In vitro and in vivo Effect of Antimicrobial Agent Combinations Against Carbapenem-Resistant Klebsiella pneumoniae with Different Resistance Mechanisms in China

Figures & data

Figure 1 The difference in MIC values between different types of carbapenemase. (A) Meropenem (MEM); (B) tigecycline (TGC); (C) colistin (COL); (D) amikacin (AK).

Notes: ***p<0.001; **p<0.01.

Table 1 Isolates Used in the Time–Kill Assay and G. mellonella Survival Assay

Strain No.	Organism	Specimen	Carbapenemase	MIC (mg/L) [Interpretation]
				MEM	TGC	COL	AK
D16	K. pneumoniae	Blood	KPC-2	64 [R]	0.5 [S]	1 [S]	8 [S]
L13	K. pneumoniae	Bile	NDM-1	64 [R]	0.25 [S]	0.5 [S]	4 [S]
L34	K. pneumoniae	Blood	IMP-4	8 [R]	1 [S]	0.5 [S]	128 [R]
P5	K. pneumoniae	Sputum	VIM-1	8 [R]	8 [R]	0.5 [S]	4 [S]
P13	K. pneumoniae	Sputum	OXA-48	4 [R]	2 [S]	0.25 [S]	2 [S]
Y105	K. pneumoniae	Blood	Negative	16 [R]	0.25 [S]	0.5 [S]	16 [R]

Abbreviations: MEM, meropenem; TGC, tigecycline; COL, colistin; AK, amikacin; R, resistant; S, susceptible.

Table 2 Susceptibility of 58 Clinical CRKps to Four Antimicrobial Agents

Antimicrobial	MIC (mg/L)			Susceptibility [n (%)]
	Range	MIC50	MIC90	S	I	R
Meropenem	2 to >256	16	64	0 (0)	4 (6.9)	54 (93.1)
Tigecycline	0.12 to 8	0.5	2	53 (91.3)	4 (6.9)	1 (1.7)
Colistin	0.06 to 128	0.5	1	54 (93.1)	–	4 (6.9)
Amikacin	1 to >256	32	>256	29 (50)	3 (5.2)	26 (44.8)

Abbreviations: MIC, minimum inhibitory concentration; S, susceptible; I, intermediate; R, resistant.

Table 3 In vitro Combination Effect of Different Regimens Against CRKps with Different Resistance Mechanisms Using the Checkerboard Assay

Resistance Mechanism	Drug Combination	Synergy	Partial Synergy	Additivity	Indifference	Antagonism	Synergy+Partial Synergy
		FICI ≤0.5	0.5<FICI<1	FICI=1	1<FICI<4	FICI ≥4
blaKPC-2 (n=21)	MEM+TGC	0 (0)	6 (28.6)	10 (47.6)*	5 (23.8)	0 (0)	6 (28.6)
	TGC+COL	1 (4.8)	7 (33.3)	3 (14.3)	10 (47.6)*	0 (0)	8 (38.1)
	MEM+AK	9 (42.9)*	6 (28.6)	4 (19.0)	2 (9.5)	0 (0)	15 (71.4)
	TGC+AK	6 (28.6)	10 (47.6)*	5 (23.8)	0 (0)	0 (0)	16 (76.2)
	MEM+COL	1 (4.8)	4 (19.0)	5 (23.8)	11 (52.4)*	0 (0)	5 (23.8)
	AK+COL	3 (14.3)	2 (9.5)	13 (61.9)*	3 (14.3)	0 (0)	5 (23.8)
blaNDM-1 (n=11)	MEM+TGC	0 (0)	4 (36.4)	5 (45.5)*	2 (18.2)	0 (0)	4 (36.4)
	TGC+COL	0 (0)	1 (9.0)	2 (18.1)	8 (72.7)*	0 (0)	1 (9.1)
	MEM+AK	3 (27.3)	6 (54.5)*	1 (9.0)	1 (9.0)	0 (0)	9 (81.8)
	TGC+AK	1 (9.0)	5 (45.5)*	2 (18.1)	3 (27.3)	0 (0)	6 (54.5)
	MEM+COL	1 (9.0)	3 (27.3)	1 (9.0)	6 (54.5)*	0 (0)	4 (36.4)
	AK+COL	0 (0)	3 (27.3)	4 (36.4)*	4 (36.4)*	0 (0)	3 (27.3)
blaIMP (n=13)	MEM+TGC	1 (7.7)	1 (7.7)	4 (30.8)	7 (53.8)*	0 (0)	2 (15.4)
	TGC+COL	0 (0)	4 (30.8)	1 (7.7)	8 (61.5)*	0 (0)	4 (30.8)
	MEM+AK	0 (0)	6 (46.2)*	1 (7.7)	6 (46.2)*	0 (0)	6 (46.2)
	TGC+AK	2 (15.4)	6 (46.2)*	4 (30.8)	1 (7.7)	0 (0)	8 (61.5)
	MEM+COL	2 (15.4)	6 (46.2)*	4 (30.8)	1 (7.7)	0 (0)	8 (61.5)
	AK+COL	1 (7.7)	5 (38.5)*	2 (15.4)	5 (38.5)*	0 (0)	6 (46.2)
blaVIM-1 (n=1)	MEM+TGC	0 (0)	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0.0)
	TGC+COL	1 (100.0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (100.0)
	MEM+AK	1 (100.0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (100.0)
	TGC+AK	0 (0)	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0.0)
	MEM+COL	0 (0)	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0.0)
	AK+COL	0 (0)	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0.0)
blaOXA-48 (n=1)	MEM+TGC	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0)	0 (0.0)
	TGC+COL	0 (0)	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0.0)
	MEM+AK	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0)	0 (0.0)
	TGC+AK	0 (0)	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0.0)
	MEM+COL	0 (0)	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0.0)
	AK+COL	0 (0)	0 (0)	0 (0)	1 (100.0)	0 (0)	0 (0.0)
Porin loss (n=11)	MEM+TGC	2 (18.2)	3 (27.3)*	3 (27.3)*	3 (27.3)*	0 (0)	5 (45.5)
	TGC+COL	2 (18.2)	5 (45.5)*	0 (0)	4 (36.4)	0 (0)	7 (63.6)
	MEM+AK	2 (18.1)	3 (27.3)	4 (36.4)*	2 (18.1)	0 (0)	5 (45.5)
	TGC+AK	0 (0)	6 (54.5)*	5 (45.5)	0 (0)	0 (0)	6 (54.5)
	MEM+COL	1 (9.0)	3 (27.3)	2 (18.1)	5 (45.5)*	0 (0)	4 (36.4)
	AK+COL	0 (0)	1 (9.0)	7 (63.6)*	3 (27.3)	0 (0)	1 (9.1)

Note: Date are presented as numbers (percentage %). *The maximum part in whole data in corresponding group.

Abbreviations: MEM, meropenem; TGC, tigecycline; COL, colistin; AK, amikacin.

Figure 2 In vitro time–kill assay using meropenem (MEM), colistin (COL), tigecycline (TGC) and amikacin (AK), either alone or in combination, against six CRKps with different resistance mechanisms

Figure 3 Survival rate of infected Galleria mellonella larvae treated with different drugs: meropenem (MEM), colistin (COL), tigecycline (TGC) and amikacin (AK), or mock-inoculated with sterile saline (controls).