Phenotypic and genotypic characterization of antibiotic resistant soil bacteria adjacent to swine production facilities

Whether there was a correlation between the antibiotic use in agriculture and the recent increase in the rate of antiotic resistant infections in humans has been highly debated. In this study, the environmental factors in the increase of antibiotic resistance were investigated. In this regard, the effect of manure application from swine confinement facility to the fields has been evaluated. Sampling was performed four times in a year: one day before manure application, one day after manure application, three weeks after manure application, and three months after manure application. Eritromycin and tetracycline susceptibility of isolates was determined by agar dilution and E-test methods. 68 per cent of the soil samples was detected as resistant to tetracycline and 94 per cent of the soil samples was found to be resistant to erithromycin. Samples were double resistant to both antibiotics. Bacteria were identified by determining the nucleotide sequence of 16S rRNA gene fragments from each organism. Polymerase chain reaction (PCR) used to amplify the V3 region of the rRNA gene. The phylogenic positions of the isolates were determined by searching the GeneBank database. 213 bacteria from 4 different phylum were isolated namely Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes. Class-specific primers were used to amplify each of the six erythromycin resistance genes erm(A,B,C,F,G,Q) in 32 samples. In 46 samples were investigated the distribution of tet genes (4 genes encoding efflux pumps tet(C), tet(H), tet(Z), tet(W) and 3 genes encoding ribosomal protection proteins tet(M), tet(O), tet(Q). While Tet(Q) gene was detected in only soil isolates, tet(W) andtet(M) were detected in only manure isolates. Other tet genes were identified both in soil and manure samples. Furthermore, erm(Q) gene was found in only two manure samples. In conclusion, the demonstration of the resistance genes of erithromycin and tetracycline in samples grabbed from soil and manure, constitutes the first phase of linking the antibiotic use with the spread of resistance to the environment.