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Phenotypic and enzymatic comparative analysis of the KPC variants, KPC-2 and its recently discovered variant KPC-15.

Wang D, Chen J, Yang L, Mou Y, Yang Y - PLoS ONE (2014)

Bottom Line: Steady-state kinetic parameters showed the catalytic efficiency of KPC-15 was higher than that of KPC-2 for all tested antibiotics in this study.The catalytic efficiency of KPC-15 caused resistance to β-lactam antibiotics was higher than that of KPC-2.Meanwhile, an evolutionary transformation changed KPC from an efficient carbapenemase to its variants (KPC-15) with better ceftazidimase catalytic efficiency, and the old antibiotics polymyxin B and colistin might play a role in the therapy for multi-resistant strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Lab Medicine, Taizhou Municipal Hospital affiliated with Taizhou University and the Institute of Molecular Diagnostics of Taizhou University, Zhejiang, China.

ABSTRACT
Sixteen different variants (KPC-2 to KPC-17) in the KPC family have been reported, and most current studies are focusing on KPC-2 and KPC-3. The KPC-15 variant, which isolated from Klebsiella pneumoniae in a Chinese hospital, was a recently discovered KPC enzyme. To compare the characteristics of KPC-15 and KPC-2, the variants were determined by susceptibility testing, PCR amplification and sequencing, and study of kinetic parameters. The strain harboring the KPC-15 showed resistance to 18 conventional antimicrobial agents, especially to cabapenem antibiotics, and the strain involving the KPC-2 also indicated resistance to cabapenem antibiotics, but both strains were susceptible to polymyxin B and colistin. The conjugation experiments showed that the changes of MIC values to the antibiotics were due to the transferred plasmids. The differences of amino acids were characterised at sites of 119 leucine and 146 lysine with KPC-15 and KPC-2. The minimum evolution tree indicated the KPC alleles evolution, and showed that the KPC-15 appeared to be homogenous with KPC-4 closely. Steady-state kinetic parameters showed the catalytic efficiency of KPC-15 was higher than that of KPC-2 for all tested antibiotics in this study. The catalytic efficiency of KPC-15 caused resistance to β-lactam antibiotics was higher than that of KPC-2. Meanwhile, an evolutionary transformation changed KPC from an efficient carbapenemase to its variants (KPC-15) with better ceftazidimase catalytic efficiency, and the old antibiotics polymyxin B and colistin might play a role in the therapy for multi-resistant strains.

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Promoter elements of the blaKPC-15 and blaTEM genes in 8.997 kb-length nucleotide sequence.The sequence provided the upstream and downstream regions of blaKPC-15 structural genes. The tnpA gene, which was upstream of the blaKPC-15 (3,020 bp) gene, was homologous to a putative ISKpn8 transposon, and the downstream region of the blaKPC-15 gene (1,320 bp) was homologous to a putative ISKpn6-like transposon. The nucleotides upstream of the blaKPC-15 and blaTEM-12 gene translational start codons were shown in the box. The putative −10 promoter elements of the blaKPC-15 gene were shown as gattaa, labeled as −10 below, and there were no obvious −35 promoter elements to be discovered in the promoter region. The putative −10 and −35 promoter elements of the blaTEM-12 gene were shown as tataac and ttattg, labeled as −10 and −35 below the promoter region. The start sites of transcription were indicated as G by +1 residue. RBS was the abbreviation of the ribosome binding site.
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pone-0111491-g003: Promoter elements of the blaKPC-15 and blaTEM genes in 8.997 kb-length nucleotide sequence.The sequence provided the upstream and downstream regions of blaKPC-15 structural genes. The tnpA gene, which was upstream of the blaKPC-15 (3,020 bp) gene, was homologous to a putative ISKpn8 transposon, and the downstream region of the blaKPC-15 gene (1,320 bp) was homologous to a putative ISKpn6-like transposon. The nucleotides upstream of the blaKPC-15 and blaTEM-12 gene translational start codons were shown in the box. The putative −10 promoter elements of the blaKPC-15 gene were shown as gattaa, labeled as −10 below, and there were no obvious −35 promoter elements to be discovered in the promoter region. The putative −10 and −35 promoter elements of the blaTEM-12 gene were shown as tataac and ttattg, labeled as −10 and −35 below the promoter region. The start sites of transcription were indicated as G by +1 residue. RBS was the abbreviation of the ribosome binding site.

Mentions: The analysis of the 8,997 bp nucleotide sequence for Kp1241 was shown in Figure 3. There were 5 different genes in the sequence, including the genes tnpR, blaTEM-12 and blaKPC-15, and the transposons ISKpn8 and ISKpn6-like [24]. The sites of the transcriptional promoters for the blaTEM-12 and blaKPC-15 genes were indicated as +1 and the −10 and −35 regions were also shown; however, there were no obvious transcriptional promoters in tnpR, ISKpn8 and ISKpn6-like transposons. Between the tnpR gene and the ISKpn8 transposon, there was a 109 bp nucleotide interval; the ISKpn8 transposon nucleotide sequence was 3,020 bp. Between the ISKpn8 transposon and the blaTEM-12 gene, there was a 206 bp nucleotide interval, which included the site of the transcriptional promoter for the blaTEM-12 gene, +1 (g, start codon), −10 region (tataac) and −35 region (ttattg). Between the blaTEM-12 and blaKPC-15 genes, there was a 223 bp nucleotide interval, which included the site of the transcriptional promoter for the blaKPC-15 gene, +1 (g, start codon), −10 region (gattac) and RBS (aaggaa, the ribosome binding site), but no obvious −35 region was shown in the blaKPC-15 gene. Between the blaKPC-15 gene and the ISKpn6-like transposon, there was a 249 bp nucleotide interval, and the ISKpn6-like transposon had a 1,320 bp nucleotide sequence.


Phenotypic and enzymatic comparative analysis of the KPC variants, KPC-2 and its recently discovered variant KPC-15.

Wang D, Chen J, Yang L, Mou Y, Yang Y - PLoS ONE (2014)

Promoter elements of the blaKPC-15 and blaTEM genes in 8.997 kb-length nucleotide sequence.The sequence provided the upstream and downstream regions of blaKPC-15 structural genes. The tnpA gene, which was upstream of the blaKPC-15 (3,020 bp) gene, was homologous to a putative ISKpn8 transposon, and the downstream region of the blaKPC-15 gene (1,320 bp) was homologous to a putative ISKpn6-like transposon. The nucleotides upstream of the blaKPC-15 and blaTEM-12 gene translational start codons were shown in the box. The putative −10 promoter elements of the blaKPC-15 gene were shown as gattaa, labeled as −10 below, and there were no obvious −35 promoter elements to be discovered in the promoter region. The putative −10 and −35 promoter elements of the blaTEM-12 gene were shown as tataac and ttattg, labeled as −10 and −35 below the promoter region. The start sites of transcription were indicated as G by +1 residue. RBS was the abbreviation of the ribosome binding site.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4216079&req=5

pone-0111491-g003: Promoter elements of the blaKPC-15 and blaTEM genes in 8.997 kb-length nucleotide sequence.The sequence provided the upstream and downstream regions of blaKPC-15 structural genes. The tnpA gene, which was upstream of the blaKPC-15 (3,020 bp) gene, was homologous to a putative ISKpn8 transposon, and the downstream region of the blaKPC-15 gene (1,320 bp) was homologous to a putative ISKpn6-like transposon. The nucleotides upstream of the blaKPC-15 and blaTEM-12 gene translational start codons were shown in the box. The putative −10 promoter elements of the blaKPC-15 gene were shown as gattaa, labeled as −10 below, and there were no obvious −35 promoter elements to be discovered in the promoter region. The putative −10 and −35 promoter elements of the blaTEM-12 gene were shown as tataac and ttattg, labeled as −10 and −35 below the promoter region. The start sites of transcription were indicated as G by +1 residue. RBS was the abbreviation of the ribosome binding site.
Mentions: The analysis of the 8,997 bp nucleotide sequence for Kp1241 was shown in Figure 3. There were 5 different genes in the sequence, including the genes tnpR, blaTEM-12 and blaKPC-15, and the transposons ISKpn8 and ISKpn6-like [24]. The sites of the transcriptional promoters for the blaTEM-12 and blaKPC-15 genes were indicated as +1 and the −10 and −35 regions were also shown; however, there were no obvious transcriptional promoters in tnpR, ISKpn8 and ISKpn6-like transposons. Between the tnpR gene and the ISKpn8 transposon, there was a 109 bp nucleotide interval; the ISKpn8 transposon nucleotide sequence was 3,020 bp. Between the ISKpn8 transposon and the blaTEM-12 gene, there was a 206 bp nucleotide interval, which included the site of the transcriptional promoter for the blaTEM-12 gene, +1 (g, start codon), −10 region (tataac) and −35 region (ttattg). Between the blaTEM-12 and blaKPC-15 genes, there was a 223 bp nucleotide interval, which included the site of the transcriptional promoter for the blaKPC-15 gene, +1 (g, start codon), −10 region (gattac) and RBS (aaggaa, the ribosome binding site), but no obvious −35 region was shown in the blaKPC-15 gene. Between the blaKPC-15 gene and the ISKpn6-like transposon, there was a 249 bp nucleotide interval, and the ISKpn6-like transposon had a 1,320 bp nucleotide sequence.

Bottom Line: Steady-state kinetic parameters showed the catalytic efficiency of KPC-15 was higher than that of KPC-2 for all tested antibiotics in this study.The catalytic efficiency of KPC-15 caused resistance to β-lactam antibiotics was higher than that of KPC-2.Meanwhile, an evolutionary transformation changed KPC from an efficient carbapenemase to its variants (KPC-15) with better ceftazidimase catalytic efficiency, and the old antibiotics polymyxin B and colistin might play a role in the therapy for multi-resistant strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Lab Medicine, Taizhou Municipal Hospital affiliated with Taizhou University and the Institute of Molecular Diagnostics of Taizhou University, Zhejiang, China.

ABSTRACT
Sixteen different variants (KPC-2 to KPC-17) in the KPC family have been reported, and most current studies are focusing on KPC-2 and KPC-3. The KPC-15 variant, which isolated from Klebsiella pneumoniae in a Chinese hospital, was a recently discovered KPC enzyme. To compare the characteristics of KPC-15 and KPC-2, the variants were determined by susceptibility testing, PCR amplification and sequencing, and study of kinetic parameters. The strain harboring the KPC-15 showed resistance to 18 conventional antimicrobial agents, especially to cabapenem antibiotics, and the strain involving the KPC-2 also indicated resistance to cabapenem antibiotics, but both strains were susceptible to polymyxin B and colistin. The conjugation experiments showed that the changes of MIC values to the antibiotics were due to the transferred plasmids. The differences of amino acids were characterised at sites of 119 leucine and 146 lysine with KPC-15 and KPC-2. The minimum evolution tree indicated the KPC alleles evolution, and showed that the KPC-15 appeared to be homogenous with KPC-4 closely. Steady-state kinetic parameters showed the catalytic efficiency of KPC-15 was higher than that of KPC-2 for all tested antibiotics in this study. The catalytic efficiency of KPC-15 caused resistance to β-lactam antibiotics was higher than that of KPC-2. Meanwhile, an evolutionary transformation changed KPC from an efficient carbapenemase to its variants (KPC-15) with better ceftazidimase catalytic efficiency, and the old antibiotics polymyxin B and colistin might play a role in the therapy for multi-resistant strains.

Show MeSH
Related in: MedlinePlus