Molecular mechanisms responsible KPC-135-mediated resistance to ceftazidime-avibactam in ST11-K47 hypervirulent Klebsiella pneumoniae

Figures & data

Figure 1. Morphological characteristics and virulence of clinical isolate K. pneumoniae HSD. (a) Transmission electron microscopy (TEM) image of K. pneumoniae HSD with negative staining using methylamine tungstate. (b) TEM image of a transverse section of embedded K. pneumoniae HSD. (c) Colony morphology of K. pneumoniae HSD on Columbia blood agar. (d) Survival rates of G. mellonella infected with K. pneumoniae HSD and biofilm formation in K. pneumoniae HSD. Statistical analysis was performed using unpaired t-tests. ****, P < 0.0001.

Table 1. Minimal inhibitory concentrations (MICs) of K. pneumoniae HSD, bla_KPC-135-positive transformants, and recipient strains.

Antibiotics	MIC (μg/mL)^a
	K. pneumonia HSD	E.coli DH5α-K. pneumoniae HSD	E.coli DH5α	K. pneumonia 13882-KPC-2	K. pneumonia 13882-KPC-135	K. pneumonia 13882-pHSG398
Aztreonam	>128	16	≤0.06	128	0.5	≤0.06
Aztreonam-avibactam	2	0.125	≤0.06	0.25	0.25	≤0.06
Ceftazidime	>128	32	0.125	16	128	≤0.25
Ceftazidime-avibactam	>128	16	≤0.25	1	32	≤0.25
Amoxicillin	>32	>32	≤0.06	128	16	8
Amoxicillin-clavulanic acid	>32	8	≤0.06	128	4	1
Imipenem	0.5	0.125	≤0.06	8	0.125	0.125
Imipenem-relebactam	0.5	0.125	≤0.06	0.25	0.125	0.125
Meropenem	4	≤0.06	≤0.06	16	≤0.06	≤0.06
Meropenem-vaborbactam	1	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
Ertapenem	32	≤0.125	≤0.125	2	≤0.125	≤0.125
Ceftriaxone	>32	>32	≤0.5	>32	16	≤0.5
Cefepime	128	0.5	≤0.06	4	2	≤0.06
Amikacin	>128	>128	≤1	≤1	≤1	≤1
Ciprofloxacin	>8	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
Levofloxacin	>16	≤0.125	≤0.125	≤0.125	≤0.125	≤0.125
Colistin	0.5	0.5	0.25	0.25	0.25	0.25
Polymyxin B	0.5	0.25	0.25	0.25	0.25	0.25
Trimethoprim-sulfamethoxazole	1	≤0.25	≤0.25	≤0.25	≤0.25	≤0.25
Tigecycline	0.5	0.125	≤0.06	≤0.06	≤0.06	≤0.06

^a E.coli DH5α-K. pneumoniae HSD; E.coli DH5α was transformed by wild-type plasmid carrying bla_KPC-135 gene from K. pneumoniae HSD. K. pneumonia 13882-pHSG398, 13882-KPC-2, and 13882-KPC-135; K. pneumonia 13882 was transformed by pHSG398 empty plasmid and recombinant plasmid carrying bla_KPC-2 and bla_KPC-135, respectively.

Figure 2. Structure and function of the β-lactamases KPC-135 and KPC-2 and their fitness costs. (a) Structure of KPC-2 (PDB ID: 3DW0) and KPC-135 enzyme (modelled by homology). The catalytic pockets of enzymes were depicted. (b) Kinetic parameters of purified KPC-2 (blue) and KPC-135 (yellow). The bar graph: MICs of bla_KPC cloning strains are shown on the left vertical axis in base-2 logarithmic scale. The line graph: kcat/Km values of the enzymes were plotted on the right vertical axis. (c) IC₅₀ of β-lactamase inhibitors against KPC-2 (blue) and KPC-135 (yellow). The bar graph: The reduced multiples of MICs of bla_KPC cloning strains after addition of inhibitors to antibiotics are shown on the left vertical axis in base-2 logarithmic scale, with ceftazidime highlighted in blue. The line graph: The IC₅₀ values were represented on the right vertical axis in an inverted base-10 logarithmic scale. (d) Relative fitness and (e) the bacterial growth curve of the K. pneumonia 13882-KPC-2 and 13882-KPC-135 cloning strains. Statistical analysis was performed using unpaired t-tests. ****, P < 0.0001.

Figure 3. Comparative plasmid map of pK. pneumoniae HSD-KPC, p2b2, and p15WZ61-KPC. From the inside to the outside circles: circle 1, scale; circle 2, GC content; circle 3, GC Skew; circle 4, ring diagram of p15WZ61-KPC (GenBank accession number ON777848); circle 5, ring diagram of p2b2 (GenBank accession number CP034125); circle 6, functionally classified genes. MDR, bla_KPC, and insertion regions of aligned sequences have been annotated.

Figure 4. Genetic environment of Tn6296-associated bla_KPC regions. Schematic structures of Tn6296 transposons surrounding the bla_KPC-135 gene in plasmid pK. pneumoniae HSD-KPC are presented, along with a comparison to related genetic elements. Plasmids encoding KPC-2 mutants are compared as follows: pK. pneumoniae HSD-KPC (Anhui, China, OP205646), pZHKPC2 (Shanghai, China, OM928503), pLWKPC (Shanghai, China, MT875328 and MT550690), pZHKPC1 (Shanghai, China, OM928502), pCK1008-KPC-123 (Hangzhou, China, ON209376), pKPC-H2 (Shanghai, China, MT550691), pR20-14 (Hangzhou, China, CP094852), pP10159-3 (Chongqing, China, MF072963), pZYPA187 (Hangzhou, China, CP133754), pE31A (Jinan, China, CP067418), and pUC331 (Temuco, Chile, KR052099). The phylogenetic tree of the bla_KPC region is constructed. Resistance genes are shown in red, resolvase genes (tnpR) in green, transposase genes (tnpA) in yellow, other genes in white, inverted repeat left (IRL) and inverted repeat right (IRR) in black boxes. Among them, the tnpA of IS26, the mutated tnpA of Tn2, and mutated tnpA and tnpR of TnAs1 are presented. Shading below the nucleotide sequences indicates nucleotide identity (grey: ≥ 99%, light grey: 85%−99%). Truncated and inserted sequences are represented by ellipses dots and triangles, respectively. Comparative analysis of the alignment of the (putative) promoter regions of bla_KPC isoforms and the amino acids they encode. Highlighted regions include the putative – 35 and – 10 boxes of the promoters, the transcription start site (TSS, + 1), the putative ribosome binding site (RBS), the bla_KPC gene start codon (ATG) and the truncated bla_TEM-1B gene. The alignment quality of the KPC is based on Blosum62 scores.

Data availability statement

The genomic information of K. pneumoniae HSD has been deposited at DDBJ/ENA/GenBank under the accession number JBBVMI000000000, with the nucleotide sequence of bla_KPC-135 deposited separately under accession number OP205646.1.