耐恩诺沙星沙门菌gyrA和gyrB基因的分子特征分析
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摘要
为了探究临床分离的沙门菌对恩诺沙星的耐药机制,通过微量肉汤稀释法测定18株临床分离的沙门菌对恩诺沙星的敏感性,然后选取其中对恩诺沙星耐药的菌株,利用PCR扩增其gyrA和gyrB基因,并将扩增产物进行序列测定,最后用DNAMAN软件将测序结果与NCBI上公布的沙门菌标准野生型菌株(LT2)的gyrA和gyrB基因序列进行比对分析。结果显示,18株临床分离的沙门菌中有4株为耐药菌株,耐药率22.2%。对4株耐药菌株的gyrA和gyrB基因比对分析发现,gyrB基因未见突变,而gyrA基因有3个氨基酸位点突变,分别是Ser83→Phe或Leu(突变率100%)、Asp87→Asn(突变率75%)、Asn200→Asp(突变率25%);其中恩诺沙星最小抑菌浓度(MIC)≥16.000μg/mL的3株菌均是在83、87位出现双突变,而恩诺沙星MIC为4.000μg/mL的1株菌则是在83、200位出现双突变。结果表明,沙门菌对恩诺沙星产生耐药性与gyrA基因的突变密切相关,gyrA基因喹诺酮耐药区(QRDR)的双突变可使沙门菌对恩诺沙星的耐药性增强,提示Asp87的突变可能增强了Ser83位点突变所介导的耐药性,但非QRDR区的Asn200不能发挥增强耐药性的作用。
To explore the resistance mechanism of clinical isolates of Salmonella to enrofloxacin,the sensitivity of eighteen clinical isolates of Salmonella to enrofloxacin was determined by broth dilution method,then the gyrA and gyrB genes of enrofloxacin-resistant strains were amplified by PCR and the amplified products were sequenced.Finally the sequencing results were compared with gyrA and gyrB genes of standard wild type strain of Salmonella(LT2) published on NCBI by DNAMAN software.The results showed that four of eighteen Salmonella strains were drug-resistant strains,and the resistance rate was 22.2%.Analysis of gyrA and gyrB genes of four drug-resistant strains showed that gyrB gene had no change,and gyrA gene had three amino acid mutations,which were Ser83→Phe or Leu(mutation rate was 100%),Asp87→Asn(mutation rate was 75%) and Asn200→Asp(mutation rate was 25%).Three strains against which the MIC of enrofloxacin was greater than or equal to 16.000 μg/mL had double mutations at 83 and 87 sites,and one strain against which the MIC of enrofloxacin was 4.000 μg/mL had double mutations at 83 and 200 sites.The results suggested that the resistance of Salmonella to enrofloxacin was closely related to the mutation of gyrA gene,and the double mutation in QRDR region of gyrA gene could induce the strong resistance of Salmonella to enrofloxacin,and the results also indicated that Asp87 but Asn200 mutation might enhance the resistance mediated by mutation at Ser83 site.
To explore the resistance mechanism of clinical isolates of Salmonella to enrofloxacin,the sensitivity of eighteen clinical isolates of Salmonella to enrofloxacin was determined by broth dilution method,then the gyrA and gyrB genes of enrofloxacin-resistant strains were amplified by PCR and the amplified products were sequenced.Finally the sequencing results were compared with gyrA and gyrB genes of standard wild type strain of Salmonella(LT2) published on NCBI by DNAMAN software.The results showed that four of eighteen Salmonella strains were drug-resistant strains,and the resistance rate was 22.2%.Analysis of gyrA and gyrB genes of four drug-resistant strains showed that gyrB gene had no change,and gyrA gene had three amino acid mutations,which were Ser83→Phe or Leu(mutation rate was 100%),Asp87→Asn(mutation rate was 75%) and Asn200→Asp(mutation rate was 25%).Three strains against which the MIC of enrofloxacin was greater than or equal to 16.000 μg/mL had double mutations at 83 and 87 sites,and one strain against which the MIC of enrofloxacin was 4.000 μg/mL had double mutations at 83 and 200 sites.The results suggested that the resistance of Salmonella to enrofloxacin was closely related to the mutation of gyrA gene,and the double mutation in QRDR region of gyrA gene could induce the strong resistance of Salmonella to enrofloxacin,and the results also indicated that Asp87 but Asn200 mutation might enhance the resistance mediated by mutation at Ser83 site.
引文
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[9] CLSI.Performance standards for antimicrobial susceptibi-lity tests for bacteria isolated from animals,approved-fourth edition,CLSI document VET01-A4[S].USA:Clinical and Laboratory Standards Institute,2013.
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[11] 闫东辉,许淑珍,苏建荣,等.肠球菌gyrA基因突变和多重耐药泵与氟喹诺酮耐药性关系研究[J].首都医科大学学报,2012,33(2):161-164.
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[13] 王忠永,郑佳音,潘钦石.环丙沙星敏感性降低甲型副伤寒沙门菌喹诺酮耐药决定区基因突变分布特征[J].浙江实用医学,2010,15(6):469-471.
[14] 谭炳乾,王红宁,叶如俊,等.PCR-RFLP检测猪源致病性沙门氏菌gyrA基因点突变的研究[J].湖北农业科学,2003(5):88-90.
[15] PIDDOCK L J.Mechanisms of fluoroquinolone resistance:An update 1994—1998[J].Drugs,1999,58(2):11-18.
[16] 杨辉,张国良,张明霞.结核分枝杆菌氟喹诺酮耐药与gyrA基因突变的关系[J].实用医学杂志,2012,28(20):3457-3459.
[17] TAMANG M D,NAM H M,CHAE M H,et al.Prevalence of plasmid-mediated quinolone resistance determinants among Escherichia coli isolated from food animals in Korea[J].Food Pathog Dis,2012,9(12):1057-1063.
[18] DE LA FUENTE C M,DAUROS S P,BELLO T H,et al.Mutations in gyrA and gyrB genes among strains of Gram-negative bacilli isolated from Chilean hospitals and their relation with resistance to fluoroquinolones[J].Revista Médica De Chile,2007,135(9):1103-1110.
[19] 张国栋,曾章锐,王莹,等.gyrA、gyrB和外派系统共同介导铜绿假单胞菌对喹诺酮类耐药机制的研究[J].中国感染与化疗杂志,2014,14(3):224-228.
[20] 李珂婷.不同(氟)喹诺酮类抗生素作用沙门氏菌时菌株变异规律[D].杨凌:西北农林科技大学,2016.
[2] 廖成水,程相朝,吴庭才,等.河南省鸡源沙门氏菌新近流行株的分离鉴定及其耐药性分析[C]//中国畜牧兽医学会动物微生态学分会.第四届第十次全国学术研会暨动物微生态企业发展论坛论文集(上册):2010年卷.沧州:[出版社不详],2010:502-506.
[3] 顾兵,秦婷婷.沙门菌感染流行病学及对喹诺酮耐药研究进展[J].传染病信息,2017,30(5):282-285.
[4] GRIGGS D J,GENSBERG K,PIDDOCK L J.Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals[J].Antimicrob Agents Chemother,1996,40(4):1009-1013.
[5] 陈晶,孙自镛.鼠伤寒沙门菌对喹诺酮类耐药机制的研究[J].中国感染与化疗杂志,2006,6(2):100-104.
[6] 谭炳乾,王红宁,叶如俊,等.PCR-RFLP检测猪源致病性沙门氏菌gyrA基因点突变的研究[J].湖北农业科学,2003(5):88-90.
[7] 邓均华,徐涤平,伍锐,等.动物沙门氏菌对氟喹诺酮类药物的耐药性及GyrA基因的突变研究[J].湖南农业科学,2009,48(7):1554-1557.
[8] MERINO L A,ALONSO J M,RUIZ J,et al.Resistance to quinolones in Salmonella infantis due to overexpression of an active efflux system and a mutation in the gyrA gene[J].Enferm Infecc Microbiol Clin,2007,25(5):357-358.
[9] CLSI.Performance standards for antimicrobial susceptibi-lity tests for bacteria isolated from animals,approved-fourth edition,CLSI document VET01-A4[S].USA:Clinical and Laboratory Standards Institute,2013.
[10] 顾兵,秦婷婷.沙门菌感染流行病学及对喹诺酮耐药研究进展[J].传染病信息,2017,30(5):282-285.
[11] 闫东辉,许淑珍,苏建荣,等.肠球菌gyrA基因突变和多重耐药泵与氟喹诺酮耐药性关系研究[J].首都医科大学学报,2012,33(2):161-164.
[12] JOHNNING A,KRISTIANSSON E,FICK J,et al.Resistance mutations in gyrA and parC are common in Escherichia communities of both fluoroquinolone-polluted and uncontaminated aquatic environments[J].Front Microbiol,2015(6):1355.
[13] 王忠永,郑佳音,潘钦石.环丙沙星敏感性降低甲型副伤寒沙门菌喹诺酮耐药决定区基因突变分布特征[J].浙江实用医学,2010,15(6):469-471.
[14] 谭炳乾,王红宁,叶如俊,等.PCR-RFLP检测猪源致病性沙门氏菌gyrA基因点突变的研究[J].湖北农业科学,2003(5):88-90.
[15] PIDDOCK L J.Mechanisms of fluoroquinolone resistance:An update 1994—1998[J].Drugs,1999,58(2):11-18.
[16] 杨辉,张国良,张明霞.结核分枝杆菌氟喹诺酮耐药与gyrA基因突变的关系[J].实用医学杂志,2012,28(20):3457-3459.
[17] TAMANG M D,NAM H M,CHAE M H,et al.Prevalence of plasmid-mediated quinolone resistance determinants among Escherichia coli isolated from food animals in Korea[J].Food Pathog Dis,2012,9(12):1057-1063.
[18] DE LA FUENTE C M,DAUROS S P,BELLO T H,et al.Mutations in gyrA and gyrB genes among strains of Gram-negative bacilli isolated from Chilean hospitals and their relation with resistance to fluoroquinolones[J].Revista Médica De Chile,2007,135(9):1103-1110.
[19] 张国栋,曾章锐,王莹,等.gyrA、gyrB和外派系统共同介导铜绿假单胞菌对喹诺酮类耐药机制的研究[J].中国感染与化疗杂志,2014,14(3):224-228.
[20] 李珂婷.不同(氟)喹诺酮类抗生素作用沙门氏菌时菌株变异规律[D].杨凌:西北农林科技大学,2016.