Tetracycline in liquid manure selects for co-occurrence of the resistance genes tet(M) and tet(L) in Enterococcus faecalis.

Causal relations between antibiotic use and selection of antibiotic resistance have been widely discussed. However, appropriate examinations have been mainly performed on phenotypic level, whereas genetic investigations, as well as researches under realistic conditions, are scarce. Therefore, the present field study aimed to accomplish a particular description of how an antibiotic contaminated environment influences microorganisms on both a phenotypic and a genetic level, using analytical, phenotypical and molecular biological methods. For this purpose, concentrations of tetracycline, chlortetracycline, oxytetracycline and doxycycline were analysed in liquid manure samples (n=179) from Bavarian (Germany) pig farms. All detected tetracyclines found in each manure sample were summed up and referred to as total tetracycline concentrations (TET). Phenotypic doxycycline resistance of Enterococcus faecalis isolated from these manure samples was determined by means of the microdilution method. After that, doxycycline resistant (n=147) and susceptible (n=32) E. faecalis were screened for tet(L), tet(M), tet(S) and tet(O) by using PCR. If despite doxycycline resistance no respective gene was detected, tet(A/B/C/D/K/L/M/W/Z) were additionally tested. The most frequent resistance determinant was tet(M) (n=128), followed by tet(L) (n=95). Tet(S) and tet(O) were present in 12 and 7 isolates; the remaining tet-genes were not detected. A correlation between the TET concentration in manure and the occurrence of tet(M) and tet(L) could be shown. In particular, strains that contained neither tet(M) nor tet(L) (n=44) were isolated from manure samples with mean TET of 0.35 mg/kg. If tet(M) was the only tetracycline resistance gene (n=40), mean concentrations were 0.51 mg/kg, and, if tet(L) was the only tet-gene (n=7), 1.18 mg/kg, respectively. On the other hand, if co-occurrence of tet(M) and tet(L) was detected (n=88, including 1 susceptible isolate), mean TET in the referring manure samples was 4.08 mg/kg. The present study demonstrates that high tetracycline concentrations in manure lead to higher doxycycline minimum inhibitory concentrations (MICs) in E. faecalis, genetically based on co-occurrence of tet(M) and tet(L).