Ferrer González, Frank X.

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    Antibiotic resistance screening in metagenomics libraries generated from cave soil of Puerto Rico
    (2015) Ferrer González, Frank X.; Rodríguez Minguela, Carlos M.; College of Arts and Sciences - Sciences; Martínez Cruzado, Juan C.; Ruíz Acevedo, Alejandro; Department of Biology; Mégret, Rémi
    After more than 70 years of their discovery, abuse and misuse of antibiotic substances, has selected for pathogens the abilities to confer antibiotic resistances, threatening modern medicine and becoming one of the main health concerns of the 21st century. The development of novel technology to help solve and understand the current trends in antibiotic resistance is a must. By the use of techniques such as metagenomics, the detection and analysis of functional microbial activity in the environment has been made possible through culture independent approaches. In our research a metagenomic library (ML) from cave Ventana’s soil in Puerto Rico was generated using the direct DNA extraction method. The cave environment was selected due to the low amount of studies on antibiotic resistance in caves. The ML was produced using the fosmid vectors delivered into Escherichia coli Epi300, averaging 40,000 clones. The ML was screened for resistance for some of the first and most broadly used antibiotics. After determining Minimal Inhibitory Concentration for the antibiotics; gentamicin (MIC 8. 0µg/ml), kanamycin (MIC 12.0µg/ml) and tetracycline (MIC 1.0µg/ml), the ML was spread on LB plates containing different antibiotic concentrations. Activity was found for kanamycin and tetracycline. In addition, the presence of fosmid insert in clones was confirmed through an enzyme restriction analysis. The ML showed one clone resistant to tetracycline (MIC 10.0µg/ml) and one clone highly resistant to kanamycin (MIC>1200.0µg/ml). Target genes were inactivated using Tn5 transposon mutagenesis, sequenced and bioinformatics analysis predicted an efflux like mechanism conferring the resistance for tetracycline. More data is needed to determine resistance mechanisms to kanamycin, literature suggests it could be related to aminoglycoside modifying enzymes. This is the first metagenomic library generated from caves in Puerto Rico and the first antibiotic resistance functional study done from caves in the island.