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EVALUATE THE UTILITY OF ENTEROCOCCI AND BACTEROIDES AS INDICATORS OF THE SOURCES OF FECAL CONTAMINATION IN IMPAIRED SUBWATERSHEDS THROUGH DNA-BASED MOLECULAR TECHNIQUES.
Impact/Purpose:
The objective of the proposed study is to evaluate and apply fast and reproducible DNA-based technology that can detect and track fecal contamination back to its source in complex environmental matrices, including recreational and drinking water resources.
Description:
Microbial source tracking (MST) is based on the assumption that specific strains of bacteria are associated with specific host species. MST methods are attractive because their application on environmental samples could help define the nature of water quality problems in impaired aquatic resources. Until recently, MST was primarily determined by comparing phenotypic fingerprints of bacterial indicator bacteria. However, with the advent of molecular biology, nucleic acid-based methods are now commonly used to distinguish between closely related strains of bacteria in food and waterborne outbreaks. Therefore, it is reasonable to assume that nucleic acid- based tools are better suited for the identification of specific sources of fecal pollution than phenotypic methods. Most bacterial source tracking methods target Escherichia coli, Enterococcus, Bacteroides and Bifidobacterium species as indicators of fecal contamination. From these four genera, E. coli has been the most common target. Nevertheless, utilization of enterococci seems to be gaining interest. Some of the advantages of using Enterococcus species include long survival periods in aquatic environments, and a limited number of animal hosts. The latter one is opposite to what has been observed with E. coli, which can be found in a large variety of hosts. While they are classically considered harmless, some enterococci strains are now known to be opportunistic pathogens causing urinary tract infection and subacute bacterial endocarditis. In addition, there is an alarming increase of antibiotic-resistant strains which are capable of horizontal and vertical transfer of genetic material.
Currently, the most common nucleic acid based methods used in MST are ribotyping, pulse field electrophoresis, and PCR based approaches, like REP- and BOX-PCR. Some of these methods are time consuming and not prone to automation. Other methods such as terminal restriction fragment length polymorphisms (T-RFLP) have been applied with an acceptable degree of success. One method that is starting to gain attention in clinical microbiology is amplified fragment length polymorphisms (AFLP). The epidemiological field has applied AFLP analysis to differentiate between strains of Enterococcus faecium obtained from a variety of hosts and hospital environments. To our knowledge, very few studies have utilized AFLP analysis in the environmental field. Although AFLP requires the use of pure cultures, its advantage over other MST methods is that it consists of a whole genomic fingerprinting method that produces a large number of traceable characters (i.e., 180 bands per isolate). The large amount of bands produced allows for the differentiation of closely related organisms, therefore its utility as a MST method. Genomic fingerprints of multiple isolates can be processed simultaneously since fluorescently-labeled AFLP bands can be detected using fluorescence-based DNA sequencers. Another methodology that will be evaluated is the utilization of 16S primers to identify cow and human specific Bacteroides. This methodology does not require culturing of microorganisms and will be used to compare results obtained with the AFLP approach. This task is intended to be a joint effort between NERL and NRMRL where researchers from both laboratories will address the utility and effectiveness of alternative bacterial indicators of fecal contamination. An active component of the task is also an ongoing collaboration with a Region IV scientist and USDA/ARS scientist. The effort involves the evaluation and application of fairly new DNA-based technology to address the causes for impairment of surface waters when fecal contamination has been identified.