Mutations in porin LamB contribute to ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae

Figures & data

Table 1. Characteristics of the strains used in this study.

Strain	Sample source	Carbapenemase types	Serotype	MLST
220	sputum	KPC-2	K47	11
1419	sputum	KPC-2	K47	11
14192	sputum	KPC-2	K47	11
BL94	sputum	KPC-2	K47	11
1295	blood	KPC-2	K64	11
BL18	blood	KPC-2	K64	11
BL152	blood	KPC-2	K64	11
84082	blood	KPC-2	K64	11
P77	urine	KPC-2	K47	11
C4	urine	KPC-12	K47	11
Q38	urine	KPC-2	K19	15
Q35	aseptic humoral	KPC-2	K19	15
P152	aseptic humoral	KPC-2	K75	1296
Q30	aseptic humoral	KPC-2	K12	37

Note: KPC, Klebsiella pneumoniae carbapenemase; MLST, multi-locus sequence typing.

Table 2. Minimum inhibitory concentration (MIC) of various antibiotics for strains before and after induction.

Strain ID	MIC value (mg/L)^b
	CAZ-AVI		MEM		AMP		AMP-AVI		ATM		ATM-AVI		PB		GM		LVX		CRO		TGC		CAZ
	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a	0^a	50^a
220	4	4	≥64	≥64	≥128	≥128	128	128	>128	>128	2	2	2	2	≥128	≥128	16	16	≥128	≥128	0.25	0.25	1024	1024
1419	4	128	≥64	2	≥128	≥128	128	256	>128	>128	4	32	64	16	≥128	≥128	32	32	≥128	≥128	0.25	0.25	1024	2048
14192	4	32	≥64	≥64	≥128	≥128	128	512	>128	≥128	4	16	64	16	≥128	≥128	32	32	≥128	≥128	0.25	0.25	1024	>4096
BL94	2	16	≥64	32	≥128	≥128	128	64	>128	>128	2	2	0.5	0.5	≥128	≥128	≥128	32	≥128	≥128	1	0.5	1024	512
1295	4	8	≥64	≥64	≥128	≥128	128	128	>128	>128	2	4	1	1	≥128	≥128	64	64	≥128	≥128	0.25	0.25	512	512
BL18	4	32	≥64	≥64	≥128	≥128	256	1024	>128	>128	4	8	0.5	0.25	≥128	≥128	64	64	≥128	≥128	1	0.5	1024	4096
BL152	4	32	≥64	≥64	≥128	≥128	128	512	>128	>128	4	32	2	4	≥128	≥128	64	32	≥128	≥128	1	2	512	>4096
84082	4	32	≥64	≥64	≥128	≥128	128	64	>128	>128	4	4	32	32	≥128	≥128	32	16	≥128	≥128	0.5	1	1024	2048
P77	4	16	≥64	16	≥128	≥128	128	64	>128	>128	2	2	1	1	≥128	≥128	32	32	≥128	≥128	0.5	0.25	512	1024
C4	2	8	32	16	≥128	≥128	64	32	>128	>128	2	2	0.5	0.5	≥128	≥128	32	64	≥128	≥128	0.25	0.25	512	512
Q38	1	128	16	≥64	≥128	≥128	128	64	>128	>128	≤0.125	2	0.5	0.25	128	≥128	4	8	≥128	≥128	0.25	0.25	128	512
Q35	0.5	16	16	16	≥128	≥128	128	64	>128	>128	1	1	1	0.5	≥128	128	4	8	≥128	≥128	0.25	0.5	128	512
P152	0.25	4	16	0.125	≥128	≥128	64	8	>128	>128	≤0.125	0.25	16	2	0.5	0.5	≤0.125	≤0.125	≥128	>128	0.5	0.25	64	128
Q30	2	16	≥64	4	≥128	≥128	128	8	>128	128	1	1	0.25	0.5	0.5	1	32	64	≥128	≥128	0.25	0.25	128	512

Note: Abbreviations: CAZ-AVI: Ceftazidime-avibactam; MEM: Meropenem; AMP: Ampicillin; AMP-AVI: Ampicillin-avibactam; ATM: Aztreonam; ATM-AVI: Aztreonam-avibactam; PB: Polymyxin B; GM: Gentamicin; LVX: Levofloxacin; CRO: Ceftriaxone; TGC: Tigecycline. ^a 0 indicates strains before in vitro induction; 50 indicates induced strains after 50 passages.^b MIC values above resistance breakpoint are shown in bold according to CLSI. The MIC values of AMP-AVI and ATM-AVI are not in bold because their resistance breakpoints are not available. The MIC results of tigecycline were determined according to the US Food and Drug Administration standard.

Table 3. Resistance to CAZ-AVI increased in a stepwise manner following passage with CAZ-AVI.

Strain ID	Minimum inhibitory concentration (MIC) of each passage (mg/L)
	0	2	4	6	8	10	12	14	16	18	20	22	24	26	28	30	32	34	36	38	40	42	44	46	48	50
ATCC 700603^a	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
220	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	8	8	8	4	8	4	8	8	8
1419	4	16	16	8	8	16	16	16	32	32	32	32	32	64	32	32	32	32	32	64	32	32	32	32	64	128
14192	4	8	16	32	16	32	32	16	32	32	32	32	32	32	32	32	32	32	32	32	32	32	32	32	32	32
BL94	2	2	2	8	8	8	8	16	16	16	16	32	16	16	16	16	16	16	16	16	16	16	16	16	16	16
1295	4	8	4	4	4	4	4	4	4	8	4	4	4	8	8	4	4	8	4	4	8	8	8	8	8	8
BL18	4	4	8	8	8	8	8	16	16	16	16	16	16	16	16	16	16	32	16	32	16	16	16	16	32	32
BL152	4	4	8	8	8	16	8	8	8	8	16	8	16	8	16	16	16	16	32	32	32	32	32	32	32	32
84082	4	4	4	8	8	8	8	8	8	8	8	16	8	16	8	8	8	16	16	16	32	32	64	128	128	128
P77	2	2	2	4	4	2	4	4	2	2	2	2	2	2	2	2	2	4	2	2	2	4	4	8	16	16
C4	2	4	2	4	4	8	4	8	8	8	8	8	8	16	8	16	8	16	16	16	16	16	16	16	16	16
Q38	1	2	64	64	64	64	64	64	64	64	64	64	64	64	64	64	128	64	128	128	64	128	128	128	128	128
Q35	0.5	1	1	2	2	2	1	2	1	2	2	1	1	2	2	2	2	2	2	4	2	2	2	2	2	16
P152	0.25	0.5	0.5	0.5	0.5	0.5	1	2	2	2	4	1	4	2	1	2	2	2	4	4	4	4	4	4	4	4
Q30	2	2	1	2	1	1	1	2	1	2	2	1	2	2	2	2	2	2	2	2	2	2	2	2	2	16

^a ATCC 700603, the recommended strain by the Clinical & Laboratory Standards Institute for the MIC determination of CAZ-AVI, was passaged alongside the test strains in LB broth without antibiotics, acting as a negative control.

Table 4. Mutations identified in KPCs, LamB and PBP3 and expression level of bla_KPC during induction.

Strains	Mutations^a				Relative expression of blaKPC (P50/P0)^b(means ± SD)	Change of MICs^c of CAZ-AVI (mg/L)
	KPC ^d		porin LamB	PBP3
	KPC-2	KPC-12
BL94	E165_L166ins	-	R374L	W	0.86 ± 0.28	2→16
C4	-	R178S	R33H	W	1.33 ± 0.63	2→8
1419	L169P + S181 ins	-	W	W	1.12 ± 0.38	4→128
P77	A172V	-	R374L	W	1.26 ± 0.15	4→16
P152	E166_L167del	-	W	W	1.96 ± 1.19	0.25→4
Q30	E166_L167del	-	W	W	1.38 ± 0.40	2→16
BL152	W	-	W	W	2.45 ± 0.22	4→32
Q35	W	-	R374S	W	3.27 ± 0.04	0.5→16
Q38	W	-	R134P	W	2.05 ± 0.11	1→128
84082	W	-	W	L367Q	0.79 ± 0.21	4→32
BL18	W	-	W	W	5.83 ± 1.48	4→32
14192	W	-	W	W	1.40 ± 0.17	4→32

^a W,wild-type.

^b P50: strain after 50 passages; P0: strain before passages; the relative expression of bla_KPC was calculate by considering the bla_KPC expression at P0 as 1 for each strain; qRT-PCR data (relative expression of bla_KPC) are given as means ± standard deviation (SD) of the results from three independent experiments.

^c Change in MIC in each strain is presented as ‘MIC of P0 → MIC of P50’.

^d -, indicates gene not present in strain.

Table 5. Complementation experiment for LamB and PBP3.

Transformed plasmid	Strain	MIC value of CAZ-AVI (mg/L)				MIC value of CAZ (mg/L)
		Mutant strain	Mutant stain + empty vector	Mutant stain complemented with wild-type gene	MIC Fold change	Mutant strain	Mutant stain + empty vector	Mutant stain complemented with wild-type gene	MIC Fold change
pAClamB	P77-IR	16	16	16	1	1024	1024	1024	1
	C4-IS	16	16	8	0.5	512	512	256	0.5
	BL94-IR	16	16	8	0.5	512	512	256	0.5
	Q38-IR	64	64	32	0.5	512	512	256	0.5
pAC ftsI	84082-IR	128	128	4	0.03	2048	2048	1024	0.5

Note: The CAZ-AVI resistant strains after induction were named following the format of ‘original strain name -IR’ (e.g. P77-IR). The induced strains that were still susceptible to CZA-AVI are named as ‘original strain name -IS’ (e.g. P152-IS).

Table 6. Complementation experiment of KPCs.

Transformed plasmid	Strain	MIC value of CAZ-AVI (mg/L)				MIC value of CAZ (mg/L)
		WT strain	WT strain + empty vector	WT strain complemented with gene of interest	MIC Fold change	WT strain	WT strain + empty vector	WT strain complemented with gene of interest	MIC Fold change
pACBL94KPC	BL94	2	2	16	8	1024	1024	1024	1
pACC4KPC	C4	2	2	8	4	512	512	512	1
pAC1419KPC	1419	4	4	128	32	1024	1024	1024	1
pACP77KPC	P77	2	2	8	4	512	512	1024	2
pACP152KPC	P152	0.25	0.25	32	128	64	64	128	2
pACQ30KPC	Q30	2	2	32	16	128	128	256	2

Note: The recombinant plasmid's target fragments containing bla_KPC were obtained from each CAZ-AVI resistant strain. WT strains, wild type strains.

Figure 1. The bla_KPC expression levels in strains without mutations in KPC. The horizontal axis is the number of passages of induction. The vertical axis is the relative expression level compared to the strain before induction (expression = 1.0). The housekeeping gene rpoB was used as the endogenous reference gene. All RT-qPCR were carried out in triplicate. *P < 0.05 (Student’s t-tests).