Tiedje Lab - Projects
ENVIRONMENTAL FATE AND IMPACT OF PHARMACEUTICAL SUBSTANCES
Molecular detection of antibiotic resistance determinants in the environment (Carlos Miguel Rodríguez Minguela, Brian Campbell)
The large scale use of antimicrobial drugs in agricultural applications and in human medicine has been implicated as a major cause of the antibiotic resistance (ABR) problem. However, most of the research on this subject has focused on the following: (i) the clinical setting, (ii) resistance mechanisms prevailing under selective conditions and, (iii) the easily culturable fraction of resistant bacteria. This narrow approach limits our understanding of the extent of the ABR problem by overlooking the pool of resistance determinants carried by non-culturable bacteria outside the clinical setting and the role they may have in future clinical problems. Additionally, knowledge on the distribution, diversity, and reservoirs of genetic elements involved in the capture and dissemination of ABR determinants is still limited. In order to address these problems a PCR-based culture independent approach was applied to the detection, characterization, tracking and quantification of ABR determinants and integrons (a genetic element implicated in the uptake of antibiotic resistance genes) in total DNA extracted from a variety environmental samples. This approach allowed the detection of four classes of tetracycline resistance determinants: tet (M), tet (O), tet (Q), and tet 36), encoding ribosomal protection proteins (RPP) conferring resistance against tetracycline in DNA from soil supplemented with manure from swine fed tetracycline as a growth promoter. A novel mosaic gene and two new putative classes of RPP genes were also detected. Tetracycline resistance genes were not detected in non-agricultural reference soils.
A quantitative real-time PCR assay was developed for the detection, tracking and quantification of the novel Tet 36 determinant and to determine if putative RPP genes were functional. This assay allowed the tracing of the target sequences to the animals feed with tetracycline and also indicated that putative sequences could be functional since their frequency increased in correlation with exposure to tetracycline. Further analyses revealed the presence of nine variants (one new) of aminoglycoside nucleotydiltransferases (ANT) encoded by class 1 integrons along with two genotypes (one new) for resistance to quaternary ammonium drugs. Integrons were prevalent and diverse among the tested soils but integrons encoding ABR genes were only detected in manured soils.
An integron-targeted screening was also applied to samples from marine sediments, permafrost and subtropical soils in order to survey the distribution of this genetic system. A total of 126 novel integron encoded integrases were uncovered although integrons were not detected in permafrost samples. These findings indicate that PCR based techniques are effective tools for the detection and tracking of novel resistance loci in environmental DNA and that the integron module is highly diverse and prevalent among environmental reservoirs and not restricted to the clinical strains.
Anaerobic microbial metabolism and soil sorption of halogenated pharmaceuticals (John Quensen and Mary Beth Leigh in collaboration with Dr. Hui Li, MSU Department of Crop and Soil Sciences [http://www.msu.edu/~lihui/]
The anaerobic microbial degradation and soil sorption of several halogenated pharmaceutical substances are being assessed to better understand their environmental fate and ecological risk. Compounds under investigation are pharmaceuticals being either currently marketed or tested for use in humans and livestock. The capacity for anaerobic microorganisms to dehalogenate and biodegrade these substances are being evaluated in both pure culture systems and in microcosms constructed with samples from a variety of anaerobic environments likely to be impacted as a result of pharmaceutical use, including swine waste, municipal wastewater treatment plant sludge, river, estuatine and wetland sediments and rumen fluid. For example, the dechlorination of Clindamycin to Lincomycin via anaerobic dechlorination is being evaluated to assess whether this transformation may account for detection of Lincomycin in the environment. The sorption and desorption of pharmaceuticals by several soils with different soil organic matter and mineral contents are being determined to measure the distribution of these two pharmaceuticals in soil-water systems, to evaluate the availability of these two pharmaceuticals in microbial metabolism experiments and to aid in predictions of environmental fate.
The aerobic degradation of several common analgesics is also being explored. Organisms capable of growing and biodegrading ibuprofen, ketoprofen and naproxen have been isolated and identified, and degradative pathways will be explored in collaboration with Mary Beth Leigh (University of Alaska Fairbanks, http://mercury.bio.uaf.edu/~mary_beth_leigh/).