The Occurrence of Antibiotic Resistant Bacteria in Manure, Manure-Applied Fields, and Pets on the Farm

Final Report for LNC02-214

Project Type: Research and Education
Funds awarded in 2002: $99,835.00
Projected End Date: 12/31/2003
Region: North Central
State: Minnesota
Project Coordinator:
Dr. Satish Gupta
University of Minneota
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Project Information

Summary:

There was greater prevalence of antibiotic resistance bacteria (ARB) in manure from farms that use antibiotics sub-therapeutically than the farms that do not. This was more so for tetracycline than for tylosin or monensin. The trend was present for all three animal species; swine, turkeys, and cattle. However, this resistance did not appear to permeate to manure applied fields or dogs. Lack of trend in soil and dog fecal samples may be due to large variability (small number of farms). Resistance profile of ARB isolates from farms that use antibiotics sub-therapeutically showed higher levels of resistance to 20 other antibiotics.

Introduction:

Since their discovery, antibiotics have been instrumental in treating infectious diseases that were previously known to kill humans and animals. However, it has now become clear that widespread use of antibiotics is not without problems (Halling-Sørensen et al., 1998; Jørgensen and Halling-Sørensen, 2000). The major concern is the development of antibiotic-resistant microorganisms, which are difficult to treat with existing antibiotics (Ford, 1994; Herron et al., 1997). Furthermore, increasingly more microorganisms are becoming resistant to multiple antibiotics (Goldburg, 1999).

According to one estimate, two million pounds of antibiotics were produced in the U. S. in 1954 compared to more than 50 million pounds being produced each year currently (Environmental Media Services, (EMS) 2000). Although most of these antibiotics are used for the treatment of infections in humans and animals, a significant portion is used as a supplement in animal feed to promote growth of food-producing animals. According to the Institute of Medicine, 32% of the antibiotics produced in the U.S are used as feed supplements (Shea, 2003). However, Union of Concerned Scientists (2001) contends that as much as 78% of the antibiotics produced are being used for non-therapeutic purposes in agriculture. The use of antibiotics in animal feed helps increase the animal’s ability to absorb feed and thus reach market weight quicker. In addition, supplementing antibiotics in animal feed counteract the effects of crowded living conditions and/or poor hygiene in intensive animal agriculture (EMS, 2000).

Antibiotics commonly used as feed additive for animals include aureomycin, bacitracin, bambermycins, erythromycin, lincomycin, monensin, oxytetracycline, penicillin, tylosin, and virginiamycin (Church and Pond, 1982). The antibiotic dose varies from 1 to 200 g per ton of feed depending upon type and size of the animal and the type of antibiotic (Kumar et al., 2005). Most of the antibiotics added to animal feed are excreted in urine and manure, and as much as 80% may pass through the animal unchanged (Levy, 1992). Several groups contend that sub-therapeutic use of antibiotics in animal production is leading to increased presence of antibiotic resistant microbes in the environment including manure, soil, and water (Levy, 1992; Union of Concerned Scientists, 2001; Shea et al., 2001).

Land application of manure is a common practice in many parts of the U.S. With emphasis on sustainable farming and demand for organic foods, there is even greater use of manure in current food production. The manure is land applied mainly because of its value in supplying nutrients to crops but in some cases it is also a means of disposing unwanted waste. According to Environmental Defense (2001), nearly 1 billion tons of animal waste is produced each year in the United States. Once land applied, the presence of antibiotics and antibiotic resistance bacteria in manure can cause selection of antibiotic resistance bacteria (ARB) in soil.

Ability of pathogens to counteract the effectiveness of antibiotics leads to higher medicare costs. For example the cost of treating a patient with tuberculosis increases from $12,000 for a patient with a drug-susceptible strain to $180,000 for a patient with a multidrug-resistant strain (www.cspinet.org). In a report prepared by the U.S. Congressional Office of Technology Assessment (1995), it was concluded that the antibiotic resistance of just six different strains of bacteria has increased hospital costs by 1.3 billions in 1992 dollars.

The groups most affected by continuous increased antibiotic resistance in microbes are the infants and children, senior citizens, and cancer, HIV/AIDS, and organ transplant patients (Shea et al., 2001). This is because their immune system is not fully developed or they have a weak immune system and thus need a full range of antibiotics to ward off infections.

The goal of this study was to determine the role of sub-therapeutic feeding of antibiotics in food animals on increased microbial resistance in manure, soil where manure has been applied, and pets on the farm. The premise underlying the inclusion of pets in this study is to assess the potential spread of ARB to rest of the environment including humans on the farm.

LITERATURE REVIEW

About two years ago, antibiotics were found in a lake in Ohio (News Report on a TV Network, Feb. 2000). It is unknown how these pharmaceuticals found their way to the surface water. A possible route may be that antibiotics came to the lake with surface runoff from fields where manure had been applied. In 2003, there has been a comprehensive report on the National Public Radio (http://americanradioworks.org/features/farm/antibiotics.html) about the use of antibiotics in animal production and public’s concern about this practice. Because of the recent anthrax terror, there is also heightened awareness of regular antibiotic use in animal production and its consequences (Hilts, 2001, http://www.nytimes.com). Since manure, soil, and ground and surface waters are not regularly tested for antibiotics in the U.S., it is unknown to what extent this type of contamination exists in manure, soils, lakes, rivers, and groundwater in the United States.

Some data are available on the occurrence, fate, and effects of pharmaceutical in the environment (Strauch 1987; Halling-Sorensen et al., 1998; Kumar et al., 2005). However, most of this work has been done in European Union countries and some of this literature is contradictory. Much of the concern has been related to pathways through which these pharmaceutical drugs find their way to the environment and their adverse effects on biological signals and the genetic structure of the ecosystem (Jørgensen and Halling-Sørensen, 2000). Another major concern is that widespread use of antibiotics may lead to new strains of bacteria that are resistant to these and other antibiotics and in turn result in untreatability of livestock diseases (Solomons, 1978; Hirsh and Wiger, 1977). During the 15 years period from 1975-1991 incidences of methicillin resistant Staphylococcus aureus (MRSA) isolates in U.S. hospitals has increased from 2.4% to 29% (www.cspinet.org). A potentially more dangerous scenario is the possible transmission of such strains to humans resulting in untreatable human diseases. Once land applied, the presence of antibiotics and antibiotic resistant microbes in manure can further lead to selection and propagation of ARB in the terrestrial environment and then to rest of the environment through non-point source pollution.

Potentially, there are three mechanisms through which antibiotic resistance might develop in bacteria in the environment: (i) Continuous pressure from sub-therapeutic feeding of antibiotics leading to selection of ARB in the guts of animals, (ii) Microbes might acquire antibiotic resistance in-situ when antibiotic laced manure is applied to farms on a continuous basis, and (iii) Transfer of antibiotic resistance genes through genetic elements such as plasmid, integrons, transposons from ARB to sensitive bacteria. Potentially, one or all three mechanisms can act together to affect the prevalence of antibiotic resistant microbes in the environment.

The limiting data on the spread of antibiotic resistance from animals that have been fed sub-therapeutic levels of antibiotics to the environment is generally conflicting. Nijsten et al. (1996a, b) reported that E. coli resistance was significantly lower in pig farmers than their pigs and the evidence for a common pool of plasmids among pig farmers and their pigs was inconclusive. Hunter et al. (1994) found a widespread dissemination of apramycin resistant plasmids in E. coli between the pigs and the stockman. These authors even found apramycin-resistant Klebsiella pneumoniae from the stockman’s wife despite the fact that she had no direct contact with the pigs. Earlier Hunter et al. (1992) reported on the possible transfer of apramycin-resistant plasmids from E. coli to Salmonella typhimurium in calves.

With the recent availability of gene tracing techniques, there has been stronger evidence on the spread of antibiotic resistance from animal farms to the environment. Chee-Sanford et al. (2001) showed that there was some transfer of tetracycline resistance gene from manure lagoons to indigenous microbiota at two swine farms in Illinois. These authors also concluded that tetracycline resistance determinants were seeping into underlying groundwater as far as 250 m downstream from the lagoons. Based on the resistance patterns of E. coli in turkeys, turkey farmers, and turkey slaughterers and in broiler, broiler farmers, and broiler slaughterers, van den Bogaard et al. (2001) showed a strong indication on the transmission of resistant clones and resistant plasmids from poultry to humans.

Perhaps a most comprehensive overview of this problem has come from The Committee on Drug Use in Food Animals from the National Research Council (1999). The Committee concluded, in part, that “a data driven scientific consensus on human health risk posed by antibiotic use in food animal is lacking”. The authors of this report recently put together a comprehensive review on the antibiotic use in agriculture and its impact in the terrestrial environment (Kumar et al., 2005). A copy of that review is attached with this report.

Project Objectives:

The objectives of this study were: (1) to quantify the extent of antibiotic resistant bacteria in manure and manure-applied fields for three different types of animal (swine, beef, and turkeys) production systems, (2) to determine whether or not microbial antibiotic resistance is higher from farms that use sub-therapeutic levels of antibiotics (AU) vs. farms that do not use sub-therapeutic levels of antibiotics (NAU), (3) to assess whether microbial antibiotic resistance permeates to domestic pets that live on these farms, and (4) to identify manure and soil management practices that may lessen the impact of antibiotic use on antibiotic resistance in microbes on the farm.

Cooperators

Click linked name(s) to expand
  • Yogesh Chander
  • Sagar Goyal
  • Kuldip Kumar
  • Helene Murray
  • Ashok Singh

Research

Materials and methods:

The procedure for determining whether or not antibiotic feeding of food animal increases antimicrobial resistance in the environment involved collecting manure, soil where manure has been applied, and dog feces samples from AU and NAU farms for three animal species (swine, turkeys, cattle); screening the samples for antibiotic resistance bacteria to three commonly used antibiotics (tetracycline, tylosin, monensin); identification of antibiotic resistance bacteria; and then determination of the minimum inhibitory concentrations (MIC) of selected species of antibiotic resistance isolates. NAU farms were those which have not used antibiotics sub-therapeutically at least for the last three years. From some AU and NAU farms, we also collected samples from their compost piles. This was to test whether or not composting helps reduce ARB in manure. In addition to AU and NAU farms, we also collected soil and dog feces samples from farms where no manure had been applied for at least 10 years. All the farms in our study were within Minnesota.

Sample collection: From each farm, samples of manure, soil (where manure was applied as fertilizer), and fresh dog feces (less than 24 hrs old) were collected in sterile polypropylene containers. Samples were brought to the laboratory and stored at 40C until screened, usually within 24 hrs of collection.

Screening of samples: The screening procedure for quantifying the presence of ARB was that of National Committee for Clinical Laboratory Standards (NCCLS, 2000). Briefly, the procedure involved making a 10% suspension by mixing 1g of sample in 9 mL of buffered peptone water (BPW, pH 7.0) followed by vortexing to homogenized the suspension. Serial 10-fold dilutions of this suspension were then made in BPW. For screening, 50 ml of each dilution (-3 to -8) were plated on solidified Muller Hinton II agar (MHA; Becton Dickinson, Sparks, MD) plates containing tetracycline (20 mg ml-1) and tylosin (10 mg ml-1) and monensin (6 mg ml-1). All antibiotics used were obtained from Sigma, St. Louis, MO. For control, same dilutions were simultaneously plated on MHA plates without antibiotics. After inoculation, plates were incubated at 370C for 24 hrs and the number of colony forming units (CFU) was recorded manually. Percentage ARB in each sample for each antimicrobial agent was calculated as the number of CFU growing on the plate containing antibiotic divided by the number of CFU growing on the control (antibiotic-free) plate X 100 (van den Bogaard et al., 2000).

Identification: After counting, phenotypically different colonies (4-5 colonies per plate) of ARB were picked from each antibiotic plate, purified, and stored on tryptic soy agar (TSA; Becton Dickinson, Sparks, MD) slants. Isolated bacteria were identified using API 20E and API 20NE identification strips (BioMereiux, Paris, France). In this study, only the Gram negative bacteria were identified.

Minimum inhibitory concentrations (MIC): Susceptibility of bacterial isolates to various antibiotics was determined using the Sensititer® CMV1 ABPF antibiotic sensitivity testing plates (TREK Diagnostics, Cleveland, OH). These are 96-well microtiter plates containing a panel of 21 antibiotics at various concentrations as per the recommendations of NCCLS (NCCLS, 2002). Plates were inoculated with 0.5 McFarland adjusted inoculum and then incubated at 370C for 24 hrs. After incubation, MIC was determined using the Sensititer® plate reader. MIC was calculated as the minimum antibiotic concentration that completely inhibited bacterial growth. MIC breakpoint concentrations were supplied by the manufacturer of the Sensititer® kits.

Statistical analysis: Statistical analysis of the antibiotic resistance data was done using the SAS statistical package (SAS Institute, 1999). The experimental design for the analysis of variance (ANOVA) was a split-plot design. Primary factors were the antibiotic use and the animal species. Secondary factor (split) was the matrix (manure, soil, dog feces). ARB data was analyzed separately for each of the three antibiotics. Significance of differences among treatments was tested at p≤0.05. To achieve normality, data was transformed by taking the arcsin of the square root of percent antibiotic resistance. The ANOVA was performed on the transformed data. Reverse transformations were also performed on the mean values obtained from the ANOVA. Difference in antibiotic resistance profile of selected bacterial isolates between the AU and the NAU farms was done using the student t-test.

Research results and discussion:

A. Prevalence of ARB

(i) Antibiotic use on different animal farms
At the time of sample collection, animal producers were surveyed for the types of antibiotics being used sub-therapeutically on their farm as well as for their manure management practices. In Table 1 is a list of commonly used antibiotics by animal type. Tylosin was the most commonly used antibiotic on the swine farms. The concentration of tylosin varied from 40g T-1 of feed in nursery and grower hogs to 400g T-1 of feed in finishing hogs. Other sub-therapeutic antibiotics used on the swine farms were chlortetracycline (60 g T-1), bacitracin (30-60 g T-1) and sulphonamide. Use of lincomycin as feed additive was reported on only one farm. All antibiotics were mixed with the feed.

On turkey farms, the most common sub-therapeutic antibiotics used were oxytetracycline, neomycin, monensin and virginiamycin. The antibiotic dose ranged from 20g T-1- 60 g T-1 of feed. In addition to mixing with the feed, tetracycline antibiotics (oxytetracycline and chlortetracycline) were also supplied with drinking water. All the AU cattle farmers surveyed in the present study used only the ionophore antibiotics for growth promotion.

(ii) Prevalence of Tetracycline Resistance Bacteria on Animal Farms
In general, prevalence of tetracycline resistant bacteria was significantly higher on AU than NAU farms (Table 2, Fig. 1). There was also some difference in tetracycline resistance between different species but only at a higher level of significance (p= 0.10). Tetracycline resistance was significantly higher at turkey farms compared to swine farms (Fig. 2). However, there was no difference in tetracycline resistance between turkey and cattle farms, or swine and cattle farms.

There were significant differences in prevalence of tetracycline resistance in bacteria from three different matrices (Table 1). Tetracycline resistance was higher in manure and dog feces bacteria compared to the bacteria from soil where manure had been applied (Fig. 3). Most of the tetracycline resistance in manure bacteria was in samples collected from the AU farms (Fig. 4). However, there was no difference in tetracycline resistance of dog fecal bacteria between the AU and NAU farms (Fig. 4). This would suggest that tetracycline resistance did not permeate to dogs on the AU farms. Lower resistance in soil bacteria (Figs. 3 and 4) could be because of the difficulty of culturing all soil bacteria and/or low survivability of manure bacteria in soil. Another possible reason may be that the development of antibiotic resistance from genetic exchange (between native soil microorganism and manure bacteria) is a slow process compared to direct selection pressure in the gut of the animal. In any case, these numbers suggest that tetracycline resistance from manure bacteria did not spread to soil bacteria when manure was land applied.

There were also 2 two-way and 1 three-way significant interactions between antibiotic use, matrix, and animal species (Table 1). Figure 5 shows the differences in the prevalence of tetracycline resistance in bacteria from three different types of samples and three different animal species. Tetracycline resistance in turkey and swine farms was mainly due to higher resistance bacteria in manure samples whereas a majority of tetracycline resistance at cattle farms was in dog fecal samples and that too from the AU farms (Table 3a). Prevalence of higher levels of tetracycline resistant bacteria in manure samples from AU swine farms can be associated with the increase use of tetracycline as feed additive on swine farms. A report by the Union of Concerned Scientist (2001) suggests that the use of tetracycline as feed additive in swine production has increased by 28% in last ten years. The reasons for the prevalence of significant higher tetracycline resistance in dog fecal bacteria from AU compare to NAU cattle farms (Table 3a) is not apparent considering that tetracycline was not used on cattle farms. It is possible that this difference in antibiotic resistance may be an effect of other antibiotics used on AU cattle farms.

Similar to swine farms, tetracycline resistance was also significantly higher in manure bacteria from AU turkey and cattle farms (Table 3a). Tetracycline (chlortetracycline and oxytetracycline) antibiotics are routinely used both in feed and water for turkeys. This is possibly the main reason for higher prevalence of tetracycline resistant bacteria in turkey manure from AU farms (Fig. 5). Although tetracycline resistance in cattle manure bacteria was also significantly higher from the AU than the NAU farms, the resistance levels in cattle manure for any given type of farms were much lower than those in swine and turkey manure (Table 3a, Fig. 5). This is expected considering that tetracycline is not as widely used as feed additives for cattle as compared to swine or turkey production systems. According to the report prepared by Union of Concerned Scientists (2001), the use of tetracyclines in cattle feed has decreased by 50% over the last couple of years. The reason for significant differences in tetracycline resistance between AU and NAU cattle farms is not apparent.

(iii) Prevalence of Tylosin Resistance Bacteria on Animal Farms

Statistical analysis on the prevalence of tylosin resistance in bacteria isolated from AU and NAU farms is given in Table 4. These results indicate that there is no difference in the prevalence of tylosin resistant bacteria between the AU and the NAU farms. This may be because tylosin has being used as a growth promoter for longtime and over the years many bacterial species may have developed resistance to this antibiotic. Except for turkey manure, tylosin resistance was generally higher than the tetracycline resistance for all three spices and three types of samples (Table 3a). These results are in line with each antibiotic’s use in swine and turkey production systems, i.e. higher tetracycline use in swine production compared to higher tylosin use in turkey production. Although tylosin is used as a feed additive in turkey production, the level of usage is lower than that of tetracycline or virginiamycin. This may be the reason for lower tylosin resistance (Fig. 7) in turkey manure than tetracycline resistance (Fig. 5) in turkey manure samples.

There was also no difference in the prevalence of tylosin resistance between different animal species. However, there were significant differences in tylosin resistance between three different matrices (p=0.04). Tylosin resistance was significantly higher in bacterial isolates from dog fecal samples compared to soil samples but there was no difference in tylosin resistance in bacterial isolates from manure and dog fecal samples (Fig. 6). Irrespective of the antibiotic use or the animal species, most of the bacterial isolates from dog fecal samples were E. coli (Table 6-8), which is intrinsically resistant to high concentrations of tylosin (> 32 mg ml-1). We postulate that this may be the reason for higher levels of tylosin resistance in dog feces as compared to manure. Tylosin resistance between manure and soil samples was different but at a higher level of significance (p=0.10, Fig. 6).

There was also a significant animal by matrix interaction. Tylosin resistance was the highest in swine manure samples followed by equal levels of tylosin resistance in turkey and cattle manure (Fig. 7). Higher levels of tylosin resistance in swine manure is well expected because of the wide spread use of tylosin in swine production systems. Tylosin is mixed both in nursery and finishing pig feeds with concentrations varying from 40-400 g T-1 of feed. Tylosin resistance in swine manure was about 1.5 times more than that of tetracycline resistance. These results are in line with respective use of each antibiotic in swine production.

There was no difference in tylosin resistance of bacteria isolated from soil samples where swine, turkey or cattle manure has been applied. This merely supports earlier observations that tylosin resistance did not transfer to soil bacteria upon manure application or we were not able to culture all soil bacteria. Tylosin resistance in bacteria from dog fecal samples was higher from turkey and cattle farms than swine farms. The reasons for these differences among different animal species farms are not clear.

(iv) Prevalence of Monensin Resistance Bacteria on Animal Farms

Monensin belongs to the ionophore group of antibiotics and is used for the control of parasitic infections. Ionophores are primarily used sub-therapeutically in poultry and cattle; however, there are some reports on ionophore (salinomycin) use in swine production systems. To keep the protocol uniform in our experiment, we screened all samples including the samples from swine farms for the presence of monensin resistant bacteria.

Analysis of variance on prevalence of monensin resistance in bacteria is given in Table 5. There was no difference in the prevalence of monensin resistant bacteria by usage or animal species. However, there was a significant interaction between antibiotic use and animal species on monensin resistance. There was a higher level of monensin resistance in bacteria from AU than NAU swine farms (Fig. 8). These results are surprising considering that monensin is not used for growth promotion on swine farms. One possibility may be that swine manure bacteria are resistance because some other similar antibiotics have been used on these farms. However, this hypothesis needs further validation.

In turkey farms, there was greater monensin resistance in samples collected from NAU than AU turkey farms while in cattle farms, there was no difference in monensin resistance in samples collected from either AU or NAU farms (Fig. 8). We are not certain of the reasons for these trends and suggest further studies to validate these observations.

There were significant (p= 0.10) differences in the prevalence of monensin resistance between three different matrices and also there was a significant (p=0.10) usage by matrix interaction (Table 5). Monensin resistance in dog fecal bacteria was significantly higher compared to soil bacteria (Fig. 9). However, there was no difference in monensin resistance between dog fecal and manure samples or manure and soil samples. Higher resistance in dog fecal samples was mainly due to higher monensin resistance collected from NAU farms (Fig. 10, Table 3a). These higher levels of monensin resistance in dog fecal samples may be because E. coli was the predominant bacterial isolate in dog feces and E. coli is known to have natural resistance at monensin concentration (6 mg ml-1) irrespective of the farm types used in our study. Higher levels of monensin resistance in dog fecal samples from NAU farms may be due to higher number of E. coli isolated from dog fecal samples collected from NAU farms than AU farms (Table 6-8).

Although monensin and other ionophores are widely used for growth promotion in poultry and cattle production, not much is known about the resistance mechanisms to these antibiotics. There are no standardized resistance breakpoint concentrations available for determining the monensin minimum inhibitory concentration (MIC) for different bacterial species. In the present study, we used monensin at a concentration of 6 mg ml-1 for isolation of resistant bacteria. This concentration was determined on the basis of some published studies (van den Bogaard and Stobberingh, 2000). We believe because of this lack of standardized MIC value for monensin, our results on prevalence of monensin resistant bacteria under different farm conditions are inconclusive.

(v) Prevalence of ARB on no Manure Farms

In addition to quantifying the differences in the prevalence of ARB in samples from AU and NAU farms, another objective of our study was to quantify the level of ARB in the samples from farms where no food animals are being produced and thus no manure has been applied to their fields. Table 3b gives the level of ARB isolated from soil and dog fecal samples collected from no manure farms. Except from one (out of 4 farms) farm, no ARB was isolated from any of the soil samples. On this one farm, only tylosin and monensin resistant bacteria were isolated from the soil sample (no tetracycline resistant bacteria). Comparison of NAU and no manure farms shows slightly higher levels of tylosin and monensin resistance in soil bacteria from NAU farms. These results suggest that antibiotic resistance profile of soil bacteria is somewhat influenced by manure application even though antibiotics have not been used sub-therapeutically on the farm. Since antibiotics are used to some extent therapeutically on many food animal farms, higher levels of ARB may be expected in manure, which on land application is contributing to higher levels of resistance in soil microflora. This increased resistance on AU farms may be because of the exchange of antibiotic resistance genes between manure and soil microflora or uptake of free genetic elements (antibiotic resistance genes, integrons, transposons, plasmids) by soil microorganism from manure. Prevalence of ARB in dog fecal samples from no manure vs. AU or NAU farms was not different (Table 3b).

B. Resistance profile of ARB

In Table 6-8 are given the distribution of ARB species isolated from manure, soil, and dog fecal samples collected from AU and NAU swine farms. The susceptibility of all these species to various antibiotics were determined as described in the procedure section. In the following sections, we describe the relative differences in the susceptibility of a few dominating species that were present in both AU and NAU farms to 20 antibiotics belonging to 10 different groups (Tables 9-15). These groups are: cephalosporin, macrolide, tetracycline, b-lactam, chloramphenicol, aminoglycoside, fluoroquinolone, lincosamide, sulphonamides, and aminocyclitols. Because of the limitation that there was only one resistance value for AU and NAU farms for each bacterial species and each antibiotic, statistical analysis (t-test) on significant differences between AU and NAU farms for each bacterial species was run over all antibiotics. In the following sections, we describe the broad differences in resistance profile of bacterial isolates which were found to be common between AU and NAU farms for different antibiotics and then a summary statement as to its resistance over all antibiotics.

(vi) Resistance profile of ARB Isolated from Swine Farms

Manure: In Table 9 are listed the percent isolates of Pasteurella sp., Moraxella sp., and Acinetobacter sp. that were resistant to a given antibiotic. Pasteurella sp. from AU farm (n=11) showed higher resistance to tetracycline, b-lactam, and sulphonamide (except trimethoprim:sulfa), group of antibiotics whereas those from NAU farm (n=7) showed higher resistance to macrolide, group of antibiotics. All the isolates from NAU farm were susceptible to b-lactam antibiotics whereas three isolates from AU farms were resistant to this group of antibiotics. Only one Pasteurella sp. isolate from AU farm showed resistance to cephalosporin (ceftiofur) and aminoglycoside (apramycin) antibiotics where as all of the isolates from NAU farm were found susceptible to cephalosporin (ceftiofur) and one isolate was resistant to aminoglycoside (gentamicin). Averaged over all antibiotics, there was no statistical difference in antibiotic resistance of Pasteurella sp. to 20 antibiotics.

There was no statistical difference in percent resistance of Moraxella sp. to various antibiotics between NAU (n=6) and AU farms (n=3). Majority of the isolates from either type of farms showed resistance to macrolide, tetracycline and sulphonamide group of antibiotics. None of the isolate was resistant to ceftiofur and enrofloxacin.

The number for Acinetobacter sp. isolated from both types of farms was very low (Table 6). All the Acinetobacter sp. isolates irrespective of the farm type (AU or NAU) were found to be susceptible to ceftiofur, erythromycin, tilmicosin, florfenicol, and to b-lactam and aminoglycoside group of antibiotics. Acinetobacter sp. isolates from AU farms showed higher resistance to clindamycin and spectinomycin and to some extent to sulphonamide group of antibiotics. However averaged over all antibiotics, there was no statistical difference in antibiotic resistance of Acinetobacter sp. between AU and NAU farms.

Soil: In Table 10 is given the comparative resistance profile of Pseudomonas sp. and Pasteurella sp. isolates from AU and NAU farms. All the Pseudomonas sp. isolates from both types of farm were resistant to macrolide group of antibiotics, and also to florfenicol and clindamycin antibiotics. Resistance to sulphonamides group of antibiotics was high in Pseudomonas sp. isolates from AU farms. Most of the Pseudomonas sp. isolates from either type of farm were susceptible to tetracycline and aminoglycoside group of antibiotic, however one Pseudomonas sp. isolate (20%) from AU farm was found to be resistant to chlortetracycline and gentamicin. Resistance to ceftiofur was high in Pseudomonas sp. isolates from NAU farm (100.0%) as compared to the AU farm (60.0%). Statistically there was no difference in resistance profile of Pseudomonas sp. between AU and NAU farms over all 20 antibiotics.

Resistance of Pasteurella sp. isolates from AU farm to sulphonamide group of antibiotics was higher as compared to those from NAU farm. Pasteurella sp. isolate from NAU farm (n=1) was susceptible to b-lactam, aminoglycoside and sulphonamide group of antibiotics but it was resistant to macrolides. Also, this isolate was found to be susceptible to ceftiofur, chlortetracycline, florfenicol and spectinomycin antibiotics but resistant to oxytetracycline, clindamycin, and tiamulin antibiotics. Although all three Pasteurella sp. isolates from AU showed resistance to tylosin, only 2 of the isolates were found resistant to other macrolides tested (erythromycin and tilmicosin). Averaged over all 20 antibiotics, there was no statistical difference in antibiotic resistance profile of Pasteurella sp. between the AU and the NAU farms.

Dog Feces: Most of the ARB isolates from dog fecal samples were E. coli (Table 6). In Table 10 are also given the comparative resistance of E. coli from AU and NAU farms to various groups of antibiotics. E. coli isolates from AU farms had slightly higher resistance to tetracycline, and sulphonamide group of antibiotics. Resistance to flouoquinolone group of antibiotics was comparable between the isolates from the AU and the NAU farms. All the E. coli isolates from AU farm were found susceptible to ceftiofur and florfenicol antibiotics, but there were few isolates from NAU farm that were resistant to these antibiotics. The isolates from the dog fecal samples collected on the AU farms showed higher resistance to ampicillin antibiotic as compared to those isolated from the NAU farms (64.3% and 21.4% respectively). Resistance to clindamycin was same for both type of farms but 42.8% of the isolates from AU farm were resistant to spectinomycin as compared to 14.3% of the isolates from NAU farm. Averaged over all 20 antibiotics, E. coli isolates from AU farms had statistically (p=0.05) higher resistance than NAU farms.

(vii) Resistance Profile of ARB Isolated from Turkey Farms

In Table 7 are given the distribution of ARB isolates in manure, soil, and dog fecal samples from AU and NAU turkey farms. In the following section, we describe the relative differences in the susceptibility of selected species for each matrix between AU and NAU turkey farms.

Manure: Comparative resistance profile of E.coli and Pasteurella sp. in turkey manure samples from AU and NAU farms is given in Table 11. E. coli isolates from the AU farms showed higher resistance to tetracycline, b-lactam (ampicillin), fluoroqinolone, and sulphonamide group of antibiotics. Some of the isolates from AU farm showed higher resistance to neomycin (43.0%) and gentamicin (57.0%) antibiotics whereas all of the isolates from NAU farm were found to be highly susceptible to these two antibiotics. Both these antibiotics along with tetracyclines are widely used as feed additives for growth promotion in turkeys and resistance to these antibiotics may be because of this widespread use. Also, E. coli isolates from AU farms showed higher resistance to spectinomycin and clindamycin antibiotics as compared to NAU farms. All isolates from either type of farms were found to be susceptible to ceftiofur antibiotic whereas resistance to florfenicol antibiotic was high in isolates from the NAU farms (47.0%) as compared to those from the AU farms (14.0%). Averaged over all 20 antibiotics, E.coli isolates from the AU farms had statistically (p=0.05) higher resistance than the NAU farms.

Pasteurella sp. isolates from the AU farms were more resistant to macrolide and tetracycline group of antibiotics than the NAU farms (Table 11). However, all the isolates from both types of farms were susceptible to b-lactam and aminoglycoside (except one isolate from AU farm) group of antibiotics. Although resistance to sulphonamides group of antibiotics was comparable, Pasteurella sp. isolates from AU farm showed higher resistance to florfenicol, clindamycin and spectinomycin than isolates from NAU farms. Averaged over all 20 antibiotics, Pasteurella sp. had slightly higher resistance (p=0.1) in AU than NAU farms.

Soil: In Table 12 is given the antibiotic resistance profile of Pseudomonas sp. in soil samples from AU and NAU turkey farms. In general, resistance to most of the antibiotics was found to be comparable in the isolates from two types of farms. All the isolates were found to be susceptible to aminoglycoside antibiotics. However, isolates from the NAU farms had higher resistance to ceftiofur, florfenicol, and spectinomycin antibiotics as compared to those from AU farm. The resistance to tetracycline group of antibiotics was also higher in the Pseudomonas sp. isolates from NAU farm. The reason for this switch is not clear however, it may be because the NAU turkeys farms are generally more diversified with other types of food animals and they are all together on the range. Thus the land (soil samples) receives not only the turkey manure but also the manure from other types of food animal. Averaged over all 20 antibiotics, there was no statistical difference in resistance profile of Pseudomonas sp. between the AU and the NAU farms.

Dog Feces: Not much difference was observed in the resistance profile of E. coli isolates from dog fecal samples (Table 12). E. coli isolates from AU farm showed higher resistance to aminoglycoside group of antibiotics whereas as resistance to tetracyclines was found to be higher in the isolates from NAU farms (Table 12). E. coli resistance to spectinomycin (17.0%) and ceftiofur (3.0%) antibiotics was higher in bacterial isolates from AU than NAU farms. All the E. coli isolates from NAU farm were susceptible to these two antibiotics. Statistically, the difference in the antibiotic resistance profile of the E. coli isolates from two types of farms was non-significant.

(viii) Resistance Profile of ARB Isolated from Cattle Farms

In Table 8 are listed the distribution of ARB isolated from different samples collected from the AU and the NAU cattle farms. In the following section, we describe the relative differences in the susceptibility of selected species for each matrix between the AU and the NAU farms.

(1) Manure: Acinetobacter sp. isolates from the AU farms showed higher resistance to most of the antibiotics used in susceptibility testing (Table 13). Acinetobacter sp. isolates for either type of farms were resistant to tylosin antibiotic but not two other macrolides (erythromycin and tilmicosin). All the Acinetobacter sp. isolates were susceptible to ceftiofur and b-lactam antibiotics. Resistant profile (20 antibiotics) of Acinetobacter sp. isolates from the AU farms was found to be statistically (p=0.05) different than those from the NAU farms.

E. coli isolates from the AU and the NAU farms showed nearly similar resistance to most of the antibiotics tested (Table 13). All of the E. coli isolates from both the AU and the NAU farms were susceptible to ceftiofur. Isolates from the NAU farms showed marginally higher resistance to sulphonamides as compared to those from AU farms. Averaged over 20 antibiotics, there was no statistical difference in resistance of E. coli from the AU and the NAU farms.

Pasteurella sp. isolates from both types of farms were susceptible to ceftiofur and florfenicol antibiotics as well as to b-lactam, aminoglycoside, and fluoroquinolone group of antibiotics (Table 13). Resistance to sulphonamides was higher in the isolates from the NAU farms whereas isolates from the AU farms showed higher resistance to clindamycin and macrolides. Averaged over all 20 antibiotics, there was no statistical difference in resistance of Pasteurella sp. between the AU and the NAU cattle farms.

(2) Soil: Except for oxytetracycline, neomycin, and two antibiotics in sulphonamide group, Pseudomonas sp. isolates in soils from AU cattle farms showed slightly higher resistance to all antibiotics (Table 14). There was one isolate (6.0%) from NAU farms that was resistant to neomycin and oxytetracycline antibiotics. Averaged over all 20 antibiotics, there was no statistical difference in antibiotic resistance of Pseudomonas sp. from the AU and the NAU farms.

Although all the Pantoea sp. isolates were found to be susceptible to cephalosporin, tetracycline, and aminoglycoside group of antibiotics (Table 14), resistance to other antibiotics was generally higher in the Pantoea sp. isolates from the AU farms compared to those from the NAU farms. Averaged over all 20 antibiotics, resistance of Pantoea sp. was statistically (p=0.05) higher from the AU than the NAU farms.

(3) Dog feces: Most of the bacterial isolates from dog fecal samples from both the AU and the NAU cattle farms were E. coli. Except for tetracycline and sulphonamides group of antibiotics, susceptibility of E. coli to various antibiotics was nearly the same between the AU and the NAU farms (Table 14). Resistance to tetracycline group of antibiotics was slightly higher in the E. coli isolates from NAU farms, whereas resistance to sulphonamide group of antibiotics was slightly higher from the AU farms. Averaged over all 20 antibiotics, there was no statistical difference in E. coli resistance for 20 antibiotics between the AU and the NAU farms.

(ix) Resistance Profile of ARB Isolated from no Manure Farms

In Table 15 is given the antibiotic resistance profile of E. coli isolates from dog fecal samples on farms where there are no food animals. Overall no difference was observed in the antibiotic resistance profile of the E. coli from no manure (Table 15) or AU or NAU farms (Tables 10, 12, 14). From this it appears that sub-therapeutic use of antibiotics in farm animals does not have any impact on the resistance profile of E. coli isolated from dogs.

(x) Resistance Profile of Other ARB

Besides the common bacterial isolates discussed above, we also isolated some multi-drug resistant pathogenic bacteria from the AU farms and the antibiotic resistant profile of these bacteria’s is given in Table 16. Citrobacter sp. is opportunistic pathogens which is part of normal fecal flora. In the present study, Citrobacter sp. was isolated from the dog fecal samples collected from AU turkey (two isolates) and AU cattle (three isolates) farms. Isolates from turkey farm were found to be resistant to penicillin, clindamycin, and tiamulin antibiotics as well as to macrolide group of antibiotics. One out of two isolates was found to be resistant to ceftiofur, apramycin, and spectionmycin antibiotics. Antibiotic resistance was found to be widespread among the Citrobacter sp. isolates from cattle farm, as the isolates were found to be resistant to most of the antibiotics against which the resistance was determined. All the three isolates were susceptible to enrofloxacin and spectinomycin antibiotics, whereas only one isolate showed resistance to ceftiofur antibiotic.

Yersinia sp. is a zoonotic pathogen causing severe diarrhea in humans especially in children. Major animal reservoir of Yersinia sp. is pigs and most common mode of transmission to humans is through eating contaminated pork. In this study Yersinia sp. were isolated (two isolates) from swine manure and turkey manure (seven isolates) samples collected from AU farms. Isolates from swine farm were found to be highly susceptible to 17 antibiotics. Both the isolates were resistant to chlortetracycline, oxytetracycline, and erythromycin antibiotics (Table 16). Resistance to various antibiotics was more widespread in the isolates from turkey manure except for neomycin, gentamicin, ceftiofur, enrofloxacin, and ampicillin. All seven isolates were found to be resistant to tetracycline group of antibiotics.

Cedecea sp. has been associated with bactericimia infections in humans however the rate of infection is very low. In the present study, three bacterial isolates from dog fecal samples from AU turkey farms were identified as Cedecea sp. All the three isolates had high resistance to multiple antibiotics except for florfenicol and trimethoprim:sulfa, for which all three isolates were found to be susceptible. Acinetobacter sp. are reported to cause nosicomial infections in immunocompromised patients and are part of normal microflora of animals. Three bacteria isolated from dog fecal samples collected from NAU turkey farms were identified as Acinetobacter sp. All three isolates were found susceptible to 19 antibiotics. The exception was tylosin for which all three were found to be resistant (Table 16). Two isolates showed resistance to oxytetracycline, whereas only one isolate was found to be resistant to chlortetracycline antibiotic and sulphonamide group of antibiotics. In addition, we also isolated Providencia sp. (n=4) from manure compost piles. Providencia sp., a Gram negative bacterium, belongs to the family Enterobacteriaceae and is responsible for gastrointestinal disorders both in humans and animals. The isolates were found to be resistant to clindamycin and to tetracycline, macrolide, and sulphonamide groups of antibiotics. The details of these findings are described in an enclosed paper by Chander et al. (2005).

CONCLUSIONS

Results obtained in the present study indicate prevalence of higher ARB in manure samples as a result of antibiotic usage but no difference in ARB levels in soil and dog fecal samples. These results suggest that antibiotic feeding of food animals increases ARB resistance in manure samples but this resistance does not permeate to manure applied fields and to pets on the farms. There was large variability in the resistance data. Lack of resistance trends in soil and dog fecal samples may be an artifact of the large variability as a result of small sample size (number of farms under each category was less than 7). Additional reasons for a lack of differences in soil samples between two types of farms may be the difficulty of culturing all soil bacteria and also low survivability of manure bacteria in soil. Furthermore, most of the NAU farms in this study had only recently (during the last 3-5 years) switched to this practice and thus a decrease in resistance levels is not expected in such a short time frame. Except for NAU swine farms, number of animals on turkey and cattle on NAU farms was far less than those on AU farms. Some of the NAU farms also had a mix of animals and thus the manure from all these animals was collectively applied on a given land parcel. Thus it was difficult to know if the resistance level in the soil from NAU farm was represented a given animal specie.

Results suggest that only tetracycline feeding increased ARB in manure for all three animal species. For tylosin antibiotic, there was no difference in the prevalence of ARB between two types of farms for a agiven animal species. Prevalence of monensin resistant bacteria was found to be significantly higher on AU than NAU swine farms. This was surprising considering that monensin is not used as a feed additive in swine production. Higher levels of monensin ARB may be because of cross-resistance or because the MIC values for monensin are not well defined. Since significantly higher ARB were present in the manure samples from AU farms, these results suggest that feeding of antibiotics helps select ARB in the animal gut. Survey of the animal producers on their manure management practices did not indicate any special practice that might lessen the development or spread of antibiotic resistance on the farm.

ACKNOWLEDGEMENTS

The authors greatly acknowledge the funding support from the USDA-NCSARE Program. We also could not have completed this study without the participation of about 50 animal producers. They and their families have been gracious in having us on their farms and help collect samples as well as provide background information on their operations. The authors also thank many county extension educators especially Brad Carlson of Rice county in making initial contact with the animal producers. We also acknowledge the assistance of Mr. Wayne Martin, Associate Director of Swine Center at the University of Minnesota for making contacts especially with non-antibiotic feeding farms and also with collection of samples. For statistical analysis, we greatly appreciate the discussion with Dr. Sandy Weisberg of the School of Statistics, Dr. Roger Moon of the Entomology department, and Dr. Nancy Ehlke of the Agronomy and Plant Genetics department.

LITERATURE CITED

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Jørgensen, S.E., and B. Halling-Sørensen. 2000. Editorial “Drugs in the Environment” Chemoshpere, 40: 691-699.

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Research conclusions:

This is the first study which quantified the differences in bacterial resistance between sub therapeutic and non-sub therapeutic using farms. Because our sample size is small, we need to increase our database before any major policy changes can be discussed/recommended in the use of antibiotics in food animals. If the above observations hold true over many other farms then there may be a need to find alternatives to sub-therapeutic use of antibiotics in animal production. Potential impact of eliminating antibiotic use in food animal production will be substantial.

Farmer Adoption

This is relatively new area of research. Further work is needed before any alternatives to sub-therapeutic use of antibiotics or manure management practices could be suggested.

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:

Chander, Y., S.C. Gupta, K. Kumar, S. Goyal, and H. Murray. 2008. Antibiotic use and the prevalence of antibiotic resistant bacteria on turkey farms. J. Sci. Food Agric. 88:714–719.

Kumar, K., S.C. Gupta, Y. Chander, and A. Singh. 2005. Antibiotics use in agriculture and their impact on the terrestrial environment. Advances in Agronomy, 87 (In press).

Chander, Y., S.M. Goyal, and S.C. Gupta. 2005. Antimicrobial resistance of Providencia sp. isolated from animal manure. The Veterinary Journal (in press)

Chander, Y., S.C. Gupta, S.M. Goyal, K. Kumar, and H. Murray. 2007. Sub-therapeutic use of antibiotics and prevalence of antibiotic resistant bacteria on swine farms. Research J. Microbiology 2(9): 654-663, 2007.

Chander, Y., S.C. Gupta, S.M. Goyal, and K. Kumar. 2007. Antibiotics: Has the magic gone? J. Sci. Food. Agric. 87: 739-742.

Chander, Y., S.M. Goyal, and S. C. Gupta. 2006. Antimicrobial resistance of Providencia spp. isolated from animal manure. The Veterinary Journal. 172:188-191.

Chander, Y., K. Kumar, S.M. Goyal, and S.C. Gupta. 2005. Antibacterial activity of soil-bound antibiotics. J. Environ. Qual. 34: 1952-1957.

Chander, Y., K. Kumar, S.C. Gupta, and S. M. Goyal. 2005. Evaluation of CHROMagar Salmonella Medium for the Isolation of Salmonella from Animal Manure. Journal of Applied Research in Veterinary Medicine. 3 (1): 35-39.

Dolliver, H.A.S., and S.C. Gupta. 2008. Antibiotic Losses from Unprotected Manure Stockpiles. J. Environ. Qual. (in press).

Dolliver, H.A.S., K. Kumar, S.C. Gupta, and A. Singh. 2008. Application of Enzyme-Linked Immunosorbent Assay Analysis for Determination of Monensin in Environmental Samples. J. Environ. Qual. (in press).

Dolliver, H.A.S., S.C. Gupta, Sally Noll. 2008. Antibiotic Degradation during Manure Composting. J. Environ. Qual. (in press).

Dolliver, H.A.S., and S.C. Gupta. 2008. Antibiotic Losses in Leaching and Surface Runoff from Manure-Amended Agricultural Land. J. Environ. Qual. (in press).

Dolliver, H.A.S., K. Kumar, S.C. Gupta. 2007. Sulfamethazine Uptake by Plants from Manure-Amended Soil. J. Environ. Qual. 36: 1224-1230.

Kumar, K., S.C. Gupta, S. Baidoo, Y. Chander, ad C.J. Rosen. 2005. Antibiotic uptake by plants from soil fertilized with animal manure. J. Environ. Qual. 34: 2082-2085.

Kumar, K., S.C. Gupta, Y. Chander, A. K. Singh. 2005. Antibiotic use in agriculture and its impact on the terrestrial environment. Advances in Agronomy. 87: 1-54.

Kumar, K. A. Thompson. A. Singh, Y. Chander, and S.C. Gupta. 2004. Enzyme-linked immunosorbent Assay for ultratrace determination of antibiotics in aqueous samples. J. Environ. Qual. 33: 250-256.

Kumar, K., S.C. Gupta, S.K. Baidoo, Y. Chander, and C.J. Rosen. 2005. Antibiotic uptake by plants from soil fertilized with animal manure. J. Environ. Quality 34: 2082-2085.

Presentations:
Chander, Y., S.C. Gupta, S.M. Goyal, and K. Kumar. 2004. Antibiotic resistance profile as influenced by land application of swine manure. Soil Science Society of America Annual Meetings. Poster 1576.

Chander, Y., S.C. Gupta, S.M. Goyal, K. Kumar. 2004. Role of antibiotics feeding in food animals on antimicrobial resistance in the environment. Soil Science Society of America Annual Meetings. Oral presentation.

Chander, Y., K. Kumar, S.C. Gupta, A.K. Singh, and S.M. Goyal. 2003. Antimicrobial activity of soil bound antibiotics. Soil Science Society of America Annual Meetings. Poster Presentation.

Antibiotics in the Environment. Science Blog. (http://www.scienceblog.com/community/article2504.html).

Gave a brief interview on our antibiotic research to a local radio station (KKOK, KMRS) and a reporter for a local newspaper. The article “Farm antibiotics research probes environmental resistance problems” appeared in Morris Sun Tribune on 17 Feb. 2004.

MN Turkey Research & Promotion Council-Impact of animal antibiotic feeding on the environment. 13 May 2003.

Participated in a 30-minute talk show on KDHL 92 at Fairbault about antibiotics. The host was Jerry Groskrentz. Also, participated in the show were Brad Carlson and Yogesh Chander (16 April 2003).

Gave a presentation on the “Role of Antibiotics Feeding in the spread of Antibiotic Resistance in the Environment” at the BALMM (Basin Alliance for the Lower Mississippi in Minnesota) meetings in Rochester, MN. 16 June 2004.

Gave a presentation on “Role of Antibiotic Feeding on the Spread of Antibiotic Resistance in the Environment” at the Crop and Soils Winter Field day at West Central Research and Outreach Center, Morris on Feb 13 2004.

Gave a talk on “Role of Antibiotic Feeding in Food Animals on the Spread of Antimicrobial Resistance in the Environment” at the Drainage Forum, Owatonna, MN, 16 September 2004.

Gave a seminar in the department. “Role of Antibiotic Feeding in Food Animals on the Spread of Antimicrobial Resistance in the Environment” 13 September, 2004.

Project Outcomes

Recommendations:

Areas needing additional study

Although we had about 50 producers participate in our study, the sample size by animal species, and antibiotic users vs. antibiotic non-user is rather small (maximum of 7 producers in each category). Work is needed to expand these databases and increase confidence in our findings. Furthermore, we also isolated some multi-drug resistance pathogenic bacteria such as Citrobacter sp., Yersinia sp., Cedecea sp., Providencia sp. from farms that use antibiotics sub-therapeutically. The implications of these findings are not apparent. Further work is needed to better understand the implications of these findings.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.