Phenotypic and Enzymatic Comparative Analysis of the KPC Variants, KPC-2 and Its Recently Discovered Variant KPC-15

<div><p>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 <i>Klebsiella pneumoniae</i> 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.</p></div

Kinetic parameters for KPC-15 and KPC-2 enzymes.

<p>Kinetic parameters for KPC-15 and KPC-2 enzymes.</p

The susceptibility for strains of <i>E. coli</i> J53Az^R, Kp1241, Kp1769, and transconjugants (J53Az^R-Kp1241 and J53Az^R-Kp1769).

<p>Clinical breakpoints of MICs for the antimicrobial agents see the reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111491#pone.0111491-CLSI1" target="_blank">[25]</a>.</p><p>The susceptibility for strains of <i>E. coli</i> J53Az^R, Kp1241, Kp1769, and transconjugants (J53Az^R-Kp1241 and J53Az^R-Kp1769).</p

Promoter elements of the <i>bla</i>_KPC-15 and <i>bla</i>_TEM genes in 8.997 kb-length nucleotide sequence.

<p>The sequence provided the upstream and downstream regions of <i>bla</i>_KPC-15 structural genes. The <i>tnpA</i> gene, which was upstream of the <i>bla</i>_KPC-15 (3,020 bp) gene, was homologous to a putative IS<i>Kpn8</i> transposon, and the downstream region of the <i>bla</i>_KPC-15 gene (1,320 bp) was homologous to a putative IS<i>Kpn6</i>-like transposon. The nucleotides upstream of the <i>bla</i>_KPC-15 and <i>bla</i>_TEM-12 gene translational start codons were shown in the box. The putative −10 promoter elements of the <i>bla</i>_KPC-15 gene were shown as <i>gattaa</i>, 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 <i>bla</i>_TEM-12 gene were shown as <i>tataac</i> and <i>ttattg</i>, 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.</p

Comparison of amino acid sequence alignments of KPC carbapenemases.

<p>KPC-15 was recently identified as a carbapenemase in <i>Klebsiella pneumonae</i> from the Taizhou Municipal Hospital of China. The data within parentheses are GenBank accession numbers.</p

Minimum Evolution tree of amino acid sequences for KPC-2 to KPC-17.

<p>KPC-15 carbapenemase was our newly discovered and appeared to be homogenous with KPC-4 closely. The amino acid sequences of KPCs based on the data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111491#pone-0111491-g001" target="_blank">Figure 1</a>. This comparison was designed and analysed using MEGA 5.05 software.</p

Sequences of primers utilised to determine the <i>bla</i>_KPC genetic environment in this study.

<p>Sequences of primers utilised to determine the <i>bla</i>_KPC genetic environment in this study.</p