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

Abstract

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.

Keywords: groundwater; metagenomic analysis; non-sterile pharmaceutical product; oligotrophic enrichment; specified microorganisms.

Figure 1

Overview of the metagenomics study.

Figure 2

Growth of total heterotrophic bacteria on 1/10 strength TSA (A) and BCC on BCC-specific medium (B) on the corresponding agar media inoculated with cultures of pre-enriched cold fountain water using various media at 23°C over sampling time course.

Figure 3

Detection of specified microorganism genera after 24 h enrichment in TSB and 1/10 strength TSB media. Results are expressed as relative sequence abundance of reads. The * symbol indicates a statistically significant difference, p < 0.05.

Figure 4

Comparing incubation times; Changes in the relative sequencing read abundance of specified microorganisms in TSB (A) and 1/10 strength TSB (1/10 × TSB) (B) media at 24 h, 48 h, and 72 h. The * symbol over each diagram indicates a statistically significant difference, p < 0.05.

Figure 5

Synergistic effects of BCC-spiked media in detecting specified microorganisms; changes in the relative sequencing read abundance of specified microorganisms in TSB and TSB co-cultured 10⁶ CFU/mL BCC (TSB + BCC) at 24 h (A), 48 h (B) and 72 h (C) and 1/10 strength TSB (1/10 × TSB) and 1/10 strength TSB co-cultured 10⁶ CFU/mL BCC (1/10 × TSB + BCC) at 24 h (D), 48 h (E) and 72 h (F). The * symbol over each diagram indicates a statistically significant difference, p < 0.05.

Figure 6

Identification of BCC metabolic pathways in BCC-spiked water. Heatmap of relative abundance of the 50 most abundant metabolic pathways in the metagenomes found in 12 water samples spiked and unspiked with BCC after 24, 48, 72 h enrichment in TSB and 1/10 TSB.