Evaluation of denaturing gradient gel electrophoresis (DGGE) and next generation sequencing (NGS) in combination with enrichment culture techniques to identify bacteria in commercial microbial-based products.

Identification of bacteria in new or existing commercial microbial-based products (MBPs) is important for compliance with government regulations and for human and environmental risk assessment. Research was performed to develop effective methods to identify bacteria present in a MBP using a combined approach of conventional enrichment culture technique and denaturing gradient gel electrophoresis (DGGE) followed by clonal sequencing or next generation sequencing (NGS). Genomic DNA obtained from un-enriched or enriched MBP in MacConkey broth, Azide Dextrose broth, Peptone Water mixed with Polymixine B and Gram Negative (GN) media under three different temperatures (22 °C, 28 °C and 37 °C) were sequenced in two methods for the V3 and V6 hypersensitive regions of 16S ribosomal DNA (rDNA) and compared. Enrichment followed by DGGE and clonal sequence analysis identified 20 bacterial genera in all enriched and un-enriched media. In contrast, NGS was able to identify 114 bacterial families and 134 genera both in V3 and V6 regions. In clonal sequence analysis, in comparison to the un-enriched MBP, the MacConkey broth enriched for Escherichia or Shigella and Morganella species, GN medium enriched for Proteus and Morganella species and Azide Dextrose broth enriched for Vagococcus and Enterococcus species at both 28 °C and 37 °C. Moreover, the enrichment facilitated NGS to record higher numbers of families and genera in all enrichment cultures, comparatively higher variations in V3 region than in V6. More prominently, NGS identified 14 genera and 9 species in the family Enterobacteriaceae compared to only 5 genera identified in the un-enriched control using V6 region variance in MacConkey broth at 28 °C. Increasing the temperature without enrichment identified specific families by V3 and V6 regions. This study indicates that the polyphasic approach with appropriate enrichment and incubation at different temperatures followed by NGS analysis is a promising method for the identification of viable, non-pathogenic or potential pathogenic bacteria in complex MBPs.