Effect of River Landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG)
Introduction
Pharmaceutical compounds including antibiotics, hormones, and steroids are widely used to prevent and/or treat diseases and to promote animal growth in livestock production facilities, such as concentrated animal feeding operations (CAFOs). In particular, one half of the 50 million pounds of antibiotics produced each year in the US is used for agriculture and 90% of these are used for growth promotion (Levy, 1998). A significant amount of these antibiotics (up to 75%; Elmund et al., 1971; Feinman and Matheson, 1978) are excreted as active metabolites. Thus, animal waste presents a major potential source of antibiotic input to the environment (Haapapuro et al., 1997; Sweeten, 1992). Discharge of treated municipal, hospital, and veterinary wastewaters present additional sources. A recent study conducted by the United States Geological Survey in 1999 and 2000 indicated that, out of a network of 139 streams across 30 states, 95 contained antibiotics (Kolpin et al., 2002). Now that antibiotic contamination in the environment has been confirmed, a growing concern is that the release of antibiotics into the environment may contribute to the emergence of strains of disease-causing bacteria that are resistant to even high doses of these drugs (ASM, 2002; Chee-Sanford et al., 2001; Goni-Urriza et al., 2000; Guardabassi et al., 1998). Therefore, there is a need to better understand the relationship between antibiotics in the environment and their ultimate impact on the emergence of microbial resistance.
Recently, the occurrence of four classes of antibiotics (tetracyclines, sulfonamides, macrolides and ionophores) has been investigated in environmental samples such as natural water, animal and human wastewaters, irrigation ditches, and river sediments (Kim and Carlson, 2005; Yang and Carlson, 2003, Yang and Carlson, 2004a, Yang and Carlson, 2004b; Yang et al., 2004). In particular, within the mixed-landscape of the Cache La Poudre (Poudre) River watershed, the presence of these four classes of antibiotics in water and sediments were found to relate to urban and agricultural activity. Generally, the number of antibiotics present and their concentrations increased as agricultural and urban activates increased along the Poudre River from its pristine origins in the Rocky Mountains, where no antibiotic contamination has yet been found. Relationships could also be identified between antibiotics known to be specific to human or agricultural use, and their most likely inputs (Yang and Carlson, 2003, Yang and Carlson, 2004a, Yang and Carlson, 2004b; Yang et al., 2004). Based on these results it is clear that studies are needed to assess the impact of antibiotics released into the environment and their potential ultimate effect on human health and the environment. In particular, there is a need for the development and application of methods to quantify actual resistance genes in the environment. The quantity of antibiotic resistance genes (ARG) may serve as a good indicator of the bioavailability of antibiotics in the environment. Also, as suggested recently by Rysz and Alvarez (Rysz and Alvarez, 2004), the genes themselves could be considered to be “pollutants,” as their wide-spread dissemination is clearly undesirable.
The purpose of this study was two-fold: (1) to develop methods for quantifying ARG in environmental samples; and (2) to apply these methods in assessing and quantifying ARG present in the sediments of the Poudre River, a model river system that is zonated with respect to pristine, urban, and agricultural impacts. In particular, quantitative real-time polymerase chain reaction (Q-PCR) methods targeting tetracycline and sulfonamide resistance genes were developed and applied. Both of these classes of antibiotics are widely used in both animals and in humans (Aminov et al., 2001; Huber, 1971; Pereten and Boerlin, 2000). A culture-based approach was also implemented as a broad method of quantifying microbial resistance, including resistance incurred by genes that may not have been previously described. Finally, various tetracyclines and sulfonamides were also quantified in the sediment samples in order to compare the concentrations of antibiotics observed with those of ARG.
Section snippets
Study area and sampling sites
The Poudre River in northern Colorado has many unique attributes that make it an excellent model watershed to compare pristine, urban, and agricultural impacts. These attributes include: (1) the semi-arid nature of the front range of Colorado which results in only a small number of tributaries to the Poudre River; (2) the predominance of point sources in the urban landscape of Fort Collins versus non-point sources in the agricultural areas outside of the city; and (3) the source of the river
Heterotrophic plate counts on antibiotic-selective media
In terms of absolute numbers of resistant CFU per gram of sediment summed for the eight antibiotic R2A media, the pristine site was found to be the lowest at 1.9×103 CFU/gram sediment for the high-flow sampling event and 5.7×104 CFU/gram sediment for the low-flow event. Sites 2 and 4 were the next lowest, with site 2 at 2.0×105 and 4.4×106 CFU/gram sediment for the high-flow and low-flow events, respectively, and site 4 at 1.0×105 and 1.6×107 CFU/gram sediment, respectively. Sites 3 and 5 were the
Applicability of the methods
The Q-PCR methods developed in this study provide a useful means to quantify ARG in the environment and thus bioassay and monitor the relative impact of antibiotic use. Q-PCR also allows for direct quantification of ARG as a new class of “pollutants” in and of themselves. While all three levels of data gathered in this study (CFU, PCR presence/absence assay, Q-PCR) revealed similar overall trends, there were some differences observed. For example, the CFUs on control plates without antibiotics
Conclusions
This study demonstrates a relationship between human and agricultural activity and elevated levels of ARG in river sediments using quantitative molecular tools. This is an important step in developing a paradigm for ARG as environmental contaminants. Further study is needed in order to better understand the relationship between the use of antibiotics in humans and animals and the spread of ARG. In particular, the relationship between the transport of antibiotics and the transport of ARG in the
Acknowledgments
This research was financially supported by the US Department of Agriculture National Research Initiative (NRI) Watersheds Program.
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