Enhanced Klebsiella pneumoniae Carbapenemase Expression from a Novel Tn4401 Deletion

The Klebsiella pneumoniae carbapenemase gene (bla(KPC)) is typically located within mobile transposon Tn4401. Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of bla(KPC). Illumina sequences from bla(KPC)-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188-bp deletion [between istB and bla(KPC)]) was present in 14% (39/281) of clinical isolates. MICs showed that Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (>= 16 and >= 16, respectively), ertapenem (>= 8 and 4, respectively), and cefepime (>= 64 and 4, respectively) than E. coli strains with Tn4401b (0.5, <= 0.5, and <= 1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn4401a had a 16-fold increase and Tn4401h a 4-fold increase in bla(KPC) mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn4401a and Tn4401h promoter sequences generated higher beta-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. The activity of the isolated P2 promoter was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicated that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics.