Differentiation of trimethoprim resistance genes among Eschericha coli strains from an environment with intensive supply of antibiotic

Trimethoprim (TMP) is a synthetic, broad-spectrum chemotherapeutic agent, which inhibits the dihydrofolate reductase (DHFR) enzyme in bacterial cells. It is mostly used to treat community-acquired urinary tract infections in medicine, but TMP is also commonly used in veterinary and during livestock production. An intense use of antibiotics in these sectors leads to the rapid selection of resistant strains which, through the environment, pose a health risk. There are multiple mechanisms by which strains become resistant to trimethoprim and one of the most frequently identified mechanism is bypass. This mechanism is associated with the presence of the genes encoding a non-allelic variants of DHFR. Dfr genes are mostly associated with mobile genetic platforms and their expression is responsible for high levels of trimethoprim resistance. The aim of the study was to assess the differentiation of dfrA genes determining resistance to trimethoprim in commensal Escherichia coli strains isolated from pigs from a breeding farm with intensive supply of antibiotics in metaphylaxis program. A total of one hundred and sixty-four E.coli strains were isolated from feces of fifty animals, from two to six non-identical isolates per animal. Trimethoprim resistance was tested by microdilution method and revealed that 92% of tested strains were resistant with high MIC values (>32 mg/L). Resistance genes were tested by PCR and PCR-RFLP methods and six different genes were detected: dfrA1, dfrA5, dfrA7, dfrA12, dfrA14, dfrA21. 37% of the strains carried two to four different dfrA genes, in different combinations. In only three cases, all the strains derived from one individual had the same pattern of resistance genes, among the others, there was significant variability. DfrA genes were also detected in three strains sensitive to TMP. The study shows the great diversity of the trimethoprim resistance genes, both within the tested animal population and in the individual host. In addition, genes sequence analysis revealed nucleotide changes within some genes, which highlight the potential for alterations leading to the emergence of new resistance gene variants.