Molecular Ecology of Integron-encoded Antibiotic Resistance and Prevalence of Fungicide Resistance

FINAL REPORT (2008-2010) SEAGRANT PROJECT: R-92-3-08 Molecular Ecology of Integron-encoded Antibiotic Resistance and Prevalence of Fungicide Resistance in Microbial Populations from Critical Coastal Habitats Impacted by Sewage, Animal Waste, and Wastewater Treatment Plant Discharges. Carlos M. Rodríguez-Minguela, Rafael Montalvo-Rodríguez, and Sandra L. MaldonadoRamírez. University of Puerto Rico Mayagüez Campus, Department of Biology, PO Box 9012, Mayagüez, PR 00681. ABSTRACT Sewage discharges are major contamination sources of ecologically and economically important coastal environments. Moreover, fecal bacteria from clinical settings have been identified as key hosts of multiple antibiotic resistance (ABR) genes encoded by integrons. Integrons are genetic elements that mediate the assimilation of DNA molecules encoding a variety of adaptive functions that may promote competence under unfavorable circumstances. Using a PCR-based approach we detected a prevalence pattern of clinical integrons and integron-encoded antibiotic resistance genes (class 1) associated with mangrove, beach and estuarine habitats exposed to high levels of wastewater pollution or anthropogenic impact. The application of these molecular techniques to the analysis of culturable bacteria also revealed the presence of similar resistance determinants among conventional fecal indicators as well as in reference isolates from hospital-related settings. We similarly detected the dominance of fluconazole resistant yeasts in association with human influence. Moreover, phylogenetic analysis of ITS sequences identified various fluconazole-resistant yeast isolates that were highly similar to opportunistic pathogens that are routinely treated with this medicament. Our data indicate that polluted coastal habitats are stable reservoirs of antimicrobialresistant bacterial and fungal populations and that cassette PCR assays targeted at class 1 integrons have the potential to serve as dual indicators of fecal contamination and inconspicuous risks to human health. INTRODUCTION The widespread use of antimicrobials in human and veterinary medicine as well as in agricultural production settings have resulted in a large scale release of these chemicals into natural environments. These observations have raised serious concerns within the clinical sector which also have environmental implications of great significance (Kümmerer, 2004). Recently, integrons, a genetic system carried by bacteria, have been implicated in the establishment and dispersal of antibiotic resistance traits which include protection mechanisms against the most important antibiotics used for the treatment of infection in humans as well as in plants and animals of economical importance. This system is based on the activity of a site-specific recombination mechanism catalyzed by an integrase which captures and inserts circular DNA molecules (gene cassettes) downstream a promoter for subsequent expression (Figure 1). Although