Enhanced Klebsiella pneumoniae Carbapenemase Expression from a Novel Tn 4401 Deletion
Cheruvanky A., Stoesser N., Sheppard AE., Crook DW., Hoffman PS., Weddle E., Carroll J., Sifri CD., Chai W., Barry K., Ramakrishnan G., Mathers AJ.
ABSTRACT The Klebsiella pneumoniae carbapenemase gene ( bla KPC ) is typically located within mobile transposon Tn 4401 . 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 Tn 4401 b reference sequence, which carries no deletions. The novel isoform Tn 4401 h (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 Tn 4401 a and Tn 4401 h 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 Tn 4401 b (0.5, ≤0.5, and ≤1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn 4401 a had a 16-fold increase and Tn 4401 h a 4-fold increase in bla KPC mRNA levels compared to the reference Tn 4401 b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn 4401 a and Tn 4401 h promoter sequences generated higher β-galactosidase activity than the corresponding Tn 4401 b 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.