The 16S rRNA lung microbiome in mechanically ventilated patients: a methodological study

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Characterizing the respiratory tract bacterial microbiome is less advanced than gut microbiome studies.
• Accurate microbiome analysis relies on robust methodology, particularly in primer selection for 16S rRNA gene amplification.
• Primer bias can significantly impact the perceived composition of microbial communities.

Purpose of the Study:

• To evaluate the impact of different 515F-806R primer pairs, targeting the V4 region of the 16S rRNA gene, on respiratory microbiome analysis.
• To compare the accuracy of the 'Stringent V4' (S-V4) and 'Relaxed V4' (R-V4) primer pairs in respiratory samples.
• To determine the optimal primer set for accurate bacterial lung microbiome profiling.

Main Methods:

• Comparison of two 515F-806R primer pairs (S-V4 and R-V4) for 16S rRNA gene amplification.
• Sequencing of respiratory samples from critically-ill ventilated patients using Illumina paired-end sequencing.
• Validation of primer pair accuracy by concordance with conventional microbiology techniques.

Main Results:

• Significant differences in bacterial microbiome composition were observed between the two primer pairs.
• The S-V4 primer pair showed higher concordance (89%) with conventional microbiology compared to R-V4 (56%) for endotracheal aspirates.
• The R-V4 primer pair exhibited poor detection of Enterobacteriaceae, a key finding impacting accuracy.

Conclusions:

• The accuracy of bacterial lung microbiome profiling is highly dependent on the chosen 16S rRNA hypervariable region primers.
• The 515F-806R S-V4 primer pair, in conjunction with Illumina MiSeq sequencing, is validated as the optimal choice for future lung microbiome studies.
• This finding is critical for advancing research into the respiratory tract microbiome and associated diseases.