Characterization of phenotypic and genotypic traits of carbapenem-resistant Acinetobacter baumannii clinical isolates recovered from a tertiary care hospital in Taif, Saudi Arabia

Figures & data

Table 1 Primers and cycling conditions used for the amplification of carbapenemase genes and the repetitive intergenic consensus

Primer	Sequence	Gene	Reference	Amplification conditions	Amplicon size (bp)
ERIC-1R	R:AACCCACGATGTGGGTAGC	-	^Citation50	Initial denaturation at 95^oC for 15 min, then 35 cycles of 95^oC for 1 min, 40^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	-
IPM	F: GGAATAGRRTGGCTTAAYT R: GGTTTAAYAAARCAMCCACC	blaIPM	^Citation51	Initial denaturation at 95^oC for 15 min, then 35 cycles of 95^oC for 1 min, 40^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	233
KPC	F: GTATCGCCGTCTAGTTCTGC R: GGTCGTGTTTCCCTTTAGCC	blaKPC	^Citation52	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 55^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	637
NDM	F: GGTTTGGCGATCTGGTTTTC R: CGGAATGGCTCATCACGATC	blaNDM	^Citation53	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 66.7^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	621
VIM	F: GTTTGGTCGCATATCGCAAC R: GAGCAAKTCYAGACCGCCC	blaVIM	^Citation51	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 63^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	591
OXA-23	F: GATCGGATTGGAGAACCAGA R: ATTTCTGACCGCATTTCCAT	blaOXA-23	^Citation54	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 66.7^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	501
OXA-40	F: GGTTAGTTGGCCCCCTTAAA R: AGTTGAGCGAAAAGGGGATT	blaOXA-40	^Citation55	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 66.6^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	249
OXA-48	F: GCTTGATCGCCCTCGATT R: GATTTGCTCCGTGGCCGAAA	blaOXA-48	^Citation56	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 60.5^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	281
OXA-51	F: TAATGCTTTGATCGGCCTTG R: TGGATTGCACTTCATCTTGG	blaOXA-51	^Citation57	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 60.5^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72 ^oC.	353
OXA-58	F: AAGTATTGGGGCTTGTGCTG R: CCCCTCTGCGCTCTACATAC	blaOXA-58	^Citation54	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 60.5^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	599
OXA-181	F: ATGCGTGTATTAGCCTTATCG R: AACTACAAGCGCATCGAGCA	blaOXA-181	^Citation22	Initial denaturation at 95^oC for 15 min, then 30 cycles of 95^oC for 1 min, 55^oC for 1min and 72^oC for 5 minutes and one cycle of final elongation at 72^oC.	888

Notes: R= (A⁄G), Y= (C⁄ T), K= (G⁄ T) and M= (A⁄C). Boldface in this  table clarifies primers and different stages of the PCR cycles.

Table 2 Susceptibility pattern of A. baumannii clinical isolates

Antibiotic	Susceptibility pattern
	Sensitive		Intermediate		Resistant
	No.	%	No.	%	No.	%
Cefotaxime	0.00	0.00	1.00	3.12	31.00	96.88
Ceftazidime	2.00	6.24	0.00	0.00	30.00	93.76
Ceftriaxone	1.00	3.12	1.00	3.12	30.00	93.76
Cefepime	0.00	0.00	3.00	9.36	29.00	90.64
Imipenem	0.00	0.00	0.00	0.00	32.00	100.00
Meropenem	0.00	0.00	2.00	6.24	30.00	93.76
Ticarcillin	0.00	0.00	0.00	0.00	32.00	100.00
Piperacillin	0.00	0.00	0.00	0.00	32.00	100.00
Piperacillin/Tazobactam	0.00	0.00	0.00	0.00	32.00	100.00
Ampicillin/Sulbactam	20.00	62.50	12.00	37.50	0.00	0.00
Gentamicin	5.00	15.63	9.00	28.13	18.00	56.25
Tobramycin	11.00	34.38	2.00	6.24	19.00	59.38
Amikacin	3.00	9.36	0.00	0.00	29.00	90.64
Netilmicin	8.00	25.00	0.00	0.00	25.00	75.00
Ciprofloxacin	0.00	0.00	0.00	0.00	32.00	100.00
Levofloxacin	0.00	0.00	7.00	21.88	25.00	78.12
Tetracycline	3.00	9.36	0.00	0.00	29.00	90.64
Tigecycline	24.00	75.00	0.00	0.00	9.00	25.00
Sulfamethoxazole/Trimethoprim	6.00	18.75	0.00	0.00	26.00	81.25
Polymyxin B	32.00	100.00	0.00	0.00	0.00	0.00
Colistin	32.00	100.00	0.00	0.00	0.00	0.00

Table 3 Resistance patterns of the A. baumannii clinical isolates

Isolate No.	Antibiotic resistance profile	Resistance markers
AC01	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, SXT	18
AC02	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV, SXT	16
AC03	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, AK, TET, CIP, LEV, SXT	13
AC04	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, TEG, SXT	19
AC05	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, TET, CIP, LEV, SXT	13
AC06	CTX, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, AK, NET, CIP, LEV	13
AC07	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV, SXT	17
AC08	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV, SXT	17
AC66	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, AK, NET, TET, CIP, LEV, SXT	15
AC10	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, CIP, LEV	13
AC11	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, TEG, SXT	19
AC12	CTX, CAZ, FEP, IPM, MEM, TIC, PIP, TPZ, CN, AK, TET, CIP, LEV, SXT	14
AC13	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, SXT	18
AC14	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV, SXT	17
AC15	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV	16
AC16	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV, SXT	17
AC17	CTX, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, AK, NET, CIP, LEV	13
AC18	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, TET, CIP, LEV, TEG, SXT	18
AC19	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, AK, NET, TET, CIP, LEV, SXT	15
AC20	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, AK, NET, TET, CIP, LEV, SXT	16
AC21	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, AK, NET, TET, CIP, LEV, SXT	16
AC22	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, TEG, SXT	19
AC23	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, TEG, SXT	19
AC24	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV, TEG	17
AC25	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV, TEG	17
AC26	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, CN, TOB, AK, NET, TET, CIP, LEV, SXT	17
AC27	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, AK, NET, TET, CIP, LEV, SXT	17
AC28	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, SXT	18
AC29	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, TEG, SXT	19
AC30	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, TET, CIP, LEV, SXT	13
AC31	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, SXT	18
AC32	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM, CN, TOB, AK, NET, TET, CIP, LEV, SXT	18

Abbreviations: CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; FEP, cefepime; IPM, imipenem; MEM, meropenem; TIC, ticarcillin; PIP, piperacillin; TPZ, piperacillin/tazobactam; SAM, ampicillin/sulbactam; CN, gentamicin; TOB, tobramycin; NET, netilmicin; CIP, ciprofloxacin; LEV, levofloxacin; TET, tetracycline; TEG, tigecycline; SXT, sulfamethoxazole/trimethoprim.

Table 4 Relationship between genotypic profiles of carbapenemases and phenotypic resistance profiles A. baumannii for β-lactam antibiotics

No	Genetic Profile	No of isolates	Profiles of detected β -lactamase genes	Phenotypic resistance profiles to β -lactams	Resistance markers
1	A	6	blaOXA-51, blaIPM, blaNDM	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM	9-10
2	B	8	blaOXA-51, blaOXA-23, blaIPM	CTX, CRO, FEP, IPM, MEM, TIC, PIP, TPZ CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM	8-10
3	C	1	blaOXA-51, blaOXA-23	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM	10
4	D	6	blaOXA-51	CTX, CAZ, FEP, IPM, MEM, TIC, PIP, TPZ CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM	8-10
5	E	1	blaOXA-51, blaOXA-40	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ	9
6	F	2	blaOXA-51, blaOXA-23, blaIPM, blaNDM, blaVIM	CTX, CRO, FEP, IPM, MEM, TIC, PIP, TPZ CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ	8-9
7	G	1	blaOXA-51, blaOXA-23, blaIPM, blaVIM	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM	10
8	H	1	blaOXA-51, blaOXA-23, blaNDM	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ	9
9	I	6	blaOXA-51, blaOXA-23, blaIPM, blaNDM	CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ CTX, CAZ, CRO, FEP, IPM, MEM, TIC, PIP, TPZ, SAM	9-10

Figure 1 DNA fingerprint pattern generated by ERIC-PCR for Acinetobacter clinical isolates. L, 100bp DNA ladder; lanes 1, 2, 3, 27,19, 26 and 32 represent the code No. of the isolates.

Figure 2 Clonal relationship between A. baumannii clinical isolates generated by UPGMA dendrogram.

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