Survey on Genetic Diversity, Biofilm Formation, and Detection of Colistin Resistance Genes in Clinical Isolates of Acinetobacter baumannii

Figures & data

Table 1 Primers Used for the Amplification of Genes

Target Genes	Sequence (5^/–3^/)	Amplicon Size (Base Pairs)	References
bap	F:ATGCCTGAGATACAAATTAT R:GTCAATCGTAAAGGTAACG	1449	^Citation21
ompA	F:GTTAAAGGCGACGTAGACG R:CCAGTGTTATCTGTGTGACC	578	^Citation21
csuE	F:CATCTTCTATTTCGGTCCC R:CGGTCTGAGCATTGGTAA	168	^Citation21
blaPER-1	F:ATGAATGTCATTATAAAAGC R:AATTTGGGCTTAGGGCAGAA	925	^Citation21
mcr-1	F:AGTCCGTTTGTTCTTGTGGC R:AGATCCTTGGTCTCGGCTTG	320	^Citation22
mcr-2	F:CAAGTGTGTTGGTCGCAGTT R:TCTAGCCCGACAAGCATACC	715	^Citation22
pmrA	F:ACTGGACATGTTGCACTCTTGT R:ATGCACTTTTATGAAGTCCCGA	757	^Citation23
pmrB	F:TCGGGACTTCATAAAAGTGCAT R:CAGTCACAGGTGTTCGTAATT	722	^Citation23
16S_rRNA	F:CAGCTCGTGTCGTGAGATGT R:CGTAAGGGCCATGATGACTT	150	^Citation23
Group1ompAF306 Group1and2ompAR660	GATGGCGTAAATCGTGGTA CAACTTTAGCGATTTCTGG	355	^Citation27
Group1csuEF Group1csuER	CTTTAGCAAACATGACCTACC TACACCCGGGTTAATCGT	702	^Citation27
Gp1OXA66F89 Gp1OXA66R647	GCGCTTCAAAATCTGATGTA GCGTATATTTTGTTTCCATTC	559	^Citation27
Group2ompAF378 Group1and2ompAR660	GACCTTTCTTATCACAACGA GGCGAACATGACCTATTT	343	^Citation27
Group2csuEF Group2csuER	GGCGAACATGACCTATTT CTTCATGGCTCGTTGGTT	580	^Citation27
Gp2OXA69F169 Gp2OXA69R330	CATCAAGGTCAAACTCAA TAGCCTTTTTTCCCCATC	162	^Citation27

Table 2 Antibiotic Susceptibility Pattern of A. baumannii Isolates

Antibiotic		LEV	CTZ	CFP	PIP	TGC	AMP/S	COL	CTX	SXT	AMK	CIP	IMI	MER
Susceptibility pattern	R N (%)	65 (92.8)	70 (100)	70 (100)	68 (97.1)	32 (45.7)	55 (78.6)	2 (2.8)	70 (100)	67 (95.7)	59 (84.2)	65 (92.8)	50 (71.4)	52 (74.2)
	I N (%)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (2.8)	0 (0.0)	3 (4.3)	0 0.0)
	S N (%)	5 (7.1)	0 (0.0)	0 (0.0)	2 (2.8)	38 (54.3)	15 (21.4)	68 (97.1)	0 (0.0)	3 (4.3)	9 (12.8)	5 (7.1)	17 (24.2)	18 (25.7)
Interpretive categories and MIC breakpoints (µg/mL)	R	≥8	≥32	≥32	≥128	≥8	≥32/16	≥4	≥64	≥4/76	64	≥4	≥8	≥8
	I	4	16	16	32–64	–	16/8	–	16–32	–	32	2	4	4
	S	≤2	≤8	≤8	≤16	<8	≤8/4	≤2	≤8	≤ 2/38	16	≤1	≤2	≤2

Abbreviations: LEV, levofloxacin; CTZ, ceftazidime; CFP, cefepime; PIP, piperacillin; TGC, tigecycline; Amp/S, ampicillin/sulbactam; COL, colistin; CTX, cefotaxime; SXT, trimethoprim-sulphamethoxazole; AMK, amikacin; CIP, ciprofloxacin; IMI, imipenem; MEM, meropenem; R, resistant; I, intermediate; S, susceptible.

Table 3 Characteristics of A. baumannii Strains

Strain ID	Biofilm	Phe	MIC			Biofilm Genes	IC	MLST
			MER	TGC	COL
Ab 1	S	XDR	16	64	0.5	bap, ompA, csuE	G1	–	–
Ab 2	S	MDR	0.25	16	0.5	bap, ompA, csuE, blaPER-1	G1	–	–
Ab 3	S	XDR	64	2	0.5	bap, ompA, csuE	G2	–	–
Ab 4	S	XDR	128	8	32	bap, ompA, csuE, blaPER-1	G2	387	–
Ab 5	W	MDR	16	16	0.25	bap,ompA	G1	–	–
Ab 6	S	MDR	0.5	4	0.25	bap, ompA, csuE, blaPER-1	G2
Ab 7	S	MDR	128	0.25	1	bap, ompA, csuE	G15	138	92
Ab 8	S	MDR	16	32	0.5	bap, ompA, csuE	G1	–	–
Ab 9	S	MDR	8	1	0.5	bap, ompA, csuE, blaPER-1	G1	–	–
Ab 10	N	XDR	32	0.5	0.25	ompA, csuE, blaPER-1	G10	–	–
Ab 11	M	MDR	0.5	64	0.125	bap, ompA, csuE	G1	–	–
Ab 12	S	MDR	128	0.25	0.25	bap, ompA, csuE, blaPER-1	G1	–	–
Ab 13	S	MDR	0.25	2	0.5	bap, ompA, csuE	G1	–	–
Ab 14	S	MDR	16	32	0.5	bap, ompA	G4	–	–
Ab 15	S	MDR	256	0.25	0.25	bap, ompA	G10	–	–
Ab 16	N	MDR	256	1	0.25	ompA, csuE, blaPER-1	G1
Ab 17	S	MDR	32	0.5	0.25	bap, ompA, csuE	G2	–	–
Ab 18	S	MDR	0.5	32	0.125	bap, ompA, csuE	G1	–	–
Ab 19	M	MDR	0.5	0.25	0.25	bap, ompA, csuE	G10	–	–
Ab 20	S	MDR	32	16	0.5	bap, ompA, csuE	G1	–	–
Ab 21	S	MDR	16	1	0.25	bap, ompA	G1	–	–
Ab 22	S	MDR	128	0.5	1	bap, ompA, csuE, blaPER-1	G1	118	92
Ab 23	S	MDR	256	16	0.25	bap, ompA, csuE, blaPER-1	G15	–	–
Ab 24	N	MDR	16	2	0.25	ompA, csuE	G 1	–	–
Ab 25	S	MDR	2	8	0.125	bap, ompA, csuE	G 4	–	–
Ab 26	S	MDR	16	4	2	bap, ompA	G 1	421	92
Ab 27	S	XDR	16	32	0.25	bap, ompA, csuE, blaPER-1	G 10	–	–
Ab 28	W	MDR	64	0.5	0.25	ompA, csuE	G 1	–	–
Ab 29	S	MDR	0.25	8	0.25	bap, ompA, csuE, blaPER-1	G 1	–	–
Ab 30	S	MDR	0.5	8	0.5	bap, ompA, csuE	G 2	–	–
Ab 31	S	MDR	8	16	0.25	bap, ompA	G 1	–	–
Ab 32	S	MDR	128	1	1	bap, ompA, csuE	G 4	216	405
Ab 33	S	MDR	0.5	0.25	0.125	bap, ompA, csuE	G 7	–	–
Ab 34	M	MDR	32	32	0.25	bap,ompA, csuE, blaPER-1	G 10	–	–
Ab 35	S	MDR	0.5	1	0.25	bap, ompA	G 1	–	–
Ab 36	M	MDR	16	0.5	0.5	bap,ompA, csuE	G 15	–	–
Ab 37	N	MDR	256	8	2	ompA, csuE, blaPER-1	G 1	735	92
Ab 38	M	MDR	0.5	32	0.5	bap, ompA, csuE, blaPER-1	G 2	–	–
Ab 39	S	MDR	0.25	4	0.25	bap, ompA, csuE	G 10	–	–
Ab 40	S	MDR	16	1	0.25	bap, ompA, csuE	G 1	–	–
Ab 41	S	XDR	64	16	8	bap, ompA, blaPER-1	G 1	1812	–
Ab 42	S	MDR	16	0.5	0.25	bap, ompA, csuE	G 1	–	–
Ab 43	S	MDR	2	4	0.25	bap, ompA, csuE	G10	–	–
Ab 44	S	MDR	16	4	0.5	bap, ompA, csuE	G 1	–	–
Ab 45	M	MDR	128	16	0.25	bap,ompA, blaPER-1	G15	–	–
Ab 46	S	MDR	16	0.5	0.5	bap, ompA, csuE	G4	–	–
Ab 47	S	MDR	2	32	0.25	bap, ompA, csuE	G 1	–	–
Ab 48	S	MDR	16	1	0.5	bap, ompA, csuE, blaPER-1	G1	–	–
Ab 49	S	MDR	16	8	1	bap, ompA	G2	133	225
Ab 50	S	MDR	32	8	0.25	bap, ompA, csuE	G1	–	–
Ab 51	S	XDR	16	0.25	0.5	bap, ompA, csuE	G1	–	–
Ab 52	M	MDR	16	2	0.25	bap,ompA	G7	–	–
Ab 53	S	XDR	0.5	16	0.25	bap, ompA, csuE	G10	–	–
Ab 54	S	MDR	16	8	0.5	bap, ompA, csuE, blaPER-1	G1	–	–
Ab 55	S	MDR	64	0.5	0.5	bap, ompA, csuE	G 1	–	–
Ab 56	S	MDR	16	4	0.25	bap, ompA, csuE, blaPER-1	G 1	–	–
Ab 57	S	MDR	32	16	0.5	bap, ompA, csuE	G 4	–	–
Ab 58	S	MDR	16	8	0.25	bap, ompA, csuE	G1	–	–
Ab 59	S	MDR	0.25	0.5	0.5	bap, ompA, csuE, blaPER-1	G4	–	–
Ab 60	S	MDR	16	1	0.25	bap, ompA	G1	–	–
Ab 61	S	MDR	128	0.5	1	bap, ompA, csuE	G7	848	92
Ab62	S	MDR	16	8	0.25	bap, ompA, csuE	G15	–	–
Ab 63	S	MDR	32	1	0.5	bap, ompA, csuE	G10	–	–
Ab 64	N	MDR	16	2	0.25	ompA, csuE, blaPER-1	G2	–	–
Ab 65	S	MDR	125	32	0.25	bap, ompA, csuE	G1	–	–
Ab 66	S	XDR	16	32	0.5	bap, ompA	G1	–	–
Ab 67	S	MDR	16	64	0.25	bap, ompA, csuE	G10	–	–
Ab 68	S	MDR	0.25	0.5	0.25	bap, ompA, blaPER-1	G1	–	–
Ab 69	W	MDR	16	2	1	bap,ompA, csuE	G10	118	92
Ab 70	S	MDR	128	16	0.25	bap, ompA, csuE	G1	–	–

Abbreviations: Phe, phenotype; MDR, multidrug-resistant; XDR, extensively drug-resistant; M, medium; N, non-biofilm-forming; W, weak; S, strong; MIC, minimum inhibitory concentration; MER, meropenem; TGC, tigecycline; COL, colistin; IC, international clonal lineage.

Table 4 The Relationship Between Biofilm Formation and Related Genes in A. baumannii Strains with MDR and XDR Phenotypes

Biofilm Intensity (No.)	bap No (%)		ompA No (%)		csuE No (%)		bla-PER1 No (%)
	Positive	Negative	Positive	Negative	Positive	Negative	Positive	Negative
Strong (55)	55 (100)	0 (0)	55(100)	0 (0)	44 (80)	11(20)	15(27.27)	40(72.73)
Moderate (7)	7 (100)	0 (0)	7 (100)	0 (0)	5(71.4)	2(28.57)	3 (42.85)	4 (57.14)
Weak (3)	2 (66.6)	1 (33.3)	3(100)	0 (0)	2 (66.6)	1 (33.3)	0 (0)	3 (100)
Non- Biofilm forming (5)	0 (0)	5 (100)	5(100)	0 (0)	5 (100)	0 (0)	4 (80)	1(20)
Total	64(91.4)	6 (8.6)	70(100)	0 (0)	56 (80)	14 (20)	22 (31.42)	48(68.58)
P value	<0.001		–		0.552			0.053

Figure 1 Gene expression comparison of pmrA and pmrB in colistin-resistant (Ab 4, Ab41) and susceptible isolates (Ab49, Ab61, Ab69, Ab22, Ab26, Ab7, Ab32, Ab37) and A. baumannii ATCC 19606.

Table 5 The Relation Between Biofilm Intensity with Antibiotic Susceptibility, Phenotype, and IC

Biofilm Intensity (No.)	Antibiotic N (%)						Phenotype N (%)		IC
	Colistin		Meropenem		Tigecycline		MDR	XDR	I	II	IV
	R	S	R	S	R	S
Strong (55)	2 (3.6)	53 (96.4)	40 (72.72)	15 (27.28)	26 (47.27)	29(52.73)	47 (85.45)	8(14.54)	6	31	18
Moderate(7)	0 (0)	7 (100)	4 (57.15)	3 (42.85)	4 (57.14)	3 (42.86)	7 (100)	0(0)	1	1	5
Weak(3)	0 (0)	3(100)	3 (100)	0 (0)	1(33.33)	2 (66.67)	3 (100)	0(0)	0	2	1
Non- Biofilm forming (5)	0 (0)	5 (100)	5(100)	0 (0)	1(20)	4(80)	4 (80)	1(20)	1	3	1
p-value	1		0.342		0.608		0.662
Total	2 (2.8)	68 (97.1)	52	18	32	38	61(87.1)	9 (12.8)	8	37	25

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