Rivera-Rivera, Michelle J.

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    Isolation and Characterization of Antimicrobial Agent Producing Microbes and Generation of Metagenomic Libraries from Diverse Forest Soils in Puerto Rico
    (2005) Rivera-Rivera, Michelle J.; Ríos-Velázquez, Carlos; College of Arts and Sciences - Sciences; Montalvo-Rodríguez, Rafael; Santos Flores, Carlos J.; Department of Biology; Schroder, Eduardo
    The misuse of antimicrobial agents is a world wide issue, since many opportunistic bacteria have acquired resistance or are becoming resistant to commercially available antibiotics. Antimicrobial resistance is not limited to clinical opportunistic bacteria but also is a concern for the agro economic sector. Many soil microbes have also obtained resistance and are less susceptible to antimicrobials and antifungals used for plague management. That is why the search for novel antimicrobial agents is of major importance. Bioprospecting is the search for novel biotic systems that will serve as a source of molecules with biotechnological potential. Many ecosystems, such as soils, are being exploited in order to bioprospect for activities such as antibiotic production. To complete this task general microbiological techniques are used but also, since not even 1% of the entire soil population can be cultured, research groups are using functional genomics to access the uncultivable microbes. The main goal of our research was the isolation and characterization of Antimicrobial Agent Producing Microbes (AAPM) and the generation of soil metagenomic libraries for the isolation of antimicrobial agent encoding genes. Soil samples were obtained from three forests in Puerto Rico. We isolated a total of 185 AAPM from soil samples by using serial dilutions and the crowded plate technique forty of the AAPM showed antagonistic capabilities toward enteric and non-enteric opportunistic bacteria used as targets which included Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus epidermidis. The capability of antimicrobial production of the isolated microbes was determined by using a modified version of the Kirby Bauer susceptibility test, the radial inhibition, and the streak technique, among others. The top ten antagonists were chosen for further molecular, microbiological and biochemical characterization. Our data indicates that a group of the top ten isolates belong to the Pseudomonas spp. and Bacillus spp. A preliminary study demonstrated that one of the isolates has biocontrol capabilities, by inhibiting the pathogenic fungi Rhizoctonia solani. In a green house experiment we determined that two of the microbes inhibited necrosis formation in the plant Phaseolus vulgaris and also had the capability of acting as a Plant Growth Promoting Rhizobacteria. Two soil metagenomic libraries of the forest soils were generated and screened for antibiosis. Even though antimicrobial activity was not detected, one of the clones was able to grow in minimal media supplemented with three different carbon sources. Through this study we have constructed the first Puerto Rican soil metagenomic library and have obtained a collection of forest soil AAPM with clinical, agricultural and biotechnological potential.