Presentation Abstract

Mechanisms of Tetracycline Resistance Development in the Environment as Detected by Real-Time PCR
David Graham
University of Kansas, Lawrence, KS

Strong evidence exists suggesting that the use of antibacterial agents in agriculture is increasing the level of antibiotic resistance among microbial pathogens. This presentation focuses on four studies assessing relationships between tetracycline exposure and antibiotic resistance development in the environment. The goal of this research project was to examine the transport pathway from the point of antibiotic use to possible downstream exposure to determine rates and mechanisms by which resistance are gained or lost in different settings. The study used laboratory-, mesocosm-, and field-scale systems (both in rivers and operating feedlot lagoons) and both classical microbiological techniques and quantitative real-time polymerase chain reaction (PCR) for tracking resistant organisms and genes in the systems. Tetracyclines were chosen for study because it and its derivatives are used extensively in clinical or veterinary applications, and the genetic basis of resistance is well established, allowing the use of quantitative real-time PCR for determining quantifying resistance gene numbers in exposed systems.

Two watersheds with variable land use, five feedlots with eight wastewater lagoons, and a series of controlled laboratory and mesocosm experiments were examined to fulfill the goals of this study. In all studies, real-time PCR probe-primer sets for the tet(O) , tet(W), tet(Q), tet(M), tet(A), and tet(B) resistance genes were used to quantify resistance gene numbers in organisms as a function of antibiotic use patterns and receiving water or other environmental conditions. ¹ The full-scale river and feedlot studies showed that elevated resistance genes in receiving waters were seasonal and primarily resulted from the transport of resistant organisms away from the point of veterinary use rather than via in situ resistance development. ^2,3 Ambient tetracycline levels in receiving waters, however, usually did not correlate with resistance gene numbers; therefore, the in situ detection of antibiotics is often a poor marker of environmental exposure.

These studies led to further work assessing how resistance genes move in the environment after release, how long genes were retained after release (and why), and ancillary environmental effects caused by tetracyclines, such as impairment of aquatic plants. ⁴ Followup laboratory and mesocosm experiments were performed to track key genes and whole-organism resistance in different types of receiving water settings, including light-exposed, dark, high-tetracycline, low-tetracycline, and waters with elevated natural bacterial communities. In general, ambient light supply dominated the fate of both resistant organisms and resistance genes in downstream waters. Resistance gene half-lives were often two to three orders of magnitude greater under dark treatments compared with all other exposures. Some individual resistant bacteria were enriched after initial release into receiving waters, although this was not the norm with most resistance genes and organisms typically dying very rapidly upon release into surface waters. Photodeactivation appeared to be a key mechanism for “resistance” die-off in most systems. tet(W) and tet(M) had the longest half-lives among all genes tested (in the order of hours), suggesting that these genes might be possible biomarkers for resistance-gene exposure in future work.

Results indicate that tetracycline resistance is developed primarily at the point of use rather than in the environment, although individual resistant organisms can be enriched in some receiving water scenarios. Generally, resistance gene “die-off” is most rapid in light-exposed aquatic systems, which suggests that photo-deactivation is important for “resistance” reduction in environmental systems. Work continues on the fate and transfer of resistance genes and organisms in aquatic systems, with the development of a new mathematical receiving-water model being underway to help predict downstream resistance impacts below point and nonpoint resistance sources.

References

1. Smith M.S., Yang R.K., Knapp C.W., Niu Y., Peak N., Hanfelt M., Galland J.S, and Graham D.W . Quantification of tetracycline resistance genes in feedlot lagoons using real-time PCR. Appl Environ Microbiol 2004; 70:7372-7377 .
2. Peak N., Knapp C.W., Yang R.K., Hanfelt M.M., Smith M.S., Aga D.S., and Graham D.W. Relationships between antibiotic use practices and resistance gene levels in feedlot wastewater lagoons as detected by real-time PCR. Environ Sci Techno (submitted, 2005).
3. Knapp C.W., Busch C., Miller J., Hanson M.L., Keen P., Hall K., and Graham D.W. In situ resistance development to oxytetracycline in aquatic systems. Antimicrob Agents Chemother (submitted, 2005).
4. Hanson M.L., Knapp C.W., and Graham D.W. Field assessment of oxytetracycline exposure to the freshwater macrophytes Elodea densa (Planch.) and Ceratophyllum demersum L. Env Poll (in revision, 2005).

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