Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae

Figures & data

Table 1. Resistance and virulence genes in three K. pneumoniae strains and mutations in the coding region of strains KP29499 and KP30086 using KP29105 as reference.

Nucleotide mutations

Amino acid change

Resistance genes

Virulence genes

Position

KP29105

KP29499

KP30086

KP29105

KP29499

KP30086

Gene

Product

KP29105

KP29499

KP30086

KP29105

KP29499

KP30086

651702

G

G

T

A

A

A→S

pduE

Glycerol dehydratase small subunit

blaCTX-M-15, qnrS1, aph(3’’)-Ib, aph [6]-Id, blaTEM-1B, sul2, blaSHV-11, oqxA, oqxB, fosA

blaCTX-M-15, qnrS1, aph(3’’)-Ib, aph [6]-Id, blaTEM-1B, sul2, blaSHV-11, oqxA, oqxB, fosA

aph(3’’)-Ib, aph [6]-Id, blaTEM-1B, sul2, blaSHV-11, oqxA, oqxB, fosA fosA

chromosome: clbB, irp1 plasmid: iroBCDN, iucABCDiutA, rmpA, rmpA2

1593899

T

G

G

L

L→R

R

ompK36

Outer membrane protein C

1682820

C

T

T

A

A→V

V

apbC

Iron-sulphur cluster carrier protein

3803414

C

C

T

V

V

V→V

mdtE

Co/Zn/Cd efflux system membrane fusion protein

4501692

G

G

T

I

I

I→I

mrcB

Penicillin-binding protein 1B

4550720

G

G

A

A

A

A→T

-

Hypothetical protein

4552605

C

C

A

A

A

A→S

lpdA

Dihydrolipoyl dehydrogenase

5406044

C

T

T

P

P→S

S

cpxA

Histidine kinase

Figure 1. Genomic profiles of these 3 clinical K. pneumoniae strains KP29105, KP29499 and KP30086. (a) Circular maps of plasmids in these 3 strains (b) Major structural features of the multi-resistance plasmids in the 3 K. pneumoniae isolates. pKP29499–2 integrated an IncR type plasmid after an insertion sequence IS6 of pKP29105–2, and bla_CTX-M-15 becomes bla_CTX-M-71 by single amino acid substitution. Whereas pKP30086–2 had a large fragment deletion compared with pKP29499–2, and the bla_CTX-M gene was located in this fragment (figure 1B). Boxed arrows indicate the positions of open reading frames (ORFs) and their directions of transcription. ORFs encoding resistance genes are portrayed by red arrows. Green arrows indicate insertion sequences. Gray areas denote more than 95% DNA identity between sequences. (c) Resistance genes in the 3 clinical K. pneumoniae strains. Asterisks represent genes with mutations.

Table 2. Antibiotic resistance characteristics (MICs, mg/L) of 3 clinical K. pneumoniae isolates and the transconjugant, transformants and mutants.

Antibiotics	KP29015	KP29499	KP30086	J53	Transconjugant	DH5α	DH5α (CTX-M-15)	DH5α (CTX-M-71)	KP30086+CTX-M-71	KP29499-ΔCTX-M-71	KP29499-ΔompK36Comp
Piperacillin	≥128	≥128	≥128	≤4	≥128	≤4	≥128	≥128	≥128	≥128	≥128
TZP	≥128	≥128	≥128	≤4	≤4	≤4	≤4	≤4	≥128	64	≥128
Ceftazidime	>512	512	≤1	≤0.5	32	≤0.5	64	64	256	≤1	256
CZA	1	32	0.5	≤0.125	1	≤0.125	0.25	1	16	2	4
Cefotaxime	>512	>512	≤0.5	≤0.5	512	≤0.5	256	256	512	≤0.5	>512
Cefepime	>512	>512	1	≤0.5	512	≤0.5	32	128	512	1	>512
Aztreonam	>256	>256	0.5	≤0.25	128	≤0.25	64	128	>256	1	>256
Imipenem	0.25	2	0.5	≤0.125	0.25	≤0.125	≤0.125	≤0.125	2	2	2
Meropenem	≤0.125	16	0.5	≤0.125	0.25	≤0.125	≤0.125	≤0.125	8	1	8
Ertapenem	≤0.125	64	2	≤0.125	0.5	≤0.125	≤0.125	≤0.125	64	4	16

TZP, Piperacillin-tazobactam; CZA, Ceftazidime-avibactam; -, Intrinsic resistance.

Table 3. Antibiotic resistance characteristics (MICs, mg/L) of K. pneumoniae strain KP29499 to ceftazidime, ceftazidime-avibactam, meropenem and ceftriaxone.

Antibiotics	PMBN(-)	PMBN(+)
Ceftazidime	512	16
Ceftazidime-avibactam	32	1
Meropenem	16	4
Cefotaxime	>512	512

PMBN, polymyxin B nonapeptide.

Figure 2. Porin profiles and growth curves of K. pneumoniae strains KP29105, KP29499 and KP30086. (a) Relative expression at the transcriptional level of genes ompK35, ompK36 and bla_CTX-M. Data were normalized to the expression levels of gene rpoB. K. pneumoniae NTUH-K2044 and KP29105 were set as the basic expression for porins and CTX-M, respectively. (b) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of outer membrane proteins. K. pneumoniae NTUH-K2044 was used as reference. (c) Growth curves of the K. pneumoniae isolates. Three independent experiments were carried out. The significance level was as follows: *p < 0.05.

Castanheira M, Doyle TB, Hubler C, et al. Ceftazidime-avibactam activity against a challenge set of carbapenem-resistant enterobacterales: Ompk36 L3 alterations and β-lactamases with ceftazidime hydrolytic activity lead to elevated MIC values. Int J Antimicrob Agents. 2020;56(1):106011. doi: 10.1016/j.ijantimicag.2020.106011

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Data Availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.