaryotes, and a Spearman test showed a tendency toward correlation, albeit not significant, in the Gram-negative/Gram-positive prokaryote ratio ( 0.3282, P 0.2146). In contrast, when comparing the qPCR and pyrosequencing results, a significant correlation was found for the Bacte-roidetes/Firmicutes ratio ( 0.6057, P 0.0130). Our study showed that the entire diversity of the human gut microbiota re-mains unknown because different techniques generate extremely different results. We found that to assess the overall composi-tion of bacterial communities, multiple techniques must be combined. The biases that exist for each technique may be useful i