Antibiotic-resistant bacteria in crop fields

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A variety of antimicrobials are added to swine feed or water by the swine industry, often at sub-therapeutic levels for growth promotion. These pharmaceuticals can boost the daily growth of pigs and reduce animal death rates at swine facilities, thereby enhancing overall production efficiency and increasing profitability.  Tylosin is among the most widely used of the macrolide class of veterinary antibiotics by the swine industry.

The occurrence of antibiotic-resistant bacteria and antibiotic-resistant genes near swine operations has been reported. However, very little is known about their release from tile-drained fields receiving swine manure application. Data from full-scale field experiments will provide valuable information about the relative retention and transport of ARB and ARGs in manure-amended, tile-drained agricultural fields in the corn/soybean cropping systems prevalent in the Upper Midwest.

Studying the Fate and Transport of Antibiotics, Antibiotic-resistant Bacteria and Resistance Genes

Four plots were sampled at Iowa State University’s Northeast Research Farm near Nashua, Iowa.  These agricultural plots are instrumented with a subsurface water-quality monitoring system operational since 1988. Each 1-acre plot is drained separately with subsurface drain lines installed in the center of the plot.   

The plots encompass two tillage practices: chisel plow and no-till. Liquid swine manure was injected on one of each tillage type while the second of each type received urea ammonium nitrate and served as a control for assessing background levels.  A summary of the plots selected for sampling is presented in Table 1. 

Table 1.  Northeast Research Farm plots selected for sample collection, including ID numbers, crop, tillage practice and nutrient-management history  

Plot Crop Tillage Nitrogen Management
23 Corn CP Fall injected swine manure at 150 lb total N/acre
24 Corn CP Spring preplant spoke injected UAN at 150 lb N/acre
25 Corn NT Fall injected swine manure at 150 lb total N/acre
34 Corn NT Spring preplant spoke injected UAN at 150 lb N/acre with cover crop

Manure was obtained from a commercial swine facility currently including tylosin in the feed at sub-therapeutic levels for growth promotion. Manure was injected with a shank, forming a band of treated soil at agronomic rates of 150 pounds nitrogen per acre. Following manure application, composite soil samples were collected from each manure plot — three from the manure band and three from the area between the manure bands — and three samples were also collected from the control plots.

A second set of soil samples was collected the following April from the same locations. The tile lines flowed from April 26, 2011, until approximately July 6, 2011, when flow had virtually ceased. Soil samples were collected on a weekly basis and following major rainfall events during this period.

Enterococci and Enterococci Resistance to Tylosin in Manure, Soil and Water Samples

Enterococci (Enterococcus species) are important organisms because they are used to indicate the potential presence of pathogens in waters by the U.S. Environmental Protection Agency. A recreational water body is considered to be contaminated when the enterococci concentration is greater than 33 colony forming units per 100 ml of water. In addition to measuring the total enterococci, tylosin-resistant enterococci also were measured.

The concentrations of tylosin-resistant enterococci were the lowest in tile-drainage water and the highest in the swine manure. The average concentrations of total and tylosin-resistant enterococci in manure samples were 565,706 (±304,059) cfu per gram and 396,529 (±292,956) cfu per gram, respectively, which shows that 70 percent of the enterococci in manure samples were resistant to tylosin. The concentrations of enterococci and tylosin-resistant enterococci in soils were generally higher in the injection zone and lowest in the control plots. On average, 8 percent of the bacteria from the soil samples collected within the manure bands in both fall and spring were resistant to tylosin.

click image to zoomCropsFigure 1. Total enterococci concentrations in tile water in cfu per 100ml. The EPA Water Quality Standard for recreational water samples (<33 cfu per 100 ml for a 30-day geometric mean) is shown for reference. Tile drainage water showed a wide array of enterococci concentrations relative to time (Figure 1). As the time between manure application and water sample collection increased, a decrease in bacteria concentrations in all tillage and manure application scenarios was expected. This was not the case, however, as the total enterococci concentrations peaked in late June in three of the four plots.

Tylosin-resistant enterococci were found in only 16 percent of the tile-drainage water samples. Of the water samples from the control plots in the study, 9 percent had detectable resistant enterococci. The first year of monitoring found that a very small fraction of tylosin-resistant enterococci were transported to tile waters.

Macrolide-resistant Genes in Manure, Soil and Water Samples

Quantitative PCR analysis was conducted on DNA extracted from manure, soil and water samples for the analysis of erythromycin resistance methylase (erm) genes which confer resistance to tylosin, erythromycin and other macrolide antibiotics. While many different genes are responsible for antibiotic resistance, the erm genes are among the most commonly acquired by many different strains of bacteria (Chen et al. 2007), including enterococci, and more than 32 erm genes have been identified.

Three target genes — ermB, ermF and ermT — were analyzed.  ermT was not detected in manure, soil or water.  The most prevalent macrolide-resistant gene in this study was ermB, followed by ermF.  Concentrations of ermB and ermF genes in manure averaged 1.31 x 107 (±4.16 x 104) copies per gram and 3.50 x 103 (±3.51 x 102) copies per gram, respectively.  The ermB concentration was four orders of magnitude higher than ermF concentration in this study. 

click image to zoomCropsFigure 2. Copy number of ermB genes per gram (dry weight) for soil samples from fall and spring samplings Figure 2 shows the copy numbers of ermB per gram of soil.  As expected, tillage did not significantly impact soil copy number concentrations of ermB  or ermF.  The difference between seasons for ermB averaged a 40 percent increase for the untreated samples, whereas the manure bands averaged a 90 percent decrease.  The difference between seasons for ermF varied greatly for the untreated samples, whereas the manure bands averaged a decrease of six orders of magnitude.

Seasonal impacts were statistically significant for ermF but not for ermB.  The occurrence in the controls could potentially be caused by naturally occurring tylosin in soil, which proliferated the development of resistance.  With the exception of the fall manure bands, all ermF copies were less than ermB, which was expected based on the results of the manure samples.  

The macrolide resistance genes in water ranged from 150 to 7.6 x106 copies per 100 ml water.   The analysis indicated the presence of the targeted genes and does not identify the microorganism in which resistance developed.  Because low counts of total and tylosin-resistant enterococci were observed, it is assumed that most of the targeted genes were not present in enterococci; however, it is unknown if the erm genes are present in other pathogenic organisms, which could potentially present a risk to the health of animals or humans. 

Conclusions

Based on the preliminary results of this study, a few encouraging conclusions can be made for pork producers regarding the presence of ARGs and tylosin-resistant enterococci in manure, soil and tile drainage water.  Tylosin-resistance genes were detected in elevated concentrations in soil in the manure injection zone, compared to soil outside the manure band or in soil not treated with manure. Future studies will include soil sampling from areas with no history of manure application. 

The transport of tylosin-resistance genes in drainage water was at concentrations much less than in the manured soil, and drainage water from manured soils and non-treated soils were similar in their concentrations of tylosin-resistance genes. 

Concentrations of enterococci in tile water are very low and only exceeded the geometric mean for recreational waters nine times, with 33 percent of these exceedances occurring in tile flow from the control plots.   While the concentration of ARGs is much higher in water relative to the concentrations of detected resistant enterococci, it is unknown in which organisms these genes reside. Future studies are recommended to further investigate the presence of antibiotic-resistant genes in other pathogenic organisms. 

The true influence of soil-management practices such as tillage on antibiotic losses is not well understood, and a longer period of study is recommended to assess the fate and transport of these contaminants over a larger range of climatic conditions as the results presented here are from a period of below average rainfall.  Future work is recommended to capture the event hydrograph for better assessment of land-management practices on contaminant transport during precipitation events. 


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