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Updated: Apr 4, 2026
Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation
Published on: January 4, 2017
Complete sequences of KPC-2-encoding plasmid p628-KPC and CTX-M-55-encoding p628-CTXM coexisted in Klebsiella
Li Wang1, Haihong Fang2, Jiao Feng2
1Department of Clinical Laboratory, The First Hospital Affiliated to Henan University Kaifeng, China.
Abstract:
A carbapenem-resistant Klebsiella pneumoniae strain 628 was isolated from a human case of intracranial infection in a Chinese teaching hospital. Strain 628 produces KPC-2 and CTX-M-55 encoded by two different conjugative plasmids, i.e., the IncFIIK plasmid p628-KPC and the IncI1 plasmid p628-CTXM respectively. bla KPC-2 is captured by a Tn1722-based unit transposon with a linear structure. ΔTn3-ISKpn27-bla KPC-2-ΔISKpn6-ΔTn1722 and this transposon together with a mercury resistance (mer) gene locus constitutes a 34 kb acquired drug-resistance region. bla KPC-2 has two transcription starts (nucleotides G and C located at 39 and 250 bp upstream of its coding region respectively) which correspond to two promoters, i.e., the intrinsic P1 and the upstream ISKpn27/Tn3-provided P2 with the core -35/-10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively. bla CTX-M-55 is mobilized in an ISEcp1-bla CTX-M-55-Δorf477 transposition unit and appears to be the sole drug-resistant determinant in p628-CTXM. bla CTX-M-55 possesses a single transcription start (nucleotides G located at 116 bp upstream of its coding region) corresponding to the ISEcp1-provided P1 promoter with the core -35/-10 element TTGAAA/TACAAT. All the above detected promoters display a characteristic of constitutive expression. Coexistence of bla KPC and bla CTX-M in K. pneumoniae has been reported many times but this is the first report to gain deep insights into genetic platforms, promoters, and expression of the two coexisting bla genes with determination of entire nucleotide sequences of the two corresponding plasmids.
Insights
This study details carbapenem-resistant Klebsiella pneumoniae producing KPC-2 and CTX-M-55. It reveals the genetic platforms, promoters, and expression of these coexisting beta-lactamase genes on two plasmids.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant global health threat.
- Co-occurrence of different beta-lactamase genes, like KPC and CTX-M, in single bacterial isolates complicates treatment options.
Purpose of the Study:
- To elucidate the genetic basis and expression mechanisms of coexisting blaKPC-2 and blaCTX-M-55 genes in a CRKP strain.
- To determine the complete nucleotide sequences of the conjugative plasmids carrying these resistance genes.
Main Methods:
- Whole-genome sequencing and plasmid sequencing.
- Bioinformatic analysis to identify resistance genes, mobile genetic elements, and promoter regions.
- Analysis of gene expression and promoter activity.
Main Results:
- The CRKP strain 628 harbored two conjugative plasmids, p628-KPC (IncFIIK) and p628-CTXM (IncI1).
- blaKPC-2 was located within a Tn1722-based transposon and associated with a mercury resistance locus, forming a 34 kb resistance region.
- blaCTX-M-55 was mobilized by ISEcp1 within a transposition unit and was the sole resistance determinant on p628-CTXM.
- Both blaKPC-2 and blaCTX-M-55 exhibited constitutive expression driven by distinct promoters (P1/P2 for blaKPC-2, P1 for blaCTX-M-55).
Conclusions:
- This study provides the first in-depth characterization of the genetic platforms, promoters, and expression of coexisting blaKPC and blaCTX-M genes in K. pneumoniae.
- Understanding these genetic mechanisms is crucial for developing strategies to combat the spread of carbapenem resistance.
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