水产动物源气单胞菌喹诺酮类耐药与PMQR基因、QRDR突变相关性分析
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摘要
为了解PMQR基因、QRDR突变与气单胞菌喹诺酮类耐药相关性,以116株水产源气单胞菌为研究对象,采用PCR法检测PMQR基因(qnrA、qnrB、qnrS、qepA、aac(6′)-Ib-cr)并分析QRDR靶基因gyrA、parC突变情况,用微量二倍稀释法测定萘啶酸(NAL)、环丙沙星(CIP)、恩诺沙星(ENR)的最小抑菌浓度(MIC)值。结果表明:116株气单胞菌中,有18株(15.52%)菌株携带PMQR基因,其中8株(6.90%)携带qnrS2基因,9株(7.76%)携带aac(6′)-Ib-cr基因,1株同时携带qnrS2、aac(6′)-Ib-cr基因,未检测到qnrA,qnrB和qepA基因;56株(48.28%)菌株发生QRDR靶位点突变,主要突变方式为GyrA:Ser~(83)Ile和GyrA:Ser~(83)Ile+ParC:Ser~(87)Ile;对NAL、CIP及ENR耐药率分别为47.41%、12.07%及11.21%;7株仅携带qnrS2基因菌株未发生QRDR突变,对喹诺酮类药物敏感性略下降,NAL、CIP及ENR的MIC值分别小于4.00、0.50和1.00mg/L;10株携带aac(6′)-Ib-cr基因的菌株均发生QRDR突变并对NAL表现耐药,MIC值均>128.00mg/L,其中8株对CIP和ENR表现耐药,MIC值均≥4.00mg/L;发生gyrA单突变或gyrA和parC双突变菌株均对NAL表现耐药,MIC值均≥64.00mg/L,CIP和ENR的MIC值有的<4.00mg/L,有的≥4.00mg/L。综上,QRDR靶基因突变可影响水产动物源气单胞菌对萘啶酸的药物敏感性,而气单胞菌对氟喹诺酮类药物耐药可能与PMQR和QRDR协同作用有关。
To analyze the correlation of plasmid-mediated quinolone resistance(PMQR)genes/quinolone resistance determining region(QRDR)mutations with quinolones resistance in Aeromonas,116 Aeromonasisolates were taken as study object.PMQR genes including qnrA,qnrB,qnrS,qepAand aac(6′)-Ib-cr in Aeromonas were detected by PCR.The target genes of gyrAand parC were also amplified by PCR to analyze the mutations in QRDR.The minimal inhibitory concentrations(MICs)of all 116isolates against nalidixic acid(NAL),enrofloxacin(ENR)and ciprofloxacin(CIP)of quinolones were evaluated by micro broth 2-fold dilution.The results showed that:Eighteen(15.52%)isolates were detected carrying PMQR gene.Among those strains,8(6.90%)and 9(7.76%)strains carried qnrS2 gene and aac(6′)-Ib-cr gene,respectively.One strain carried both of these two genes;qnrA,qnrB and qepA genes were not detected in this study;Mutations in the QRDRs of gyrAand parC were found in 56 strains(48.28%)of all isolates with GyrA:Ser~(83)Ile and GyrA:Ser~(83)Ile+ParC:Ser~(87)Ile combination being the most common mutations.The resistance rates of 116 Aeromonasisolates to NAL,CIP and ENR were 47.41%,12.07%and 11.21%,respectively;With the exception of one strain carrying qnrS2 gene alone was resistant to CIP,7strains were susceptible to the 3 drugs of quinolones examined;No mutations in QRDR.The MICs of NAL,CIP and ENR were less than 4.00,0.50and 1.00 mg/L,respectively;Ten aac(6′)-Ib-cr positive strains were found mutations in QRDR and resistant to NAL(MIC>128.00mg/L),and eight strains were resistant to CIP and ENR(MIC≥4.00mg/L).Strains with either gyrAsingle mutation or gyrA+parCdouble mutation were resistant to NAL(MIC≥64.00mg/L).The MICs of CIP and ENR for the strains with QRDR mutation,some strains were less than 4.00mg/L,and there were more than 4.00mg/L.In conclusion,QRDR target gene mutation may contribute to the susceptibility decrease of NAL,and the fluoroquinolone resistance in Aeromonas might be attributed to the mutual cooperation between PMQR and QRDR mutations.
To analyze the correlation of plasmid-mediated quinolone resistance(PMQR)genes/quinolone resistance determining region(QRDR)mutations with quinolones resistance in Aeromonas,116 Aeromonasisolates were taken as study object.PMQR genes including qnrA,qnrB,qnrS,qepAand aac(6′)-Ib-cr in Aeromonas were detected by PCR.The target genes of gyrAand parC were also amplified by PCR to analyze the mutations in QRDR.The minimal inhibitory concentrations(MICs)of all 116isolates against nalidixic acid(NAL),enrofloxacin(ENR)and ciprofloxacin(CIP)of quinolones were evaluated by micro broth 2-fold dilution.The results showed that:Eighteen(15.52%)isolates were detected carrying PMQR gene.Among those strains,8(6.90%)and 9(7.76%)strains carried qnrS2 gene and aac(6′)-Ib-cr gene,respectively.One strain carried both of these two genes;qnrA,qnrB and qepA genes were not detected in this study;Mutations in the QRDRs of gyrAand parC were found in 56 strains(48.28%)of all isolates with GyrA:Ser~(83)Ile and GyrA:Ser~(83)Ile+ParC:Ser~(87)Ile combination being the most common mutations.The resistance rates of 116 Aeromonasisolates to NAL,CIP and ENR were 47.41%,12.07%and 11.21%,respectively;With the exception of one strain carrying qnrS2 gene alone was resistant to CIP,7strains were susceptible to the 3 drugs of quinolones examined;No mutations in QRDR.The MICs of NAL,CIP and ENR were less than 4.00,0.50and 1.00 mg/L,respectively;Ten aac(6′)-Ib-cr positive strains were found mutations in QRDR and resistant to NAL(MIC>128.00mg/L),and eight strains were resistant to CIP and ENR(MIC≥4.00mg/L).Strains with either gyrAsingle mutation or gyrA+parCdouble mutation were resistant to NAL(MIC≥64.00mg/L).The MICs of CIP and ENR for the strains with QRDR mutation,some strains were less than 4.00mg/L,and there were more than 4.00mg/L.In conclusion,QRDR target gene mutation may contribute to the susceptibility decrease of NAL,and the fluoroquinolone resistance in Aeromonas might be attributed to the mutual cooperation between PMQR and QRDR mutations.
引文
[1]Gibotti A,Saridakis H O,Pelayo J S,Tagliari K C,Falcao D P.Prevalence and virulence properties of Vibrio cholerae non-O1,Aeromonas spp and Plesiomonas shigelloides isolated from Cambe Stream(State of Parana,Brazil)[J].Journal of Applied Microbiology,2000,89(1):70-75
[2]Kim E S,Hooper D C.Clinical importance and epidemiology of quinolone resistance[J].Infection&Chemotherapy,2014,46(4):226-238
[3]杨雨辉,佟恒敏,卢彤岩,赵吉伟.恩诺沙星在水生动物疾病防治上应用的研究进展[J].水产学杂志,2003:16(1):80-82Yang Y H,Tong H M,Lu T Y,Zhao J W.The study progress on the usage of enrofloxacin in the aquaculme[J].Chinese Journal of Fisheries,2003:16(1):80-82(in Chinese)
[4]Strahilevitz J,Jacoby G A,Hooper D C,Robicsek A.Plasmidmediated quinolone resistance:A multifaceted threat[J].Clinical Microbiology Reviews,2009,22(4):664-689
[5]崔佳佳,王荻,卢彤岩,李绍戊.养殖鱼源嗜水气单胞菌对氟喹诺酮类药物的耐药机制[J].水产学报,2016,40(3):495-502Cui J J,Wang D,Lu T Y,Li S W.In vitro study on fluoroquinolone resistance mechanism of Aeromonas hydrophila from cultured fish[J].Journal of Fisheries of China,2016,40(3):495-502(in Chinese)
[6]薛慧娟,邓玉婷,姜兰,吴雅丽,谭爱萍,王伟利,罗理,赵飞.水产动物源嗜水气单胞菌药物敏感性及QRDR基因突变分析.广东农业科学,2012:39(23):149-153Xue H J,Deng Y T,Jiang L,Wu Y L,Tan A P,Wang W L,Luo L,Zhao F.Antimicrobial susceptibility and mutations of QRDR in Aeromonas hydrophilaisolated from aquatic animals[J].Guangdong Agricultural Sciences,2012:39(23):149-153(in Chinese)
[7]Liu J H,Deng Y T,Zeng Z L,Gao J H,Chen L,Arakawa Y,Chen Z L.Coprevalence of plasmid-mediated quinolone resistance determinants QepA,Qnr,and Aac(6′)-Ib-cr among16SrRNA methylase RmtB-producing Escherichia coli isolates from pigs[J].Antimicrobial Agents and Chemotherapy,2008,52(8):2992-2993
[8]Ruiz J,Gómez J,Navia M M,Ribera A,Sierra J M,Marco F,Mensa J,Vila J.High prevalence of nalidixic acid resistant,ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae[J].Diagnostic Microbiology and Infectious Disease,2002,42(4):257-261
[9]Tavío M D M,Vila J,Ruiz J,Ruiz J,Martín-Snchez A M,de Anta M T J.Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates[J].Journal of Antimicrobial Chemotherapy,1999,44(6):735-742
[10]夏蕊蕊,国宪虎,张玉臻,徐海.喹诺酮类药物及细菌对其耐药性机制研究进展.中国抗生素杂志,2010,35(4):255-261Xia R R,Guo X H,Zhang Y Z,Xu H.Quinolones and the mechanism of quinolone resistance in bacteria[J].Chinese Journal of Antibiotics,2010,35(4):255-261(in Chinese)
[11]Han J E,Kim J H,Cheresca C H,Shin S P,Jun J W,Chai J Y,Han S Y,Park S C.First description of the qnrS-like(qnrS5)gene and analysis of quinolone resistance-determining regions in motile Aeromonas spp from diseased fish and water[J].Research in Microbiology,2012,163(1):73-79
[12]牛志伟,吕小丽,范华龙,梁静真,蒙兰丽,韩书煜,黄钧.我国耐喹诺酮类嗜水气单胞菌毒力基因及耐药基因检出情况的系统评价[J].南方农业学报,2016,47(9):1587-1595Niu Z W,Lv X L,Fan H L,Liang J Z,Meng L L,Han S Y,Huang J.Systematic review on virulence genes of quinolonesresistant Aeromonas hydrophila and detection of drugresistant genes in China[J].Journal of Southern Agriculture,2016,47(9):1587-1595(in Chinese)
[13]刘贵深,于涛.食源性沙门氏菌耐药性及质粒介导喹诺酮耐药基因检测[J].生物技术通报,2014(8):202-207Liu G S,Yu T.Study on antimicrobial resistance and plasmidmediated quinolone resistance of foodborne Salmonellaisolates[J].Biotechnology Bulletin,2014(8):202-207(in Chinese)
[14]何秋丽.气单胞菌的耐药现状及对喹诺酮类抗生素耐药机制的研究[D].杭州:浙江大学,2015He Q L.The current situation of antibiotic resistance and the mechanism study of quinolones resistance in Aeromonas strains[D].Hangzhou:Zhejiang University School,2015(in Chinese)
[15]Han J E,Kim J H,Choresca C H Jr,Shin S P,Jun J W,Chai JY,Park S C.A small IncQ-type plasmid carrying the quinolone resistance(qnrS2)gene from Aeromonas hydrophila[J].Letters in Applied Microbiology,2012,54(4):374-376
[16]Xia R R,Guo X H,Zhang Y Z,Xu H.qnrVC-like gene located in a novel complex class 1integron harboring the ISCR1element in an Aeromonas punctata strain from an aquatic environment in Shandong Province,China[J].Antimicrobial Agents and Chemotherapy,2010,54(8):3471-3474
[17]Arsène S,Leclercq R.Role of a qnr-like gene in the intrinsic resistance of Enterococcusfaecalisto fluoroquinolones[J].Antimicrobial Agents and Chemotherapy,2007,51(9):3254-3258
[18]Martínez-Martínez L,Pascual A,Jacoby G A.Quinolone resistance from a transferable plasmid[J].The Lancet,1998,351(9105):797-799
[19]李德喜,刘建华,张素梅,李新生,陈玉霞,杜向党,胡功政,苑丽.猪源大肠杆菌质粒和染色体介导的喹诺酮类药的耐药机制[J].中国兽医学报,2011,31(9):1262-1265Li D X,Liu J H,Zhang S M,Li X S,Chen Y X,Du X D,Hu,GZ,Yuan L.Mechanism of plasmid-and chromosome-mediated quinolone resistance in Escherichia coli isolated from pigs[J].Chinese Journal of Veterinary Science,2011,31(9):1262-1265(in Chinese)
[20]Arsene S,Leclercq R.Role of a qnr-like gene in the intrinsic resistance of Enterococcus faecalis to fluoroquinolones[J].Antimicrobial Agents and Chemotherapy,2007,51(9):3254-3258.
[21]Robicsek A,Strahilevitz J,Jacoby G A,Macielag M,Abbanat D,Hye Park C,Bush K,Hooper D C.Fluoroquinolonemodifying enzyme:A new adaptation of a common aminoglycoside acetyltransferase[J].Nature Medicine,2006,12(1):83-88
[22]Cesaro A,Bettoni R R D,Lascols C,Mérens A,Soussy C J,Cambau E.Low selection of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes[J].Journal of Antimicrobial Chemotherapy,2008,61(5):1007-1015
[23]张文慧.aac(6′)-Ib-cr基因对氟喹诺酮类药物耐药的作用研究[D].广州:华南农业大学,2016Zhang W H.Characteristic of the aac(6′)-Ib-cr gene on fluoroquinolone resistance[D].Guangzhou:South China Agricultural University,2016(in Chinese)
[2]Kim E S,Hooper D C.Clinical importance and epidemiology of quinolone resistance[J].Infection&Chemotherapy,2014,46(4):226-238
[3]杨雨辉,佟恒敏,卢彤岩,赵吉伟.恩诺沙星在水生动物疾病防治上应用的研究进展[J].水产学杂志,2003:16(1):80-82Yang Y H,Tong H M,Lu T Y,Zhao J W.The study progress on the usage of enrofloxacin in the aquaculme[J].Chinese Journal of Fisheries,2003:16(1):80-82(in Chinese)
[4]Strahilevitz J,Jacoby G A,Hooper D C,Robicsek A.Plasmidmediated quinolone resistance:A multifaceted threat[J].Clinical Microbiology Reviews,2009,22(4):664-689
[5]崔佳佳,王荻,卢彤岩,李绍戊.养殖鱼源嗜水气单胞菌对氟喹诺酮类药物的耐药机制[J].水产学报,2016,40(3):495-502Cui J J,Wang D,Lu T Y,Li S W.In vitro study on fluoroquinolone resistance mechanism of Aeromonas hydrophila from cultured fish[J].Journal of Fisheries of China,2016,40(3):495-502(in Chinese)
[6]薛慧娟,邓玉婷,姜兰,吴雅丽,谭爱萍,王伟利,罗理,赵飞.水产动物源嗜水气单胞菌药物敏感性及QRDR基因突变分析.广东农业科学,2012:39(23):149-153Xue H J,Deng Y T,Jiang L,Wu Y L,Tan A P,Wang W L,Luo L,Zhao F.Antimicrobial susceptibility and mutations of QRDR in Aeromonas hydrophilaisolated from aquatic animals[J].Guangdong Agricultural Sciences,2012:39(23):149-153(in Chinese)
[7]Liu J H,Deng Y T,Zeng Z L,Gao J H,Chen L,Arakawa Y,Chen Z L.Coprevalence of plasmid-mediated quinolone resistance determinants QepA,Qnr,and Aac(6′)-Ib-cr among16SrRNA methylase RmtB-producing Escherichia coli isolates from pigs[J].Antimicrobial Agents and Chemotherapy,2008,52(8):2992-2993
[8]Ruiz J,Gómez J,Navia M M,Ribera A,Sierra J M,Marco F,Mensa J,Vila J.High prevalence of nalidixic acid resistant,ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae[J].Diagnostic Microbiology and Infectious Disease,2002,42(4):257-261
[9]Tavío M D M,Vila J,Ruiz J,Ruiz J,Martín-Snchez A M,de Anta M T J.Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates[J].Journal of Antimicrobial Chemotherapy,1999,44(6):735-742
[10]夏蕊蕊,国宪虎,张玉臻,徐海.喹诺酮类药物及细菌对其耐药性机制研究进展.中国抗生素杂志,2010,35(4):255-261Xia R R,Guo X H,Zhang Y Z,Xu H.Quinolones and the mechanism of quinolone resistance in bacteria[J].Chinese Journal of Antibiotics,2010,35(4):255-261(in Chinese)
[11]Han J E,Kim J H,Cheresca C H,Shin S P,Jun J W,Chai J Y,Han S Y,Park S C.First description of the qnrS-like(qnrS5)gene and analysis of quinolone resistance-determining regions in motile Aeromonas spp from diseased fish and water[J].Research in Microbiology,2012,163(1):73-79
[12]牛志伟,吕小丽,范华龙,梁静真,蒙兰丽,韩书煜,黄钧.我国耐喹诺酮类嗜水气单胞菌毒力基因及耐药基因检出情况的系统评价[J].南方农业学报,2016,47(9):1587-1595Niu Z W,Lv X L,Fan H L,Liang J Z,Meng L L,Han S Y,Huang J.Systematic review on virulence genes of quinolonesresistant Aeromonas hydrophila and detection of drugresistant genes in China[J].Journal of Southern Agriculture,2016,47(9):1587-1595(in Chinese)
[13]刘贵深,于涛.食源性沙门氏菌耐药性及质粒介导喹诺酮耐药基因检测[J].生物技术通报,2014(8):202-207Liu G S,Yu T.Study on antimicrobial resistance and plasmidmediated quinolone resistance of foodborne Salmonellaisolates[J].Biotechnology Bulletin,2014(8):202-207(in Chinese)
[14]何秋丽.气单胞菌的耐药现状及对喹诺酮类抗生素耐药机制的研究[D].杭州:浙江大学,2015He Q L.The current situation of antibiotic resistance and the mechanism study of quinolones resistance in Aeromonas strains[D].Hangzhou:Zhejiang University School,2015(in Chinese)
[15]Han J E,Kim J H,Choresca C H Jr,Shin S P,Jun J W,Chai JY,Park S C.A small IncQ-type plasmid carrying the quinolone resistance(qnrS2)gene from Aeromonas hydrophila[J].Letters in Applied Microbiology,2012,54(4):374-376
[16]Xia R R,Guo X H,Zhang Y Z,Xu H.qnrVC-like gene located in a novel complex class 1integron harboring the ISCR1element in an Aeromonas punctata strain from an aquatic environment in Shandong Province,China[J].Antimicrobial Agents and Chemotherapy,2010,54(8):3471-3474
[17]Arsène S,Leclercq R.Role of a qnr-like gene in the intrinsic resistance of Enterococcusfaecalisto fluoroquinolones[J].Antimicrobial Agents and Chemotherapy,2007,51(9):3254-3258
[18]Martínez-Martínez L,Pascual A,Jacoby G A.Quinolone resistance from a transferable plasmid[J].The Lancet,1998,351(9105):797-799
[19]李德喜,刘建华,张素梅,李新生,陈玉霞,杜向党,胡功政,苑丽.猪源大肠杆菌质粒和染色体介导的喹诺酮类药的耐药机制[J].中国兽医学报,2011,31(9):1262-1265Li D X,Liu J H,Zhang S M,Li X S,Chen Y X,Du X D,Hu,GZ,Yuan L.Mechanism of plasmid-and chromosome-mediated quinolone resistance in Escherichia coli isolated from pigs[J].Chinese Journal of Veterinary Science,2011,31(9):1262-1265(in Chinese)
[20]Arsene S,Leclercq R.Role of a qnr-like gene in the intrinsic resistance of Enterococcus faecalis to fluoroquinolones[J].Antimicrobial Agents and Chemotherapy,2007,51(9):3254-3258.
[21]Robicsek A,Strahilevitz J,Jacoby G A,Macielag M,Abbanat D,Hye Park C,Bush K,Hooper D C.Fluoroquinolonemodifying enzyme:A new adaptation of a common aminoglycoside acetyltransferase[J].Nature Medicine,2006,12(1):83-88
[22]Cesaro A,Bettoni R R D,Lascols C,Mérens A,Soussy C J,Cambau E.Low selection of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes[J].Journal of Antimicrobial Chemotherapy,2008,61(5):1007-1015
[23]张文慧.aac(6′)-Ib-cr基因对氟喹诺酮类药物耐药的作用研究[D].广州:华南农业大学,2016Zhang W H.Characteristic of the aac(6′)-Ib-cr gene on fluoroquinolone resistance[D].Guangzhou:South China Agricultural University,2016(in Chinese)