Rabelo Fernández, Robert J.

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    Unraveling the microbiome of the snail Caracolus marginella using metagenomics
    (2018) Rabelo Fernández, Robert J.; Ríos Velázquez, Carlos; College of Arts and Sciences – Sciences; Martínez Cruzado, Juan C.; Santos Flores, Carlos J.; Department of Biology; Morales Payán, José P.
    The increased of Antibiotic Resistance (AR) is currently one of the most important public health threats faced worldwide. Increasing rates on AR has been observed globally. The use and analysis of non-traditional techniques are required to solve this problem. Based on the fact that culture independent techniques as metagenomics can access the 100% of the genomes present in the environment. Metagenomics, as a culture independent approach, has become a powerful technique used in the analysis of microbiomes to facilitate the discovery of novel activities, including AR in diverse environments. Recent studies underestimate the microbial diversity and genotypic traits in the snails’ microbiome. Caracolus marginella, a land snail native to Caribbean islands, has the ability to adapt to different environments where biomedical potential has not been explored. Thity (30) specimens of C. marginella were collected from five collection sites in the four regions of the island. Our research focused the cultivable and uncultivable studies of C. marginella microbiome. To reach these goals, thirty-four isolates (bacteria and yeast) were identified from the snail gut. These were microbiologically characterized (macro and microscopically), and biotechnological applications for cellulose and starch were searched. Of these isolates, 61.7% and 8.8% of the organisms were positive for cellulose and starch degradation, respectively. Also, four metagenomic libraries from C. marginella gut microbiomes, from four different regions of Puerto Rico were generated and the AR activity assessed. High molecular weight (40kb) metagenomic libraries were generated from C. marginella snails collected from different regions in Puerto Rico using a direct DNA isolation method. In addition, the presence of a fosmid and insert in the clones was confirmed by restriction analysis. The libraries generated from the East, South, North and West regions of Puerto Rico averaged, respectively, 11,000 clones with 60% DNA insert, 16,700 clones with 90% DNA insert, 16,500 clones with 90% DNA insert and 16,000 clones with 100% of insert. Clones in the four libraries had a 70% insert variation/region. After determining the Minimal Inhibitory Concentration (MIC) to Ampicillin (Amp, 12μg/mL), Gentamycin (Gm, 8μg/ml) and Tetracycline (Tet, 1μg/mL) of Escherichia coli Epi300 with pCC1FOS vector with no insert as control; the Metagenomic Libraries generated were spread on LB plates with MICs ranging from 1X-7X. Clones from all four libraries showed AR to Amp (too numerous to count) for 1X and 2X, AR for 1X Tetracycline in the North, East, West libraries, and 1X-2X in the South; and for Gm, resistant clones were found only in MIC 1X for the east region. The clones from the four libraries resistant to Ampicillin showed similar band patterns. After transposon mutagenesis, the in silico analysis at amino acid level suggested the presence of sequences with homology to: Histidine Kinase, Thymidylate Synthase, and Phosphoenolpyruvate Carboxykinase. These findings proposed functional multidrug resistance in C. marginella clones is due to membrane transport mechanism by efflux pump. This study is the first metagenomic AR study in a snail gut in Puerto Rico and Caribbean, and represents the first steps in gaining a better understanding of C. marginella gut microbiome resistome.