Comparative Analysis of bla _KPC Expression in Tn 4401 Transposons and the Tn 3 -Tn 4401 Chimera

Abstract

Two basic structures that carry the bla KPC gene, the Tn4401 transposon and the Tn3-Tn4401 chimera, have been identified within and outside China. However, the different bla KPC expression levels and promoter activities of these two structures are not completely understood. We constructed Tn4401a, Tn4401b, and Tn3-Tn4401 chimera recombinants and found that the imipenem (IPM) and meropenem (MEM) MICs for the Escherichia coli transformants carrying the chimera were 2-fold higher than for those carrying Tn4401b but 2-fold lower than for those carrying Tn4401a. In addition to the promoter P1, we characterized a novel potential promoter sequence (PX) in the chimera using 5= rapid amplification of cDNA ends (5= RACE), of which the Ϫ35 and Ϫ10 sequences were TTCAAA and TGAGACAAT, respectively. Although mutation of P1, P2, or PX significantly downregulated bla KPC mRNA expression in each structure (P Ͻ 0.05), the P2 mutation resulted in 2-and 3-fold greater decreases than the P1 mutation in Tn4401a and Tn4401b, respectively. Similarly, despite no significant difference in the PX and P1 mutations in the chimera, the carbapenem MIC and Klebsiella pneumoniae carbapenemase (KPC) production resulting from P2 mutations were significantly lower than those of P1 (P Ͻ 0.01) in the Tn4401 transposons. These studies indicate that the Tn3-Tn4401 chimera contains a novel potential bla KPC promoter, PX, and that its carbapenem resistance falls in between those of Tn4401a and Tn4401b.

Genetic Factors Associated with Enhanced bla KPC Expression in Tn 3 /Tn 4401 Chimeras

“…Besides, a novel isoform (CTC) with elongated noncoding and/or promoter regions was detected in 1 Klebsiella oxytoca strain in 2018. DNA sequencing revealed a 121-bp deletion (corresponding to positions 115379 to 115499 of the plasmid pLSH-KPN148-1; GenBank accession number MK396843 [17]) immediately upstream of the P1 promoter, causing the deletion of the PX promoter in CTA compared with CTB. However, an additional 624-bp insertion containing a truncated bla TEM gene was added, and a 127-bp sequence carrying the P1 promoter was deleted at 143 bp downstream of the PX promoter in CTC ( Fig.…”

“…Recombined plasmids. Taking into account pCTB, pM1CTB, pMXCTB, and pSDKPC, which had been generated previously (17), a total of 19 plasmids were systematically constructed in this study (Table 1). Among all the plasmid transformants, no significant difference in the bla KPC gene copy numbers were observed (data not shown).…”

“…In China, nearly all of the bla KPC genes are located in the Tn3-Tn4401 chimera CTB with the gene order Tn3 transposase, Tn3 resolvase, ISKpn8, bla KPC , ISKpn6-like element, Tn1721 resolvase, and Tn1721 transposase (14,16). However, the chimera lacks both the P2 and P3 promoters and carries the P1 promoter (17). Although the wild-type chimera CTB was identified in 2009, only one isoform (named CTD in this study), in which a 671-bp fragment is inserted between ISKpn8 and the bla KPC-3 gene, has been characterized in two KPC-3-producing E. coli and Citrobacter freundii strains thus far (16,24).…”

“…Fortunately, a 4-fold reduction in the MICs of imipenem (IPM), meropenem (MEM), cefepime (FEP), and cefotaxime was observed in the E. coli and C. freundii strains carrying the insertion isoform (24). Previously, we comparatively analyzed bla KPC expression in Tn4401 transposons and the Tn3-Tn4401 chimera CTB and characterized a novel promoter, PX, that drives bla KPC expression (17). However, mutations in PX or the P1 promoter still preserved half of the mRNA transcription, indicating unknown regulators of bla KPC gene expression in the Tn3-Tn4401 chimera (17).…”

“…Previously, we comparatively analyzed bla KPC expression in Tn4401 transposons and the Tn3-Tn4401 chimera CTB and characterized a novel promoter, PX, that drives bla KPC expression (17). However, mutations in PX or the P1 promoter still preserved half of the mRNA transcription, indicating unknown regulators of bla KPC gene expression in the Tn3-Tn4401 chimera (17).…”