#05-039

Complete

Date Full Report Received

05/12/2008

Date Abstract Report Received

05/12/2008

Investigation

Institution:
Primary Investigator:

Using antibiotics to manipulate the intestinal microbiota to improve growth efficiency raises a number of concerns. First, utilization of broad-spectrum antibiotics inhibits indigenous strains of bacteria in the intestinal tract that are beneficial to the host animals while increasing the potential for colonization of the gut by pathogens. Second, the presence of antibiotic residues, as well as resistant bacteria, in animal products and waste is likely to have an impact on the development of bacterial resistance in the wider environment. Given these concerns, it is not surprising that the rise in the appearance of antibiotic resistant microbes has been attributed, at least partly, to the abundant use of antibiotics in agriculture. An important link between farm animals and the environment is on-farm waste treatment systems.

Another limitation to these studies is that antibiotic resistance research has been focused on cultivable clinical isolates, which may represent only a small fraction (<1.0%) of the actual microbial diversity especially in environmental samples. In the present study, we utilized a cultivation-independent molecular ecology approach to: 1) evaluate and quantify the impact of antibiotic usage in the farms for growth promotion and prophylaxis on the dissemination of tetracycline resistance genes in the feed, feces and treated waste; 2) estimate the potential of different waste treatment systems to reduce the level of antibiotic resistance, in six farms included four commercial farms (HF, LF, SP:On-Site, SP:Off-Site), one research farm and one organic farm. To the best of our knowledge, this is the first molecular ecology based study for quantification of tetracycline determinants in environmental samples, which would help to determine if the suggested link between the use of antibiotics in farm animal diets and the spread of antibiotic resistance in the environment is real.
Conventional PCR was used to determine the genotype of tetracycline resistance genes in total DNA of swine feed, fecal and environmental samples. This was followed with real-time PCR analysis to calculate the copy number of different tetracycline resistance genes. As an internal control, a primer set targeting a conserved region of 16S rRNA gene molecule was used to determine the bacterial concentration in each sample. Feed was found to be genetically contaminated at very low levels with a diversity of tetracycline resistance genes on all farms, including the organic farm.
Considerable variation was observed among different tetracycline resistance genes when comparing 1)- the range of genes within farms 2)- a single gene between farms and 3)- the whole chain (food  well  lagoon  soil). Tetracycline resistance genes within the RPP class tetQ and tetM were most frequently distributed. Groundwater in SP:On was significantly contaminated with a wide diversity of tetracycline resistance determinants whereas for HF and OF there was less diversity of determinants with very low levels. Our results demonstrated that land application of swine waste diluted the levels of different tetracycline resistance genes sometimes to undetectable levels.
In addition, a wide range of isolates was obtained from soil samples after land application of swine manure. Antibiotic sensitivity of these isolates was initially evaluated using broth dilution and then followed using the E-test sensitivity strips. These antibiotic resistant isolates were then identified by PCR amplification of the V3 region of the 16S rRNA gene to provide rapid and sensitive identification to genus and species level. A diverse range of isolates was described that includes members of the alpha-, beta- and gamma- Proteobacteria, Bacteroidetes and Firmicutes from both the high and low Gram-positive bacterial groups. Species identification was linked to an MIC value for each of the isolates. These isolates differed considerably in their sensitivity to erythromycin and tetracycline. Our initial screening for the erythromycin genotype that results in the antibiotic resistance phenotype has been initiated using a family of genes from the clinically relevant methylase genes but so far only one isolate has given a positive signal for any of the erm genes screened. It is likely that some of these genotypes may represent novel resistance genes that have not been described previously.
In conclusion, these two studies applying both molecular cultivation-independent, as well as classical cultivation, based approaches confirm that a wide variety of tetracycline resistance determinants exist in swine manure from animals fed with growth promoting levels of antibiotics although considerable variation exists in both the frequency and level of detection. Swine waste can be managed to reduce levels of resistance determinants. However, these resistance determinants are able to disseminate after land application of swine manure and ultimately can penetrate into surface and underlying groundwater supplies. This constitutes a potential risk since groundwater provides a substantial part of the public water supply in the United States. Thus, along with other ways of acquiring antibiotic resistance, such as consumption of tainted food, the occurrence of antibiotic resistance genes in drinking water provides a possible way for antibiotic resistance to enter the animal and human food chain.
Contact information: Dr Rod Mackie, 458 Animal Sciences Laboratory, University of Illinois, 1207 W. Gregory Drive, Urbana, IL 61801.  Tel: (217) 244-2526 Fax: (217) 333-8286 E-mail: r-mackie@uiuc.edu