Carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa: characterization of carbapenemase genes and E-test evaluation of colistin-based combinations

Figures & data

Table 1 bla genes detected by PCR in carbapenem resistant isolates

	blagenes	Class A		Class B			Class D				Total + ve
Isolates		blaKPC	blaGES	blaIMP	blaVIM	blaNDM	blaOXA-23	blaOXA-24	blaOXA-58	blaOXA-143	30 (100)
A baumannii	No	0	15	0	0	20	27	0	0	0
	(%)	(0)	(50)	(0)	(0)	(66.7)	(90)	(0)	(0)	(0)
	Total	15 (50%)		20 (66.7%)			27 (90%)
P aeruginosa	No	0	9	4	11	6	NA	NA	NA	NA	21 (95.5)
	(%)	(0)	(40.9)	(18.2)	(50)	(27.3)
	Total	9 (40.9%)		18 (81.8%)			Na (not applicable)

Abbreviations: A baumannii, Acinetobacter baumannii; P aeruginosa, Pseudomonas aeruginosa; PCR, polymerase chain reaction.

Table 2 Carbapenemase gene distribution among isolates, the corresponding meropenem MIC range and the effect of antimicrobial combination

No. of isolates	Carbapenemase genes detected	Meropenem MIC range^a	FIC (range)/effect of combination
			Colistin/meropenem	Colistin/tigecycline
Acinetobacter baumannii
15	OXA-23 + NDM	32-512	(0.75-1.17) add/indiff	(0.43-1.17) syn/add/indiff
9	OXA-23 + GES	64-512	(0.75-1.17) add/indiff	(0.46-1.26) syn/add/indiff
2	NDM + GES	256	(0.83) additive	(0.49-1) syn/add
3	OXA-23 + GEs + NDM	128-256	(1-1.17) add/indiff	(0.49-0.92) syn/add
1	GEs only	32	(0.87) add	(1.16) indiff
Pseudomonas aeruginosa
5	VIM only	64-512	(0.36-0.81) syn/add	NA (not applicable)
3	GEs only	64-512	(0.28-0.58) syn/add	NA
4	NDM only	32-128	(0.22-0.56) syn/add	NA
4	VIM + GEs	128-256	(0.31-0.75) syn/add	NA
2	IMP + GEs	64-128	(0.36-0.51) syn/add	NA
1	NDM + IMP	128	(0.73) add	NA
1	NDM + VIM	128	(0.28) syn	NA
1	VIM + IMP	128	(0.41) syn	NA
1	None detected	32	(0.36) syn	NA

Notes: ^aMeasured by broth microdilution method.

Abbreviations: FIC, fractional inhibitory concentration; MIC, minimum inhibitory concentration; add, additive; indiff, indifference; syn, synergy.

Figure 1 Colistin/tigecycline combination test (Acinetobacter baumannii).

Notes: The MIC of colistin was 0.75 µg/mL when tested alone but was 0.25 µg/mL when tested in combination. The MIC of tigecycline was 16 µg/mL when tested alone but was 3 µg/mL when tested in combination. This was an additive effect (isolate no. 12; Table S1).
Abbreviation: MIC, minimum inhibitory concentration.

Table 3 The effect of colistin based combinations against Acinetobacter baumannii and Pseudomonas aeruginosa

Antimicrobial combination	Synergy No (%)	Additive No (%)	Indifferent No (%)	Antagonism No (%)	Mean FIC (SD)	Min/Max FIC
COL/MEM
A. baumannii	0/30	25/30	5/30	0/30	0.95	0.75/1.17
	0%	(83.3%)	(16.7%)	0%	(0.18)
P. aeruginosa	14/22	8/22	0/22	0/22	0.49	0.22/0.81
	(63.6%)	(36.4%)	0%	0%	(0.26)
COL/TGC
A. baumannii	6/30	18/30	6/30	0/30	0.8	0.43/1.26
	(20%)	(60%)	(20%)	(0%)	(0.24)

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

Figure 2 Colistin/meropenem combination test (Pseudomonas aeruginosa).

Notes: The MIC of colistin was 0.75 µg/mL when tested alone but was 0.25 µg/mL when tested in combination. The MIC of meropenem was >32 µg/mL when tested alone but was 2 µg/mL when tested in combination. This was a synergistic effect (isolate no. 1; Table S2).
Abbreviation: MIC, minimum inhibitory concentration.

Table S1 Individual MIC values of tested agents, FIC values of tested combinations and corresponding carbapenemase gene/s for all Acinetobacter baumannii isolates

	MIC (μg/mL)			Colistin + tigecycline				Colistin + meropenem				blagene/s
	MEM	CL	TGC	CL	TGC	FIC	Combin.	CL	MEM	FIC	Combin.
1	256	0.5	12	0.125	3	0.5	synergy	0.25	32	1	additive	OXA-GES-NDM
2	128	1.5	6	0.75	3	1	additive	0.75	32	1	additive	OXA-GES
3	256	0.38	16	0.125	8	0.83	additive	0.125	32	0.83	additive	OXA-NDM
4	128	1	16	0.5	8	1	additive	0.38	32	0.88	additive	OXA-GES
5	128	0.5	8	0.125	3	0.625	additive	0.25	32	1	additive	OXA-NDM
6	64	0.75	6	0.5	1	0.83	additive	0.25	32	0.83	additive	OXA-NDM
7	64	1.5	2	0.5	1	0.83	additive	1.0	32	1.17	indifferent	OXA-NDM
8	512	0.75	3	0.25	1	0.66	additive	0.25	32	0.83	additive	OXA-GES
9	32	0.5	3	0.125	1.5	0.75	additive	0.25	16	1	additive	OXA-NDM
10	64	0.75	16	0.25	2	0.46	synergy	0.25	32	0.83	additive	OXA-GES
11	128	0.75	8	0.38	2	0.75	additive	0.25	32	0.83	additive	OXA-NDM
12	512	0.75	16	0.25	3	0.52	additive	0.25	32	0.83	additive	OXA-NDM
13	128	1	3	0.25	2	0.92	additive	0.50	32	1	additive	OXA-GES
14	512	1	16	0.25	3	0.43	synergy	0.25	32	0.75	additive	OXA-NDM
15	128	1	3	0.38	1	0.71	additive	0.38	32	0.88	additive	OXA-NDM
16	256	0.38	6	0.19	3	1	additive	0.125	32	0.83	additive	GES-NDM
17	32	1.5	8	1	4	1.16	indifferent	0.75	12	0.87	additive	GES
18	512	0.5	6	0.19	3	0.88	additive	0.25	32	1	additive	OXA-NDM
19	128	0.5	8	0.38	4	1.26	indifferent	0.25	32	1	additive	OXA-GES
20	128	0.75	6	0.38	4	1.17	indifferent	0.25	32	0.83	additive	OXA-NDM
21	128	1	6	0.38	4	1.05	indifferent	0.38	32	0.88	additive	OXA-GES
22	128	0.75	8	0.5	2	0.92	additive	0.50	32	1.17	indifferent	OXA-GES-NDM
23	256	0.75	6	0.25	1	0.49	synergy	0.25	32	0.83	additive	GES-NDM
24	128	0.75	2	0.25	0.75	0.71	additive	0.25	32	0.83	additive	OXA-NDM
25	64	0.75	16	0.5	1	0.73	additive	0.50	32	1.17	indifferent	OXA-GES
26	512	0.75	16	0.5	6	1.04	indifferent	0.38	32	1.01	indifferent	OXA-NDM
27	256	0.75	6	0.25	1	0.49	synergy	0.38	32	1.01	indifferent	OXA-GES-NDM
28	64	1	12	0.25	3	0.5	synergy	0.25	32	0.75	additive	OXA-GES
29	256	0.38	8	0.19	3	0.875	additive	0.125	32	0.83	additive	OXA-NDM
30	64	1.5	2	1	1	1.16	indifferent	0.75	32	1	additive	OXA-NDM

Abbreviations: FIC, fractional inhibitory concentration; MIC, minimum inhibitory concentration; CL, colistin; MEM, meropenem; TGC, tigecycline.

Table S2 Individual MIC values of tested agents, FIC values of tested combination and corresponding carbapenemase gene/s for all Pseudomonas aeruginosa isolates

	MIC (μg/mL)		Colistin + meropenem combination				blagene/s
	Meropenem	Colistin	Colistin MIC	Meropenem MIC	FIC	Combination
1	128	0.75	0.25	2	0.36	synergy	GES-VIM
2	256	16	3	8	0.31	synergy	GES-VIM
3	128	1	0.38	2	0.41	synergy	VIM-IMP
4	128	1	0.5	4	0.56	additive	NDM
5	64	2	1	1	0.51	additive	GES-IMP
6	128	1.5	0.38	2	0.28	synergy	NDM
7	256	1	0.25	8	0.38	synergy	VIM
8	128	0.75	0.5	4	0.73	additive	IMP-NDM
9	128	0.75	0.25	2	0.36	synergy	GES-IMP
10	512	2	1	16	0.75	additive	VIM
11	32	2	0.38	1	0.22	synergy	NDM
12	256	1.5	0.75	16	0.75	additive	GES-VIM
13	128	2	1	4	0.56	additive	NDM
14	512	0.75	0.25	16	0.58	additive	GES
15	128	0.5	0.125	2	0.28	synergy	VIM-NDM
16	64	0.38	0.125	1	0.34	synergy	GES
17	256	1	0.25	8	0.38	synergy	VIM
18	64	1.5	0.50	2	0.36	synergy	VIM
19	256	8	2	8	0.38	synergy	GES-VIM
20	128	2	1.5	4	0.81	additive	VIM
21	32	0.75	0.25	1	0.36	synergy	-
22	128	0.5	0.125	2	0.28	synergy	GES

Notes: Meropenem MIC values >32 µg/mL (highest concentration on E-test strip) were calculated as 64 µg/mL (two-fold dilution above highest concentration on E-test strip).

Abbreviations: FIC, fractional inhibitory concentration; MIC, minimum inhibitory concentration.