design: DNA amplicon of the V3-V4 region of the 16S gene was extracted using an optimized and standardized 16S amplicon library preparation protocol (Metabiote, GenoScreen, Lille, France). 16S DNA PCR was performed with 5ng of genomic DNA with bar-coded primers (Metabiote MiSeq Primers) according to the manufacturers protocol (Metabiote) and PCR product purification was performed with Agencourt AMPure XP-PCR purification system (Beckman Coulter, Brea, CA, USA)