16s rRNA Lung Microbiota Study in Mechanically Ventilated Patient: A Pilot Study

Abstract BackgroundCharacterization of the respiratory tract bacterial microbiota is in its infancy when compared to the gut microbiota knowledge. As key methodological steps can directly affect the accuracy of the results, it is crucial to determine a robust methodology in order to limit bias.Two different pairs of primers 515F-806R targeting the V4 hypervariable region of the 16SrRNA gene, “S-V4” for “Stringent V4” primer pair and “R-V4” for “Relaxed V4” primer pair, are respectively used in two ongoing international projects “the Human microbiome project” and “the Earth microbiome project”. We compared two methods of sample processing using these two different primer pairs for bacterial microbiota analyses of respiratory samples from critically-ill ventilated patients. For the later, Illumina 250 paired-end sequencing was done on a MiSeq platform after DNA extraction using mechanical lysis (bead-beating) and NucliSENS easyMAG. The concordance with conventional microbiology is the criterion of choice to determine the optimal method.ResultsTwenty samples from seven patients and four controls were sequenced. The two primer pair provided highly different results. Only 54% of the samples had a similar microbial composition with both pairs of primers. “S-V4” gave the best agreement with the conventional microbiology for ETA: 89% as compared to 44% for the “R-V4” primer pair. The main difference related to Enterobacteriaceae, which were concordant between conventional cultures and microbiota analyses using “S-V4”. Enterobacteriaceae detection was poor for “R-V4”. Among patients with VAP, a decrease in alpha diversity in ETA was observed. The mean of pairwise Unifrac distance was higher inside this group of patients at the time of VAP diagnosis as compared to control patients.ConclusionAccuracy of the bacterial lung microbiota composition was highly correlated to the pair of primers used for amplification of the 16s rRNA hypervariable sequence. Comparison of microbiota results obtained by sequencing and conventional microbiology allowed us to select the best option for further studies. This work validates our methodology based on 16SrRNA gene amplification with 515F-806R “S-V4” primer pair associated to Illumina® MiSeq 250 paired-end sequencing.