Two types of colistin heteroresistance in Acinetobacter baumannii isolates

Figures & data

Table 1. Antibiotic susceptibility profiles and genotypes of A. baumannii isolates included in this study.

Isolate No.	Genotype	Minimum inhibitory concentration (MIC) (mg/L) ^a
		COL	A/S	CAZ	FEP	CIP	GEN	IMI	MER	SXT	P/T	T/C	TGC
Type I heteroresistance
SCH2	ST681	1 (S)	2/1 (S)	16 (S)	8 (S)	0.25 (S)	1 (S)	0.25 (S)	0.5 (S)	2/38 (S)	128/4 (R)	16/2 (S)	0.5 (S)
SCH39	ST229	1 (S)	32/16 (R)	64 (R)	64 (R)	4 (R)	16 (R)	16 (R)	16 (R)	32 /608 (R)	128/4 (R)	128/2 (R)	1 (S)
SCH105	ST191	1 (S)	32/16 (R)	64 (R)	64 (R)	4 (R)	16 (R)	16 (R)	16 (R)	16/1216 (R)	128/4 (R)	128/2 (R)	2 (S)
Type II heteroresistance
SCH91	ST191	1 (S)	32/16 (R)	64 (R)	64 (R)	4 (R)	16 (R)	16 (R)	16 (R)	32/608 (R)	128/4 (R)	128/2 (R)	2 (S)
Sch113	ST191	1 (S)	16/8 (I)	64 (R)	64 (R)	4 (R)	16 (R)	16 (R)	16 (R)	16/304 (R)	128/4 (R)	128/2 (R)	2 (S)
K20-B-871	ST191	2 (S)	64/32 (R)	>64 (R)	>64 (R)	>64 (R)	>64 (R)	>64 (R)	>64 (R)	64/1216 (R)	>256/4 (R)	128/2 (R)	2 (S)

^a CST, colistin; A/S, ampicillin/sulbactam; CAZ, ceftazidime; FEP, cefepime; CIP, ciprofloxacin; GEN, gentamicin; IMI, imipenem; MER, meropenem; SXT, trimethoprim/sulfamethoxazole; P/T, piperacillin/tazobactam; T/C, ticarcillin/clavulanate; TGC, tigecycline.

Figure 1. Illustrations of methods used to measure survival rates of total and colistin-resistant populations after exposure to various colistin concentrations. (a, Experiment 1) Bacteria were cultured in MH broth containing different colistin concentrations. After incubation for 18 h at 37°C with shaking, the total surviving colonies on MH agar plates without colistin and resistant colonies on plates with 4 mg/L colistin were counted. (b, Experiment 2) Unlike experiment 1, bacterial culture exposed to colistin was diluted every hour, and plated on MH agar with or without colistin. (c, Experiment 3) The bacterial culture was transferred to fresh media at each time point, and then cultivated.

Figure 2. The results of population analysis profiling (PAP) of Type I colistin-heteroresistant A. baumannii isolates (SCH2, SCH39, and SCH105) (A) and Type II colistin-heteroresistant A. baumannii isolates (SCH91, SCH113, and K20-B-871) (B). LOQ: limit of quantification.

Figure 3. The results of experiment 1. Total and colistin-resistant populations with respect to colistin concentration. The total surviving populations were identified in media without antibiotics, and colistin-resistant populations were identified in media including 4 mg/L colistin. (a) The total and colistin-resistant, surviving populations in Type I A. baumannii isolates, (b) the total and colistin-resistant, surviving populations in Type II A. baumannii isolates, (c) the rates of colistin-resistant population within the total populations in Type I A. baumannii isolates, and (d) the rates of colistin-resistant population within the total populations in Type II A. baumannii isolates.

Figure 4. The results of experiment 2. Total and colistin-resistant populations with respect to colistin concentration. (a) SCH2, a Type I A. baumannii isolates, (b) SCH91, Type II A. baumannii isolates.

Figure 5. The results of experiment 3. Total and colistin-resistant populations with respect to colistin concentration. (a) SCH2, a Type I A. baumannii isolates, (b) SCH91, Type II A. baumannii isolates.

Figure 6. (a) Colistin MIC distributions of strains in media containing 4 mg/L colistin under exposure to colistin as described in experiment 1. The grey highlighted area indicates the MIC range corresponding to colistin resistance. (b) The results of stability test for colistin resistance.

Table 2. Amino acid substitutions in PmrCAB and LpxACD genes in colonies that survived in media with 4 mg/L colistin without antibiotic exposure.

Strain	Colony No.	Colistin MIC (mg/L)	PmrA	PmrB	LpxA	LpxC	LpxD
Type I heteroresistance
SCH2	1	2
	2	4
	3	64
	4	64
	5	64
	6	8
	7	4
	8	64
	9	4
	10	4
SCH39	1	32
	2	1
	3	1
	4	16
	5	8
	6	8
	7	1
	8	8	A14V
	9	32		E301D
	10	8
SCH105	1	16			M169T
	2	16
	3	>64
	4	32
	5	8			R258H
	6	4			T81M
	7	16					T290I
	8	16
	9	32					G231V
	10	17
Type II heteroresistance
SCH91	1	16
	2	>64		T235I
	3	16		L271F
	4	64		P170L
	5	4				M62I
	6	8		L271F
	7	64
	8	64		S17R
	9	8				C63Y
	10	64		G260D
SCH113	1	8					G309V
	2	8
	3	32
	4	32	S16L
	5	4
	6	4
	7	32
	8	>64
	9	32	S16L
	10	32	S16L
K20-B-871	1	2
	2	4
	3	64		T232I
	4	2
	5	8
	6	2
	7	16
	8	32
	9	32		L168S
	10	2