Progress 08/15/03 to 08/14/05
Outputs
During this project, we sampled 248 cattle on 4 dairies. Each dairy was been visited 9 times, and therefore, some of the individual cattle have been sampled over 9 consecutive visits. We recorded antibiotic usage information for each of these animals as well as for each herd. In addition, all morbidity in the herd was recorded, including any clinical mastitis. We hypothesized that individual animals that had been treated with antimicrobials would possess a higher proportion of antimicrobial resistant E. coli in their feces, and that these resistant E. coli would persist in the animal over time. We also hypothesized that changes in the resistance levels in the E. coli of one animal would influence the E. coli populations of other animals in the same age cohort. The minimum inhibitory concentration (MIC) to various antimicrobials was determined for 3 to 6 randomly selected E. coli colonies from each sample. The main factor associated with increased resistance levels was
age. Young calves had a greater diversity of E. coli MIC phenotypes, and many of these E. coli isolates had elevated MICs to multiple antibiotics. However, regardless of treatment history, the animals had E. coli with lower MIC levels after 6 to 9 months of age. Other animals in the same age cohort had similar MIC patterns. Preliminary results show that individual animal antibiotic treatments are not highly selective for resistant phenotypes over extended periods of time. We have optimized two different multiplex PCR protocols for the detection of specific antibiotic resistance genes. One protocol detects the resistance gene flo, which confers resistance against florfenicol, and the gene cmlA, which confers resistance against chloramphenicol. To date, we have found 42 of 611 (6.9%) of the E. coli from feces with the flo gene, and only 3 of 611 with the cmlA gene. These genes are likely on plasmids, and we are currently determining their precise location within the bacterium. The E.
coli in which these genes were detected are of different DNA fingerprint patterns suggesting that the gene has been transferred to multiple E. coli types. The other PCR we have optimized detects the cmy-2 and the cmy-1 gene families, which both confer resistance against third-generation cephalosporins. The cmy-2 gene has been detected in 13 of 562 (2.3%) E. coli tested to date, but we have not detected the cmy-1 gene family. Again, the E. coli that possess the cmy-2 gene are of differing DNA fingerprints suggesting the transfer of this gene among multiple E. coli types. We also optimized a method to detect specific antibiotic resistance genes in DNA extracted directly from feces (total community DNA). This approach enabled us to determine more precisely whether genes exist in bacteria other than those that we culture in the laboratory. This could be important if there are multiple types of bacteria that possess the genes and serve as reservoirs of these genes. We have validated the
detection limits of this approach in order to better interpret the meaning of a negative result.
Impacts
This study aims to determine the relationship between bacteria that cause disease (pathogens) and those that do not (commensals) in animals as it pertains to antibiotic resistance. Specifically, we hypothesized that this type of reservoir would explain the apparent persistence of antibiotic resistance genes, even after antibiotic use has ceased.
Publications
- Patterson, S.K., and R.S. Singer. (2005). Evaluating the biases associated with community DNA approaches to antibiotic resistance detection. Journal of Veterinary Diagnostic Investigation (Accepted).
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Progress 01/01/04 to 12/31/04
Outputs
During this project, we have sampled 224 cattle on 4 dairies. Each dairy has been visited 8 times, and therefore, some of the individual cattle have been sampled over 8 consecutive visits. We have recorded antibiotic usage information for each of these animals as well as for each herd. In addition, all morbidity in the herd has been recorded, including any clinical mastitis. We hypothesized that individual animals that had been treated with antimicrobials would possess a higher proportion of antimicrobial resistant E. coli in their feces, and that these resistant E. coli would persist in the animal over time. We also hypothesized that changes in the resistance levels in the E. coli of one animal would influence the E. coli populations of other animals in the same age cohort. The minimum inhibitory concentration (MIC) to various antimicrobials was determined for 3 to 6 randomly selected E. coli colonies from each sample. The main factor associated with increased
resistance levels was age. Young calves had a greater diversity of E. coli MIC phenotypes, and many of these E. coli isolates had elevated MICs to multiple antibiotics. However, regardless of treatment history, the animals had E. coli with lower MIC levels after 6 to 9 months of age. Other animals in the same age cohort had similar MIC patterns. Preliminary results show that individual animal antibiotic treatments are not highly selective for resistant phenotypes over extended periods of time. We have optimized two different multiplex PCR protocols for the detection of specific antibiotic resistance genes. One protocol detects the resistance gene flo, which confers resistance against florfenicol, and the gene cmlA, which confers resistance against chloramphenicol. To date, we have found 42 of 611 (6.9%) of the E. coli from feces with the flo gene, and only 3 of 611 with the cmlA gene. These genes are likely on plasmids, and we are currently determining their precise location within
the bacterium. The E. coli in which these genes were detected are of different DNA fingerprint patterns suggesting that the gene has been transferred to multiple E. coli types. The other PCR we have optimized detects the cmy-2 and the cmy-1 gene families, which both confer resistance against third-generation cephalosporins. The cmy-2 gene has been detected in 13 of 562 (2.3%) E. coli tested to date, but we have not detected the cmy-1 gene family. Again, the E. coli that possess the cmy-2 gene are of differing DNA fingerprints suggesting the transfer of this gene among multiple E. coli types. We are also working on a method to detect specific antibiotic resistance genes in DNA extracted directly from feces (total community DNA). This approach will enable us to determine more precisely whether genes exist in bacteria other than those that we culture in the laboratory. This could be important if there are multiple types of bacteria that possess the genes and serve as reservoirs of these
genes. We have validated the detection limits of this approach in order to better interpret the meaning of a negative result.
Impacts
This study aims to determine the relationship between bacteria that cause disease (pathogens) and those that do not (commensals) in animals as it pertains to antibiotic resistance. Specifically, we hypothesized that this type of reservoir would explain the apparent persistence of antibiotic resistance genes, even after antibiotic use has ceased.
Publications
- Singer, R.S., S.K. Patterson, A.E. Meier, J.K. Gibson, H.L. Lee, and C.W. Maddox. (2004). Relationship between phenotypic and genotypic florfenicol resistance in Escherichia coli. Antimicrobial Agents and Chemotherapy. 48:4047-4049.
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Progress 01/01/03 to 12/31/03
Outputs
During this project, we have sampled 224 cattle on 4 dairies. Each dairy has been visited 8 times, and therefore, some of the individual cattle have been sampled over 8 consecutive visits. We have recorded antibiotic usage information for each of these animals as well as for each herd. In addition, all morbidity in the herd has been recorded, including any clinical mastitis. We have optimized two different multiplex PCR protocols for the detection of specific antibiotic resistance genes. One protocol detects the resistance gene flo, which confers resistance against florfenicol, and the gene cmlA, which confers resistance against chloramphenicol. To date, we have found 42 of 611 (6.9%) of the E. coli from feces with the flo gene, and only 3 of 611 with the cmlA gene. These genes are likely on plasmids, and we are currently determining their precise location within the bacterium. The E. coli in which these genes were detected are of different DNA fingerprint patterns
suggesting that the gene has been transferred to multiple E. coli types. The other PCR we have optimized detects the cmy-2 and the cmy-1 gene families, which both confer resistance against third-generation cephalosporins. The cmy-2 gene has been detected in 13 of 562 (2.3%) E. coli tested to date, but we have not detected the cmy-1 gene family. Again, the E. coli that possess the cmy-2 gene are of differing DNA fingerprints suggesting the transfer of this gene among multiple E. coli types. We are also working on a method to detect specific antibiotic resistance genes in DNA extracted directly from feces (total community DNA). This approach will enable us to determine more precisely whether genes exist in bacteria other than those that we culture in the laboratory. This could be important if there are multiple types of bacteria that possess the genes and serve as reservoirs of these genes. We have validated the detection limits of this approach in order to better interpret the meaning
of a negative result. We have completed the development and optimization of a Salmonella detection PCR from pooled bovine feces. The end result of this project is that the process of culturing for Salmonella from a large number of samples is now much more time and cost efficient. The results of this development were presented at two different scientific meetings and have now been accepted for publication in the Journal of Veterinary Diagnostic Investigation.
Impacts
This study aims to determine the relationship between bacteria that cause disease (pathogens) and those that do not (commensals) in animals as it pertains to antibiotic resistance. Specifically, we hypothesized that this type of reservoir would explain the apparent persistence of antibiotic resistance genes, even after antibiotic use has ceased.
Publications
- Singer, R.S., R. Finch, H.C. Wegener, R. Bywater, J. Walters, and M. Lipsitch. (2003). Antibiotic resistance--the interplay between antibiotic use in animals and human beings. The Lancet: Infectious Diseases. 3:47-51.
- Singer, R.S. (2003). Epidemiology and Ecology of Antibiotic Resistance in Agriculture. In: M. Torrence and R. Isaacson (eds.), Current Topics in Microbial Food Safety in Animal Agriculture. Iowa State Press.
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