Novel Antibiotic-Resistant Bacteria Formed in the Environment as a Result of Fecal PollutionEPA Grant Number: CR830396
Title: Novel Antibiotic-Resistant Bacteria Formed in the Environment as a Result of Fecal Pollution
Investigators: Field, Katharine G.
Institution: Oregon State University
EPA Project Officer: Carleton, James N
Project Period: January 1, 2002 through December 31, 2003
Project Amount: $207,483
RFA: Futures Research in Natural Sciences (2001) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Ecological Indicators/Assessment/Restoration , Land and Waste Management
Although the appearance of novel strains of antibiotic-resistant bacteria is a major public health concern, the extent to which these strains arise in the environment, providing a serious future threat to public health, is not known. We propose to investigate the formation of novel antibiotic-resistant bacteria by transfer of antibiotic-resistance genes from fecal bacteria released in the environment with fecal pollution. As a model system we will use the fecal anaerobic Bacteroides group. Our previous research using genetic markers from Bacteroides to diagnose the source of fecal pollution in estuarine waters showed that Bacteroides spp. are widespread pollutants in these waters. Furthermore, we found that tetQ, a gene for tetracycline resistance commonly carried by Bacteroides, is also widely distributed in estuarine and river waters. In addition, we have shown that Bacteroides is able to transfer antibiotic resistance from one strain to another when added to 15° seawater microcosms, when oysters are present. Bacteroides spp. contain conjugative transposons, which can pass into distantly-related bacteria via conjugation. These conjugative transposons carry genes for antibiotic resistance. Low levels of antibiotics increase the rate of conjugation.
We propose to investigate the formation of novel antibiotic-resistant bacteria by transfer of antibiotic-resistance genes from fecal bacteria released in the environment with fecal pollution. As a model system we will use the fecal anaerobic Bacteroides group.
To determine the extent to which this provides an important source of antibiotic resistance and poses a future environmental threat, we will: - 1) determine the survival and growth of Bacteroides in estuarine waters, suspended and bottom sediments; 2) measure the rate of conjugation with environmental bacteria under natural conditions; and 3) survey the current level of biopollution by Bacteroides and tetQ, its tetracycline resistance gene. We will measure survival and growth (Objective 1) in seawater mesocosms by labeling with bromodeoxyuridine (BrdU), a thymidine analog. BrdU-containing DNAs will be separated immunochemically and labeled (growing) and unlabeled (surviving but not growing) DNAs will be analyzed with Bacteroides-specific PCR primers. To measure conjugation to defined and native recipients (Objective 2), we will construct a strain of B. thetaiotaomicron with the green fluorescent protein (gfp) reporter gene in a conjugative transposon, introduce it into seawater mesocosms, and separate out fluorescent transconjugants by fluorescence automated cell sorting. The transconjugants will be identified by 16S rDNA sequences. We will vary environmental parameters and measure changes in the rate of conjugation. For objective 3, we will conduct seasonal sampling in river and estuarine sites in Tillamook Bay, OR, and monitor the presence of Bacteroides and tetQ by means of specific PCR primers. We will also monitor tetracycline levels. This research will assess the future risk to human health posed by the spread of antibiotic resistance in the environment from fecal Bacteroides.