Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics

Abstract

Background: Environmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA.

Results: The concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments.

Conclusion: The abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.

Figure 1

Quantification of 16S-rRNA in cattle fecal deposits under field conditions. The treatments were (N = 3; plus standard error): Control, no antimicrobial agents added to the diets of steers from which fecal deposits originated; A44, chlortetracycline (44 ppm); AS700, chlortetracycline and sulfamethazine (each at 44 ppm); T11, tylosin (11 ppm).

Figure 2

Persistence of tetracycline resistance genes in cattle fecal deposits under field conditions. The treatments were (N = 3; plus standard error): Control, no antimicrobial agents added to the diets of steers from which fecal deposits originated; A44, chlortetracycline (44 ppm); AS700, chlortetracycline and sulfamethazine (each at 44 ppm); T11, tylosin (11 ppm).

Figure 3

Persistence of sulfonamide resistance genes in cattle fecal deposits under field conditions. The treatments were (N = 3; plus standard error): Control, no antimicrobial agents added to the diets of steers from which fecal deposits originated; A44, chlortetracycline (44 ppm); AS700, chlortetracycline and sulfamethazine (each at 44 ppm); T11, tylosin (11 ppm).

Figure 4

Persistence of erythromycin resistance genes in cattle fecal depostis under field conditions. The treatments were (N = 3; plus standard error): Control, no antimicrobial agents added to the diets of steers from which fecal deposits originated; A44, chlortetracycline (44 ppm); AS700, chlortetracycline and sulfamethazine (each at 44 ppm); T11, tylosin (11 ppm).

Figure 5

Representative DGGE profiles generated from PCR-amplified 16S-rRNA in fecal deposits from the control group of cattle. DNA from replicate fecal deposits (N = 3) were pooled for analysis. The time points were days (d) 7, 28, 56, 98, 112, and 175. M, marker used to normalize gels consisted of pooled DNA from all treatments on days 7 and 175.

Figure 6

Similarity of DGGE profiles generated from PCR-amplified 16S-rRNA in cattle fecal deposits under field conditions. DNA from replicate fecal deposits (N = 3) were pooled for analysis. The time points were days (d) 7, 28, 56, 98, 112, and 175. The treatments were: Control, no antimicrobial agents added to the diets of steers from which fecal deposits originated; A44, chlortetracycline (44 ppm); AS700, chlortetracycline and sulfamethazine (each at 44 ppm); T11, tylosin (11 ppm).