Del Valle-Pérez, Laura M.

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    Detection and analysis of antimicrobial agents using metagenomic libraries generated from aquatic bodies of Puerto Rico
    (2016) Del Valle-Pérez, Laura M.; Ríos-Velázquez, Carlos; College of Arts and Sciences - Sciences; Martínez-Cruzado, Juan C.; Santos-Flores, Carlos J.; Department of Biology; Sotomayor-Ramírez, David
    Antimicrobials have been used for hundreds of years, being effective in the reduction and complete control of microbial growth. Increasing incidences of antimicrobial resistance (AR) has been observed globally. Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus are some of the microorganisms that have shown resistance to one antibiotic in the 50% of the cases reported by the World Health Organization. In spite of the antimicrobial resistance increases, the number of novel antimicrobial agents that have been discovered or developed has decreased. Antibiotics rediscovery is approximately 99.9%. Knowing that emerging disciplines such as metagenomics have become effective in understanding AR and in the discovery of molecules with antimicrobial activity, we sought in this study to find antibiosis as well as genes involved in antimicrobial activity in aquatic metagenomic libraries (AMLs). To reach this goal, samples from the water reservoir Lago Guajataca (GWR), the river Río Grande de Añasco (RGA), and the beach Playuela (PB) at Puerto Rico, were analyzed using culture dependent and independent approaches. Among them, an antibiosis assay for cultivable microorganisms, in which no antimicrobial production in any of the sampling sites was observed. A total of four high molecular weight AMLs were generated. Two were generated from two different sites at GWR; AML from sampling site 1 in GWR had approximately 2,500,000 clones with a 75% of DNA insert, while library from sampling site 2 had approximately 3,000,000 clones with 100% DNA insert. The AMLs from RGA and PB had approximately 87,000 clones and 1,500 clones, with an average of 70% and 80% of DNA insert, respectively. These libraries were screened for functional antimicrobial agents production by a modified Kirby-Bauer method and double-layer agar assay. Also, the libraries were screened to identify gene(s) associated with antimicrobial production using PCR specific primers for polyketide synthases (PKSs) and analyzed by in silico tools. Functional analysis revealed antimicrobial agents production against Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus by the AMLs. Meanwhile, PKSs similar to those in Tolypothrix sp., Anabaena sp., Oscillatoria sp., Streptomyces sp., Kutzneria sp., Aspergillus sp., Sorangium sp., Shigella sp., Enterobacter sp., Vibrio sp., Tistrella sp., and Salinispora sp. were found; the majority of these genera were producers of antimicrobial agents. The in silico analysis at amino acid level showed the presence of sequences with homology to enzymes such as hybrids PKS/NRPS, transposases, synthases, and hypothetical proteins; some of them being related to antibiotic production as well as other biological activities. Therefore, the set of approaches taken to screen AMLs for antimicrobial agents production suggested that they were present in the studied environments and probably are polyketides. These findings represent an alternative to understand the distribution of genes with antimicrobial activity in aquatic bodies, as well as their prevalence. Beyond that, the data will help in the discovery or development of novel antimicrobials with the potential to reduce the AR global issue.