Mechanism of resistance nodulation division efflux pumps mediated tigecycline-insensitive multidrug-resistant Acinetobacter baumannii

doi:10.3969/j.issn.1673-8640.2020.01.012

Abstract

Abstract:

Objective To investigate the mechanism of resistance nodulation division（RND） efflux pumps mediated tigecycline-insensitive multidrug-resistant Acinetobacter baumannii（MDR-AB）. Methods A total of 21 isolates of tigecycline-insensitive Acinetobacter baumannii and 39 isolates of tigecycline-sensitive MDR-AB in the Fourth Affiliated Hospital of Harbin Medical University from 2015 to 2018 were collected. Using broth microdilution method as the standard method,tigecycline-insensitive isolates [minimun inhibitory concentration（MIC）≥2 μg/mL] were used as experimental group,and tigecycline-sensitive isolates（MIC≤1 μg/mL） were used as control group. Polymerase chain reaction（PCR） was used to determine RND efflux pump genes for tigecycline sensitivity decreasing of Acinetobacter baumannii（adeB,adeG and adeJ）and upstream regulatory genes（adeS,baeR and baeS）,and adeS amplification products were sequenced to find insertion sequence. Real-time quantitative polymerase chain reaction（RT-qPCR） was used to determine and compare the transcript levels of efflux pump adeABC,adeFGH,adeIJK,adeRS and baeRS in experimental and control groups. Results In the 21 isolates of Acinetobacter baumannii,12 isolates of tigecycline-insensitive MDR-AB were screened. The RND gene detection rate associated with decreased tigecycline sensitivity was 100%,and the ISAbaⅠ insertion mutation of adeS was found in the 2 isolates. The expression of adeABC in experimental group was higher than that in control group,which were 3.3,3.5 and 2.7 times of the standard isolate（ATCC 19606）,respectively. The expression level of adeFGH was increased,and the levels of adeIJK,adeRS and baeRS were slightly up-regulated in some isolates. Conclusions RND efflux pump mediated MDR-AB with decreased tigecycline sensitivity is related to high expressions of adeABC and adeFGH. The high expression of adeABC is mainly related to the presence of ISAbal insertion mutation in the upstream regulatory gene adeS,and there may be other tigecycline resistance mechanisms.

Key words: Acinetobacter baumannii, Multi-drug resistance, Tigecycline, Resistance nodulation division

CLC Number:

R378.99

ZHANG Xinyu, DU Xuefei, ZOU Guiling, JIANG Xiaofeng. Mechanism of resistance nodulation division efflux pumps mediated tigecycline-insensitive multidrug-resistant Acinetobacter baumannii[J]. Laboratory Medicine, 2020, 35(1): 56-60.

Figures/Tables 5

References 23

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基因	引物序列（5’~3’）	DNA熔解温度/ ℃	参考文献
adeB	F:ATGAAGGGTTGCCACAAG	52	[16]
adeB	R:GCAATACTCAAAGATGGTCGT
adeG	F:CAATGCGCTATCACTGTTC	52	[16]
adeG	R:ATCGGTCCACTGACTTCTCG
adeJ	F:GTCGTTAATTGTAGCGTTGA	49	[16]
adeJ	R:TAAGAATGAAGACGGGAGTT
adeS	F:TAGACTGGATCTGGTTAGCC	53	[16]
adeS	R:AATATACAGGAGTGGACGTTAG
baeR	F:GGATGGTTTAACGATTTGCC	53	[17]
baeR	R:TCCACTCATGACGGTTACCA
baeS	F:CTTTCCCAGTGGTGGTTACG	55	[17]
baeS	R:GAGCCAAGTCTGCCAAATCT
qbaeS	F:GCCATTCAGCAGCATTCTTTC	55	[17]
qbaeS	R:ATTTACCGTTCCCGCATCTG
qbaeR	F:TGACAGCACGTACCGAAGAAA	55	[17]
qbaeR	R:CATAATCATCTGCCCCCATGT
adeB	F:TATTCGTCGTCCCGTTTTT	54	[16]
adeB	R:GCTGTACCGGAGGTATCTGTT
adeG	F:CCGATCTTTGCTGGTGTGC	56	[16]
adeG	R:TTACCGTCGCTGTTTGCTT
adeJ	F:GCCCTTTTAGTCTTCTTGTTC	50	[16]
adeJ	R:CCACGGTATCTTTTTCATTC
adeR	F:ACGCCAAAAAGCTCAGACT	54	[16]
adeR	R:TATATCCCACGCCACGCAC
gyrBnc	F:TAACAACCGAAACACCTACGCC	56	[18]
gyrBnc	R:ATTCCAACCGATATTCACCCTG

基因	引物序列（5’~3’）	DNA熔解温度/ ℃	参考文献
adeB	F:ATGAAGGGTTGCCACAAG	52	[16]
adeB	R:GCAATACTCAAAGATGGTCGT
adeG	F:CAATGCGCTATCACTGTTC	52	[16]
adeG	R:ATCGGTCCACTGACTTCTCG
adeJ	F:GTCGTTAATTGTAGCGTTGA	49	[16]
adeJ	R:TAAGAATGAAGACGGGAGTT
adeS	F:TAGACTGGATCTGGTTAGCC	53	[16]
adeS	R:AATATACAGGAGTGGACGTTAG
baeR	F:GGATGGTTTAACGATTTGCC	53	[17]
baeR	R:TCCACTCATGACGGTTACCA
baeS	F:CTTTCCCAGTGGTGGTTACG	55	[17]
baeS	R:GAGCCAAGTCTGCCAAATCT
qbaeS	F:GCCATTCAGCAGCATTCTTTC	55	[17]
qbaeS	R:ATTTACCGTTCCCGCATCTG
qbaeR	F:TGACAGCACGTACCGAAGAAA	55	[17]
qbaeR	R:CATAATCATCTGCCCCCATGT
adeB	F:TATTCGTCGTCCCGTTTTT	54	[16]
adeB	R:GCTGTACCGGAGGTATCTGTT
adeG	F:CCGATCTTTGCTGGTGTGC	56	[16]
adeG	R:TTACCGTCGCTGTTTGCTT
adeJ	F:GCCCTTTTAGTCTTCTTGTTC	50	[16]
adeJ	R:CCACGGTATCTTTTTCATTC
adeR	F:ACGCCAAAAAGCTCAGACT	54	[16]
adeR	R:TATATCCCACGCCACGCAC
gyrBnc	F:TAACAACCGAAACACCTACGCC	56	[18]
gyrBnc	R:ATTCCAACCGATATTCACCCTG

菌株	MIC/（μg/mL）										抑菌圈直径/mm
菌株	头孢曲松	头孢吡肟	亚胺培南	庆大霉素	妥布霉素	环丙沙星	左氧氟沙星	替加环素（Vitek2法）	替加环素（微量肉汤稀释法）	复方磺胺甲噁唑	替加环素（纸片扩散法）	头孢哌酮舒巴坦	米诺环素	阿米卡星	头孢他啶
TR1	≥64	≥64	≥16	≥16	≤1	≥4	≥8	≥8	4	≥320	10.5	6	14	14	6
TR2	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	11.0	12	14	6	6
IA6	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	10.0	6	12	6	6
TR5	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≤20	6.0	14	9	6	6
TR7	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	9.0	10	13	6	10
TR9	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	10.0	8	10	6	6
TR12	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	2	≥320	13.5	6	6	6	6
TR13	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	13.5	16	9	6	6
TR14	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	8.5	6	10	6	6
TR15	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	6.0	8	8	6	6
TR20	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≤20	10.0	16	10	6	6
TR21	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	10.0	15	11	6	6

菌株	MIC/（μg/mL）										抑菌圈直径/mm
菌株	头孢曲松	头孢吡肟	亚胺培南	庆大霉素	妥布霉素	环丙沙星	左氧氟沙星	替加环素（Vitek2法）	替加环素（微量肉汤稀释法）	复方磺胺甲噁唑	替加环素（纸片扩散法）	头孢哌酮舒巴坦	米诺环素	阿米卡星	头孢他啶
TR1	≥64	≥64	≥16	≥16	≤1	≥4	≥8	≥8	4	≥320	10.5	6	14	14	6
TR2	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	11.0	12	14	6	6
IA6	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	10.0	6	12	6	6
TR5	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≤20	6.0	14	9	6	6
TR7	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	9.0	10	13	6	10
TR9	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	10.0	8	10	6	6
TR12	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	2	≥320	13.5	6	6	6	6
TR13	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	13.5	16	9	6	6
TR14	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	8.5	6	10	6	6
TR15	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	6.0	8	8	6	6
TR20	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≤20	10.0	16	10	6	6
TR21	≥64	≥64	≥16	≥16	≥16	≥4	≥8	≥8	4	≥320	10.0	15	11	6	6