A metagenomic analysis coupled with oligotrophic enrichment approach for detecting specified microorganisms in potable groundwater samples

In pharmaceutical manufacturing, there is a significant need for the detection and identification of specified microorganisms ( i.e. , Burkholderia cepacia complex (BCC), E. coli, Pseudomonas aeruginosa , Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma ), which are often missed or not identified by traditional culture-dependent methods. We employed a metagenomic analysis coupled with oligotrophic enrichment to identify specified microorganisms and evaluate tryptic soy broth (TSB) and 1/10 strength TSB for the recovery of specific microorganisms in potable groundwater samples. A total of 589–996 genera were identified in 12 water samples taken from a cold water fountain, with Bacillus spp. (97%) in TSB and Stenotrophomonas spp. (97%) in 1/10 strength TSB, representing the primary recovered genera after a 72-h pre-enrichment at 23°C. Likewise, we also detected lower abundance of specific organisms, Clostridium spp., Burkholderia spp., and Staphylococcus spp. (0.04–0.07%) in TSB and Burkholderia spp., Pseudomonas spp., Salmonella spp., Staphylococcus spp. and Escherichia spp. (0.01–1.73%) in 1/10 strength TSB. Co-inoculation with Burkholderia cepacia complex (BCC) yielded a higher recovery rate of Pseudomonas spp. compared to uninoculated controls in 1/10 strength TSB. Further functional analyses indicated that, toluene degradation (PWY-5180 and PWY-5182) was chiefly contributed by BCC in co-cultures of TSB + BCC-24 h and TSB + BCC-48 h. Our results demonstrate the potential value of the metagenomic approach during enrichment in detecting specified microorganisms, including oligotrophs such as BCC in non-sterile pharmaceutical products.