Functional Metagenomics: A Tool to Study Antimicrobial Resistance in Saliva

Objectives: While most part of the oral microbiome community provides health beneficial functions, the high density of microorganisms within this ecosystem can also facilitate horizontal transfer of antimicrobial resistance genes (ARGs). Differently from targeted metagenomics, which compares hits to known databases, functional metagenomics (FM) allows identification of novel ARGs and potential novel antimicrobial compounds. This approach is based on cloning metagenomic DNA from samples into an expression vector which is transformed into an indicator strain that can be tested for different phenotypes, and has been used mainly for ARGs analysis of fecal samples. Here, we aimed to optimize FM methodology in order to investigate the effect antibiotics on the emergence of new ARGs in saliva.
Methods: Total bacterial DNA was extracted with different methods from saliva of volunteers treated and non-treated with antibiotics. Purified DNA was size-selected for 3-5kb-long products, end-repaired and ligated to the expression vector pZE21/MCS-1 later transformed into E.coli by either heat-shock or electroporation.
Results: Use of phenol-chloroform DNA extraction method as employed for fecal samples in FM studies failed in generating high yields in saliva samples. The use of a commercial kit for DNA extraction of Gram-positive bacteria resulted in approximately five times higher DNA yields and metagenomic DNA was of high purity by spectrophotometry, with A260/A280 ratio above 1.8 compared to 1.5 provided by phenol-chloroform method. Transformation of DNA ligated to pZE21/MCS-1 vector into E.coli by electroporation was four times more efficient than by heat-shock.
Conclusions: Our results indicate that different DNA extraction methods for saliva samples affected the final DNA yield and purity for downstream application in FM. In the study of the oral microbiome and resistome, optimization of FM methodology may grant culture-unbiased information of the entirety of ARGs and contribute to a better understanding of the potential impact of the human oral microbiome as a reservoir of ARGs.