Studies of 16S rRNA methylase gene and aminoglycoside modifying enzyme gene in Pseudomonas aeruginosa

doi:10.3969/j.issn.1673-8640.2015.06.017

Abstract

Abstract: Objective

To investigate 16S rRNA methylase gene and aminoglycoside modifying enzyme gene in aminoglycoside-resistant clinical isolates of Pseudomonas aeruginosa, and to investigate the mechanism of drug resistance to aminoglycosides.

Methods

A total of 35 isolates of aminoglycoside-resistant clinical isolates of Pseudomonas aeruginosa were collected, and the identification and susceptibility testing in vitro were performed by VITEK 2 Compact automatic microbial analysis system. Five kinds of 16S rRNA methylase genes(arm-A, rmt-A, rmt-B, rmt-C and rmt-D) and 6 kinds of aminoglycoside modifying enzyme genes [aac(3)-Ⅰ, aac(3)-Ⅱ, aac (6')-Ⅰb, aac(6')-Ⅱ, ant (3″)-Ⅰand ant (2″)-Ⅰ] were detected by polymerase chain reaction (PCR).

Results

Aminoglycoside-resistant Pseudomonas aeruginosa showed being multi-drug resistant to a variety of antibiotics. Only the resistance rate to imipenem was low (5.7%). There were 21 isolates of Pseudomonas aeruginosa carried rmt-B 16S rRNA methylase gene, accounting for 60.0%, and 30 isolates of Pseudomonas aeruginosa carried aminoglycoside modifying enzyme genes, accounting for 85.7%, among them, 28 isolates carried aac(3)-Ⅱ aminoglycoside modifying enzyme genes, accounting for 80.0%, and 18 isolates carried ant (3″)-Ⅰ aminoglycoside modifying enzyme genes, accounting for 51.4%. The other 8 isolates were not detected any genes.

Conclusions

Aminoglycoside-resistant isolates of Pseudomonas aeruginosa carry rmt-B 16S rRNA methylase gene and aac(3)-Ⅱ and ant (3″)-Ⅰ aminoglycoside modifying enzyme genes. Furthermore, the multi-drug resistances of Pseudomonas aeruginosa are serious.

Key words: Pseudomonas aeruginosa, 16S rRNA methylase gene, Aminoglycoside modifying enzyme gene, Drug resistance

CLC Number:

R378.99

PENG Jinghong, HOU Yanqiang, XIE Duoshuang, LIU Jun, ZHENG Rong. Studies of 16S rRNA methylase gene and aminoglycoside modifying enzyme gene in Pseudomonas aeruginosa[J]. Laboratory Medicine, 2015, 30(6): 613-616.

Figures/Tables 3

References 13

[1]	LEE SK,PARK DC,KIM MG, et al.Rate of isolation and trends of antimicrobial resistance of multidrug resistant Pseudomonas aeruginosa from otorrhea in chronic suppurative otitis media[J]. Clin Exp Otorhinolaryngol,2012,5(1):17-22.
[2]	赵祝香,陈惠玲,魏树全,等.多药耐药铜绿假单胞菌耐药性及相关耐药基因分析[J].中国实验诊断学,2011,15(7):1159-1162.
[3]	王娟,王前,兰海丽,等.多重耐药铜绿假单胞菌氨基糖苷类修饰酶基因的检测及分析[J]. 国际检验医学杂志,2013, 34(3):266-268.
[4]	由然,孙立颖.耐阿米卡星铜绿假单胞菌氨基糖苷类耐药基因及其基因型研究[J].检验医学,2010, 25(5):341-344.
[5]	林宁,孙海平.耐药铜绿假单胞菌临床分离株发现aac(6')-Ⅰb-cr基因[J]. 中华临床感染病杂志,2011, 4(3):163-167.
[6]	POOLE K.Aminoglycoside resistance in Pseudomonas aeruginosa[J]. Antimicrob Agents Chemother, 2005,49(2):479-487.
[7]	VAZIRI F,PEERAYEH SN,NEJAD QB, et al.The prevalence of aminoglycoside-modifying enzyme genes[aac(6') -Ⅰ,aac(6') -Ⅱ,ant(2″) -Ⅰ, aph(3') -Ⅵ] in Pseudomonas aeruginosa[J]. Clinics(Sao Paulo),2011,66(9):1519-1522.
[8]	王苏建,王家平,张晓梅,等.两所医院铜绿假单胞菌连续分离株16S rRNA甲基化酶、氨基糖苷类修饰酶基因研究[J].中华医院感染学杂志,2008, 18(11):1501-1504.
[9]	糜祖煌,秦玲.泛耐药铜绿假单胞菌16S rRNA甲基化酶、氨基糖苷类修饰酶基因研究[J].中华医院感染学杂志,2008,18(12):1656-1658.
[10]	DOI Y,DE OLIVEIRA GARCIA D,ADAMS J,et al.Coproduction of novel 16S rRNA methylase RmtD and metallo-beta-lactamase SPM-1 in a panresistant Pseudomonas aeruginosa isolate from Brazil[J].Antimicrob Agents Chemother,2007,51(3):852-856.
[11]	YOKOYAMA K,DOI Y,YAMANE K,et al.Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa[J].Lancet,2003,362(9399):1888-1893.
[12]	叶惠芬,陈惠玲,刘平,等. 广州地区多重耐药铜绿假单胞菌相关耐药基因的研究[J].检验医学,2010, 25(4):288-291.
[13]	DOI Y,YOKOYAMA K,YAMANE K,et al.Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring high-level resistance to aminoglycosides[J].Antimicrob Agents Chemother,2004,48(2):491-496.

基因名称	引物序列	产物长度 (bp)
aac(3)-Ⅰ	P1:ACCTACTCCCAACATCAGCC P2:ATATAGATCTCACTACGCGC	169
aac(3)-Ⅱ	P1:ACTGTGATGGGATACGCGTC P2:CTCCGTCAGCGTTTCAGCTA	237
aac(6')-Ⅰb	P1:ATGACTGAGCATGACCTTGC P2:TTAGGCATCACTGCGTGTTC	519
aac(6')-Ⅱ	P1:TTCATGTCCGCGAGCACCCC P2:GACTCTTCCGCCATCGCTCT	178
ant(3″)-Ⅰ	P1:TGATTTGCTGGTTACGGTGAC P2:CGCTATGTTCTCTTGCTTTTG	284
ant(2″)-Ⅰ	P1:GAGCGAAATCTGCCGCTCTGG P2:CTGTTACAACGGACTGGCCGC	320
arm-A	P1:ATGGATAAGAATGATGTTGTTAAG P2:TTATTTCTGAAATCCACTAGTAATTA	774
rmt-A	P1:CCTAGCGTCCATCCTTTCCTC P2:AGCGATATCCAACACACGATGG	315
rmt-B	P1:ATGAACATCAACGATGCCCTC P2:TTATCCATTCTTTTTTATCAAGTATAT	756
rmt-C	P1:ATGAAAACCAACGATAATTATC P2:TTACAATCTCGATACGATAAAATAC	846
rmt-D	P1:ATGAGCGAACTGAAGGAAAAACTGCT P2:TCATTTTCGTTTCAGCACGTAAAACAG	744

基因名称	引物序列	产物长度 (bp)
aac(3)-Ⅰ	P1:ACCTACTCCCAACATCAGCC P2:ATATAGATCTCACTACGCGC	169
aac(3)-Ⅱ	P1:ACTGTGATGGGATACGCGTC P2:CTCCGTCAGCGTTTCAGCTA	237
aac(6')-Ⅰb	P1:ATGACTGAGCATGACCTTGC P2:TTAGGCATCACTGCGTGTTC	519
aac(6')-Ⅱ	P1:TTCATGTCCGCGAGCACCCC P2:GACTCTTCCGCCATCGCTCT	178
ant(3″)-Ⅰ	P1:TGATTTGCTGGTTACGGTGAC P2:CGCTATGTTCTCTTGCTTTTG	284
ant(2″)-Ⅰ	P1:GAGCGAAATCTGCCGCTCTGG P2:CTGTTACAACGGACTGGCCGC	320
arm-A	P1:ATGGATAAGAATGATGTTGTTAAG P2:TTATTTCTGAAATCCACTAGTAATTA	774
rmt-A	P1:CCTAGCGTCCATCCTTTCCTC P2:AGCGATATCCAACACACGATGG	315
rmt-B	P1:ATGAACATCAACGATGCCCTC P2:TTATCCATTCTTTTTTATCAAGTATAT	756
rmt-C	P1:ATGAAAACCAACGATAATTATC P2:TTACAATCTCGATACGATAAAATAC	846
rmt-D	P1:ATGAGCGAACTGAAGGAAAAACTGCT P2:TCATTTTCGTTTCAGCACGTAAAACAG	744

抗菌药物	耐药率	中介率	敏感率
氨苄西林	100.0	0.0	0.0
氨苄西林-舒巴坦	100.0	0.0	0.0
哌拉西林-他唑巴坦	65.7	0.0	34.3
头孢唑啉	100.0	0.0	0.0
头孢替坦	97.1	0.0	2.9
头孢他啶	80.0	2.9	17.1
头孢曲松	100.0	0.0	0.0
头孢吡肟	34.3	57.1	8.6
氨曲南	51.5	31.4	17.1
亚胺培南	5.7	2.9	91.4
阿米卡星	97.1	0.0	2.9
庆大霉素	100.0	0.0	0.0
妥布霉素	100.0	0.0	0.0
环丙沙星	82.9	14.2	2.9
左氧氟沙星	82.9	5.7	11.4
复方磺胺甲噁唑	97.1	0.0	2.9

抗菌药物	耐药率	中介率	敏感率
氨苄西林	100.0	0.0	0.0
氨苄西林-舒巴坦	100.0	0.0	0.0
哌拉西林-他唑巴坦	65.7	0.0	34.3
头孢唑啉	100.0	0.0	0.0
头孢替坦	97.1	0.0	2.9
头孢他啶	80.0	2.9	17.1
头孢曲松	100.0	0.0	0.0
头孢吡肟	34.3	57.1	8.6
氨曲南	51.5	31.4	17.1
亚胺培南	5.7	2.9	91.4
阿米卡星	97.1	0.0	2.9
庆大霉素	100.0	0.0	0.0
妥布霉素	100.0	0.0	0.0
环丙沙星	82.9	14.2	2.9
左氧氟沙星	82.9	5.7	11.4
复方磺胺甲噁唑	97.1	0.0	2.9

模式	耐药基因			株数	构成比 (%)
模式	aac(3)-Ⅱ			ant(3″)-Ⅰ	rmt-B
1	+	+	+	11	31.4
2	+	-	+	8	22.9
3	+	+	-	5	14.3
4	+	-	-	4	11.4
5	-	+	+	2	5.7
6	-	-	-	5	14.3