Novel trimethoprim resistance gene, dfrA35, in IncC plasmids from Australia.

Background: In Gram-negative bacteria, over 30 different genes are known to encode a trimethoprim-insensitive dihydrofolate reductase that confers resistance to trimethoprim.

Objectives: To determine whether a gene encoding a putative dihydrofolate reductase found in type 2 IncC plasmids isolated between 2002 and 2013 in healthcare facilities in Melbourne, Australia, confers trimethoprim resistance.

Methods: Conjugation was used to transfer plasmids into a laboratory Escherichia coli. A PCR-amplified fragment was cloned into pUC19 using Gibson Assembly and transformed into E. coli. The level of resistance to trimethoprim was determined using broth microdilution. MEGA (7.0.26) and Geneious Prime (7.0.9) were used to examine the relationship to known Dfr proteins.

Results: The conjugative IncC plasmid pEc158 from a 2002 Melbourne clinical E. coli isolate was shown to transfer trimethoprim resistance. The putative DfrA protein encoded by a dfrA gene in pEc158 shares <40% amino acid identity with any previously identified DfrA protein. This gene was cloned and found to confer trimethoprim resistance. The gene and protein were named dfrA35/DfrA35. In pEc158 the dfrA35 gene is located near the ori end of a partial copy of the CR1 element, within a complex resistance island. It is found in the same location in further closely-related type 2 IncC plasmids from Klebsiella pneumoniae (Melbourne, 2013), which were not transfer proficient.

Conclusions: Resistance determinants continue to be found and will be missed using website-associated databases to infer phenotypes from genome sequences rather than direct phenotypic testing.