Macrolide resistance in microorganisms at antimicrobial-free Swine farms

Abstract

To investigate the relationship between agricultural antimicrobial use and resistance, a variety of methods for quantification of macrolide-lincosamide-streptogramin B (MLS(B)) resistance were applied to organic swine farm manure samples. Fluorescence in situ hybridization was used to indirectly quantify the specific rRNA methylation resulting in MLS(B) resistance. Using this method, an unexpectedly high prevalence of ribosomal methylation and, hence, predicted MLS(B) resistance was observed in manure samples from two swine finisher farms that reported no antimicrobial use (37.6% +/- 6.3% and 40.5% +/- 5.4%, respectively). A culture-based method targeting relatively abundant clostridia showed a lower but still unexpectedly high prevalence of resistance at both farms (27.7% +/- 11.3% and 11.7% +/- 8.6%, respectively), while the prevalence of resistance in cultured fecal streptococci was low at both farms (4.0%). These differences in the prevalence of resistance across microorganisms suggest the need for caution when extrapolating from data obtained with indicator organisms. A third antimicrobial-free swine farm, a breeder-to-finisher operation, had low levels of MLS(B) resistance in manure samples with all methods used (<9%). Tetracycline antimicrobials were detected in manure samples from one of the finisher farms and may provide a partial explanation for the high level of MLS(B) resistance. Taken together, these findings highlight the need for a more fundamental understanding of the relationship between antimicrobial use and the prevalence of antimicrobial resistance.

FIG. 1.

Optimization of hybridization stringency for the MLS_B probe. •, sensitive strain (E. faecalis JH2-2); Δ, resistant strain (E. faecalis JH2-2 pAMβ1); □, mismatch strain (R. coprophilus containing G2057). The optimal formamide concentration used to differentiate a methylated or mismatch strain from an unmethylated (sensitive) strain was 12.5%.

FIG. 2.

Microbial community analysis of swine manure samples obtained from LF and OF2. The average half-range for duplicate FISH analysis was 1.8%.

FIG. 3.

Prevalence of ribosomal methylation and tylosin resistance in building samples obtained from LF and manure samples obtained from three organic farms (OF, OF2, and OF3), quantified using different methods. Membrane hybridizations and FISH quantified the prevalence of the specific ribosomal methylation leading to MLS_B resistance and were normalized to hybridization results obtained with a general bacterial probe, while the culture-based methods quantified resistance to the macrolide tylosin and were normalized to results under the same conditions in the absence of tylosin. All of the membrane hybridization data and the culture-based fecal streptococci results for LF were previously reported (18). Membrane hybridization data are not available for OF2 and OF3. Error bars show standard deviations, except for with the FISH quantification of ribosomal methylation on OF2 manure, for which the half-range of duplicate analyses is reported.