Unraveling tetracycline resistance in puerto rican soils thru metagenomics

Traditional methods in microbiology rely on cultivable methods to study microbes in any environment. This requires growing the isolates in culture media to perform the analysis, limiting the study to cultivable microbes which represent only 1% of the total population. Metagenomics provides access to 99% of the total microbiota using culture independent techniques. This emerging field has provided strategies to functionally screen activities such as antimicrobial resistance mechanisms, one of today’s world wide public health concerns, due to the prescriptions abuse and public misused. The main focus of this research is to study antibiotic resistance from cave’s soil in Puerto Rico.   A metagenomic library (ML) from Cueva Ventana soil in Arecibo, Puerto Rico, was generated using indirect DNA extraction method. Minimal Inhibitory Concentrations (MIC) for tetracycline (Tc), was performed to Escherichia coli Epi300 with an empty pCCFOS1vector.  Finally, functional Tc resistance selection was done by growing the ML clones in Luria Bertani Agar plates containing tetracycline at 4xMIC’s, 10xMIC and 20xMIC. The cave metagenomic library generated has approximately 118,000 clones.  The Tc minimal inhibitory concentration for the host was 0.5ug/mL (1xMIC) . The functional selection preliminary results suggest .0123% of clones from the ML were able to grow at 4xMIC, 1.4x10^-5 % at 10xMIC and 3.0x10^-6 % at 20xMIC.  Ongoing research is in process to determine the identity of the gene(s) responsible for the resistance. The data obtained will provide valuable information in the resistome project, to determine the mechanisms of the resistance and microorganism responsible for such activity.