Rodríguez-Polanco, Wilmer R.

Profile Picture

Filters

20171
Reset filters

Settings

Citations

Publication Search Results

Now showing 1 - 1 of 1
  • Thumbnail Image
    Publication
    Culture dependent and functional genomic analysis of bioprospects capable of producing antibacterial agents
    (2017) Rodríguez-Polanco, Wilmer R.; Ríos-Velázquez, Carlos; College of Arts and Sciences - Sciences; Rodríguez-Minguela, Carlos; Vargas, Roberto; Department of Biology; Román Pérez, Rosa I.
    Regardless of new technologies, increasing availability of vaccines, and novel medicine advances, infectious diseases are one of the principal causes of human mortality. Infectious disease control has become even more difficult with the emergence of drug resistant microorganisms, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), carbapenem-resistant Klebsiella pneumoniae (NDM-1), and multidrug-resistant (MDR) Pseudomonas aeruginosa. To counteract this issue, this research aims to search for cultivable antimicrobial agents producing microbes (CAAPM) from the Microbial Biotechnology and Bioprospecting Laboratory´s (MBBL) collection isolated from the Guánica Dry Forest, Guajataca Forest and Toro Negro Forest, and from the hypersaline microbial mats (MM) from Cabo Rojo’s salterns. The microbial targets included Escherichia coli (ATCC 8677), Pseudomonas aeruginosa (27853), Klebsiella pneumoniae (ATCC 13883), Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus (ATCC 25923), and Bacillus subtilis (ATCC 6633). The MBBL’s collection and the MM were screened for antimicrobial production with a modified Kirby Bauer method and for polyketide synthase (PKS) by PCR using specific primers. One CAAPM was isolated from the MBBL’s collection and another from the MM, and named WJ and 7M respectively. Both CAAPMs produced an inhibition halo against S. aureus and B. subtilis by bioprospect direct contact and both amplified for PKS. Their 16S rDNA was amplified by PCR and analyzed with in silico tools suggesting 7M and WJ belong to Bacillus and Pseudomonas genus respectively. Macroscopic and microscopic characterization confirmed the morphology described for both genus and suggested by the in silico analysis. A 7M genomic library composed of 350 clones with 80% insert, and a WJ genomic library composed of 37,960 clones with 70% insert were constructed using the CopyControl™ Fosmid Library Production Kit using pCC1FOS™ Vector. No antimicrobial activity was detected with the overlay method, but PKS PCR amplification and in silico analysis from 7M and WJ genomic DNA and genomic libraries suggested they have PKS related genes. After extracting WJ genomic DNA by a direct method, a combinatorial chemistry Phage Display Library (PDL) was generated using the T7 Select 10-3 cloning kit (Novagen). Two different types of screening were done to the PDL. The first screening was a double layer assay, where a lawn of Escherichia coli with individual T7-Bioprospect PDL plaques were exposed to the target by pouring top agar inoculated with Staphylococcus aureus over the plaques. The second screening consisted in combining the T7-Bioprospect PDL with E. coli and S. aureus in the same top agar solution. After screening 0.0001% of the T7 PDL no positive results were detected, but a larger screening batch has to be done in order to test this technique’s full potential. While more library screening and optimization processes are in progress, this is a promising strategy to find molecules with antibiosis activity. The combination of cultivable dependent and functional genomics approaches have proved to be a challenging but strong resource to unravel antimicrobial agents from the microbial flora of different environments. The inhibition obtained from WJ and 7M could represent a possible solution against MRSA.