The choice of 16S rRNA gene sequence analysis impacted characterization of highly variable surface microbiota in dairy processing environments

Accurate knowledge of the microbiota collected from surfaces in food processing environments is important for food quality and safety. This study assessed discrepancies in taxonomic composition and alpha and beta diversity values generated from eight different bioinformatic workflows for the analysis of 16S rRNA gene sequences extracted from the microbiota collected from surfaces in dairy processing environments. We found that the microbiota collected from environmental surfaces varied widely in density (0–9.09 log₁₀ CFU/cm²) and Shannon alpha diversity (0.01–3.40). Consequently, depending on the sequence analysis method used, characterization of low-abundance genera (i.e., below 1% relative abundance) and the number of genera identified (114–173 genera) varied considerably. Some low-abundance genera, including Listeria, varied between the amplicon sequence variant (ASV) and operational taxonomic unit (OTU) methods. Centered log-ratio transformation inflated alpha and beta diversity values compared to rarefaction. Furthermore, the ASV method also inflated alpha and beta diversity values compared to the OTU method (P < 0.05). Therefore, for sparse, uneven, low-density data sets, the OTU method and rarefaction are better for taxonomic and ecological characterization of surface microbiota.