Molecular studies neglect apparently gram-negative populations in the human gut microbiota.
Level 4 - case-series / case-control
Cross-sectional comparative laboratory evaluation on human stool samples
PubMed 23885002 · doi:10.1128/JCM.00473-13
What was done
The authors compared microscopy, staining, and molecular approaches for characterizing gut microbiota in 16 stool samples collected from diverse human subjects (healthy individuals from different areas, patients with metabolic disorders, immunocompromised patients, and individuals receiving antibiotics). Fecal samples were evaluated using Gram staining, flow cytometry, transmission electron microscopy (TEM), quantitative real-time PCR (qPCR) targeting the Bacteroidetes and Firmicutes phyla, and 16S rRNA gene amplicon pyrosequencing targeting the V6 hypervariable region.
What was found
Prokaryote abundance was quantified at 10^10 per gram of feces. The measured proportion of Gram-negative prokaryotes differed significantly depending on the analytical method used (Mann-Whitney test): Gram staining yielded a median of 70.6%, TEM yielded 31.0%, and 16S pyrosequencing yielded 16.4%. TEM and pyrosequencing showed a 14.6% difference in Gram-negative identification with a non-significant correlation for the Gram-negative to Gram-positive ratio (Spearman rho = 0.3282, P = 0.2146). In contrast, molecular methods correlated significantly: qPCR and pyrosequencing showed a significant correlation for the Bacteroidetes/Firmicutes ratio (Spearman rho = 0.6057, P = 0.0130).
Why it matters
Standard 16S rRNA amplicon sequencing can heavily underestimate Gram-negative bacterial abundance compared to direct visual and physical methods. Single-platform metagenomic approaches risk substantial systematic bias, indicating that multi-method validation is necessary to accurately assess human gut microbial diversity.
Limits
The sample size was very small (n = 16) and pooled across highly heterogeneous clinical conditions without separate subgroup analyses. Sequencing was limited to the V6 hypervariable region rather than multi-region 16S rRNA profiling or shotgun metagenomics, and microscopic methods lack the high-resolution taxonomic identification provided by molecular sequencing.
Cited by
- supports Approximately 40% to 50% of the microbes in the human gut are made up of gram-negative bacteria.