Enhanced Klebsiella pneumoniae Carbapenemase Expression from a Novel Tn 4401 Deletion

<jats:title>ABSTRACT</jats:title> <jats:p> The <jats:named-content content-type="genus-species">Klebsiella pneumoniae</jats:named-content> carbapenemase gene ( <jats:italic>bla</jats:italic> <jats:sub>KPC</jats:sub> ) is typically located within mobile transposon Tn <jats:italic>4401</jats:italic> . Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of <jats:italic>bla</jats:italic> <jats:sub>KPC</jats:sub> . Illumina sequences from <jats:italic>bla</jats:italic> <jats:sub>KPC</jats:sub> -positive clinical isolates from a single institution were mapped to a Tn <jats:italic>4401</jats:italic> b reference sequence, which carries no deletions. The novel isoform Tn <jats:italic>4401</jats:italic> h (188-bp deletion [between <jats:italic>istB</jats:italic> and <jats:italic>bla</jats:italic> <jats:sub>KPC</jats:sub> ]) was present in 14% (39/281) of clinical isolates. MICs showed that <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content> strains containing plasmids with Tn <jats:italic>4401</jats:italic> a and Tn <jats:italic>4401</jats:italic> h were more resistant to meropenem (≥16 and ≥16, respectively), ertapenem (≥8 and 4, respectively), and cefepime (≥64 and 4, respectively) than <jats:named-content content-type="genus-species">E. coli</jats:named-content> strains with Tn <jats:italic>4401</jats:italic> b (0.5, ≤0.5, and ≤1, respectively). Quantitative real-time PCR (qRT-PCR) demonstrated that Tn <jats:italic>4401</jats:italic> a had a 16-fold increase and Tn <jats:italic>4401</jats:italic> h a 4-fold increase in <jats:italic>bla</jats:italic> <jats:sub>KPC</jats:sub> mRNA levels compared to the reference Tn <jats:italic>4401</jats:italic> b. A <jats:italic>lacZ</jats:italic> reporter plasmid was used to test the activity of the promoter regions from the different variants, and the results showed that the Tn <jats:italic>4401</jats:italic> a and Tn <jats:italic>4401</jats:italic> h promoter sequences generated higher β-galactosidase activity than the corresponding Tn <jats:italic>4401</jats:italic> 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. </jats:p>