Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/16/2014
Publication Date: 12/30/2014
Citation: Irwin, P.L., Nguyen, L.T., He, Y., Paoli, G., Gehring, A.G., Chen, C. 2014. Near-quantitative extraction of genomic DNA from various food-borne eubacteria. BMC Microbiology. DOI: 10.1186/s12866-014-0326-z.
Interpretive Summary: In nature, bacteria exist in intricate microbial communities. Some of the bacteria in these communities are known to form elaborate assemblies and biofilms even at low temperatures. Part of our mandated research project has to do with determining the total microbial content (culturable as well as total) of any niche we choose from within various food processing environments in order to determine what organisms are present, their relative abundance, and determine which ones form biofilms, or other edifices, incorporating E. coli and other foodborne pathogens within. To accurately perform this metagenomic analysis requires that bacterial DNA be completely isolated with a minimum of bias between the multitude of genetically distinct organisms. In this work we have tested more than a dozen relatively new commercial genomic DNA extraction technologies and normalized the resultant 16S rRNA gene copy number data to colony forming units (CFU) in order to better assess performance inasmuch as we know how many copies should exist per genome for many common true bacteria. We additionally tested the 3 best performers of the 12 against an additional 15 foodborne isolates and found that one device consistently (12 out of 15 isolates) provided something close to quantitative results. A low-bias quantitative extraction of DNA will allow us to use 16S rRNA “gene” amplification and next generation sequencing of fragments derived therefrom to assign the relative composition of virtually any microbial community including those found in food processing plants. Once quantities of each of the isolates in a particular community are known, we can begin to determine which organisms can form mixed biofilms, or other structures, with pathogens and determine if such heteogeneous bacterial architectures play a role in protecting pathogens.
Technical Abstract: In this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70E6 (± 9.05%), 4.77E8 (± 31.0%), and 5.93E8 (± 4.69%) colony forming units (CFU) associated with 3 cultures (n = 3) each of Brochothrix thermosphacta (Bt), Shigella sonnei (Ss), and Escherichia coli O79 (Ec), respectively. We have utilized real-time PCR (qPCR) quantitation with two specific sets of primers (one for Bt and one other for Ss and Ec) associated with the 16S rRNA “gene” to determine the number of copies per CFU by comparing the unknown target DNA qPCR results with standards (purified PCR Bt and Ss 16S rRNA gene products) for each primer set. Based upon statistical analyses of all our results, we determined that the Agencourt Genfind v2 (Beckman Coulter), High Pure PCR Template Prep Kit (Roche Diagnostics), and Omnilyse (Claremont BioSolutions) methods consistently provided the greatest yield of genomic DNA ranging from 141-934, 8-21, and 16-27 16S rDNA copies per CFU for Bt, Ss, and Ec. If one assumes 6-7 copies of 16S rRNA gene per genome, 1-2 genomes per cell (Ss and Ec were in mid-Log phase) and 100-150 cells per CFU for Bt (this range found to be reasonable using an optical method expounded upon herein) or 1-2 cells per CFU for either Ss or Ec, then the Omnilyse procedure provided near-quantitative extraction of genomic DNA from these isolates (934 ± 19.9 copies per CFU for Bt; 20.8 ± 2.68 copies per CFU for Ss; 26.9 ± 3.39 copies per CFU for Ec). The 3 best methods (Agencourt, High Pure, Omnilyse) were subsequently assessed using 5 additional Gram-positive and 9 Gram-negative isolates (n = 3) using a set of “universal” 16S rDNA primers. Overall, the preeminent method was found to be the Omnilyse procedure (a “bead blender” technique involving high frequency agitation in the presence of zirconium silicate beads) inasmuch as 73% of the isolates tested gave the greatest recovery. In 20% of the isolates tested, Omnilyse was statistically equivalent to the best method (Agencourt). For only Serratia proteamaculans was Omnilyse (1.18 ± 0.788 copies per CFU) observed to yield significantly less genomic DNA than the other 2 procedures (Agencourt, 5.83 ± 0.650 copies per CFU; High Pure, 4.39 ± 1.89 copies per CFU).