Role of IS Kpn7 and Deletions in bla KPC Gene Expression

作者：Thierry Naas, Gaëlle Cuzon, H. Tuan Truong, Patrice Nordmann · 发表于：Antimicrobial Agents and Chemotherapy · 年份：2012 · DOI：10.1128/aac.00334-12 · 被引用次数：119 · 研究领域：Antibiotic Resistance in Bacteria、Antibiotics Pharmacokinetics and Efficacy、Bacterial Identification and Susceptibility Testing

The carbapenemase-encoding bla(KPC) gene, which is rapidly spreading in Gram-negative rods, is located on a Tn3-based transposon, Tn4401, which carries a polymorphic region giving rise to five isoforms (a, b, c, d, and e) that is located immediately upstream of the bla(KPC) gene and thus likely involved in its expression. Using 5' rapid amplification of cDNA ends (5'RACE), we identified three potential promoter sequences (P1, P2, and P3) upstream of the bla(KPC) gene, of which only P1 (absent from isoforms c and d) and P2 (present in all isoforms, with a -35 box located inside the right inverted repeat of ISKpn7) were shown to be true promoters involved in expression. One representative of each different promoter combination of Tn4401, i.e., P2 alone (isoform c), P1-P2 (isoform a), and P1-P2-P3 (isoform b), was cloned into an Escherichia coli plasmid vector. Using reverse transcription-PCR (RT-PCR), the highest level of expression was obtained with isoform a (P1 and P2), which is also the most commonly encountered form in enterobacterial clinical isolates, followed by isoforms b (P1, P2, and P3) and c (P2 only). These differences in expression led to slight differences in MIC values of carbapenems. In silico analysis of the DNA sequence of isoform b revealed a stem-loop structure that is likely responsible for strong stops observed in 5'RACE experiments and for decreased expression compared to that with isoform a (P1 and P2). In addition, such structures could also be at the ...