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Whole-genome sequencing of trimethoprim-resistant Escherichia coli strains MF2165 and PF9285 from healthy Swiss fattening calves revealed a so far uncharacterized dihydrofolate reductase gene, dfrA35 Functionality and association with trimethoprim resistance were demonstrated by cloning and expressing dfrA35 in E. coli The DfrA35 protein showed the closest amino acid identity (49.4%) to DfrA20 from Pasteurella multocida and to the Dfr determinants DfrG (41.2%), DfrD (40.8%), and DfrK (40.0%) found in Gram-positive bacteria. The dfrA35 gene was integrated within a florfenicol/chloramphenicol-sulfonamide resistance IS CR2 element ( floR -IS CR2 - dfrA35 - sul2 ) next to a Tn 21 -like transposon that contained genes with resistance to sulfonamides ( sul1 ), streptomycin ( aadA1 ), gentamicin/tobramycin/kanamycin ( aadB ), and quaternary ammonium compounds ( qacE Δ 1 ). A search of GenBank databases revealed that dfrA35 was present in 26 other E. coli strains from different origins as well as in Acinetobacter  IMPORTANCE The presence of dfrA35 associated with IS CR2 in Escherichia coli from animals, as well as its presence in other E. coli strains from different sources and countries and in Acinetobacter , highlights the global spread of this gene and its potential for further dissemination. The genetic link of IS CR2 - dfrA35 with other antibiotic and disinfectant resistance genes showed that multidrug-resistant E. coli may be selected and maintained by the use of either one of several antimicrobials.

A Novel Trimethoprim Resistance Gene, dfrA36 , Characterized from Escherichia coli from Calves