嗜麦芽寡养单胞菌获得性耐药的机制研究及整合子分布调查
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摘要
目的了解嗜麦芽寡养单胞菌的临床感染特点和药敏性,探讨嗜麦芽寡养单胞菌的获得性耐药机制并检测Ⅰ、Ⅱ、Ⅲ类整合酶基因(intI1,intI2和intI3)及对应的整合子在菌株中的分布,揭示基因分布与细菌耐药性之间的关系。方法收集2015年1-12月自临床分离的嗜麦芽寡养单胞菌400株,剔除重复菌株,对其临床感染特点进行总结分析,煮沸法提取总DNA,采用PCR法扩增qacE△1-sul1、sul2、sul3、intI1、intI2、intI3、dfrA1、dfrA5、dfrA12、dfrA13和Ⅰ、Ⅱ、Ⅲ类整合子可变区。结果临床分离的400株嗜麦芽寡养单胞菌主要来自于痰标本(78.8%),其次是咽拭子(7.5%);分离率最高的科室是ICU(31.3%)、其次是老年科(20.0%);感染病例以男性居多,年龄>60岁者占89%;400株嗜麦芽寡养单胞菌中有49株检出Ⅰ类整合酶基因(12.3%),2株检出Ⅲ类整合酶基因(0.5%),49株检出qacE△1-sul1基因(12.3%),15株检出sul2基因(3.8%);而在40株耐磺胺甲噁唑/甲氧苄啶嗜麦芽寡养单胞菌中有22株检出Ⅰ类整合酶基因(55.0%),13株检出qacE△1-sul1基因(26.5%),6株检出sul2基因(15.0%);对Ⅰ类整合子可变区进行扩增后获得3个大小不同的条带,测序后发现携带是aacA4,ant(3″)和aac(6′)-Ib耐药基因盒。结论Ⅰ类整合子及sul、dfrA基因的存在可以增加嗜麦芽寡养单胞菌对磺胺甲噁唑/甲氧苄啶的耐药性,这是多方面因素协同作用的结果,此外耐磺胺甲噁唑/甲氧苄啶嗜麦芽寡养单胞菌大部分携带有耐季胺盐消毒基因qacE△1,提示随时监测细菌耐药情况并开发新型消毒剂迫在眉睫。
OBJECTIVE To understand the clinical characteristics of infections caused by Stenotrophomonas maltophilia,analyze the drug susceptibility,explore the acquired drug resistance mechanisms in the S.maltophilia strains,and detect the type I,II,III integrase genes(intI1,intI2,and intI3)and their corresponding integrons in the strains so as to define the relationship between the distribution of genes and the bacterial drug resistance.METHODS A total of 400 clinical S.maltophiliaisolates were collected from Jan 2015 to Dec 2015,the repetitive strains were excluded,the clinical characteristics of the S.maltophiliainfection were analyzed,the total DNA was extracted by using boiling method,and PCR method was employed to amplify qacE△1-sul1,sul2,sul3,intI1,intI2,intI3,dfrA1,dfrA5,dfrA12,and dfrA13 as well as variable regions of class I,II,and III integrons.RESULTS Of the 400 clinical S.maltophiliaisolates,78.8% were isolated from sputum specimens,and 7.5%were isolated from throat swab;the isolation rate was highest in ICUs(31.3%),followed by the geriatric department(20.0%).The male patients with the infection were dominant,and the patients aged more than 60 years old accounted for 89%.Of the 400 strains of S.maltophilia,49(12.3%)were detected positive for class I integrase gene,2(0.5%)were detected positive for class III integrase gene,49(12.3%)were detected positive for qacE△1-sul1 gene,15(3.8%)were detected positive for sul2 gene.Of 40 strains of sulfamethoxazole-trimethoprimresistant S.maltophilia,22(55.0%)were detected positive for class I integrase gene,13(26.5%)were detected positive for qacE△1-sul1 gene,and 6(15.0%)were detected positive for sul2 gene.Totally 3different sizes of bands were acquired after the variable regions of class I integrons were amplified,and aacA4,ant(3″),and aac(6′)-Ib drug resistance gene cassettes were identified after bands were sequenced.CONCLUSION The presence of class I integrons and sul,dfrAgenes in the S.maltophiliastrains may increase the drug resistance to sulfamethoxazole-trimethoprim,which is shown of a synergistic effect of a variety of factors,in addition,most of the sulfamethoxazole-trimethoprim-resistant S.maltophilia carry with quaternary ammonium salt resistance gene qacE△1,suggesting that it is urgent to monitor the bacterial resistance at any time and develop novel disinfectants.
OBJECTIVE To understand the clinical characteristics of infections caused by Stenotrophomonas maltophilia,analyze the drug susceptibility,explore the acquired drug resistance mechanisms in the S.maltophilia strains,and detect the type I,II,III integrase genes(intI1,intI2,and intI3)and their corresponding integrons in the strains so as to define the relationship between the distribution of genes and the bacterial drug resistance.METHODS A total of 400 clinical S.maltophiliaisolates were collected from Jan 2015 to Dec 2015,the repetitive strains were excluded,the clinical characteristics of the S.maltophiliainfection were analyzed,the total DNA was extracted by using boiling method,and PCR method was employed to amplify qacE△1-sul1,sul2,sul3,intI1,intI2,intI3,dfrA1,dfrA5,dfrA12,and dfrA13 as well as variable regions of class I,II,and III integrons.RESULTS Of the 400 clinical S.maltophiliaisolates,78.8% were isolated from sputum specimens,and 7.5%were isolated from throat swab;the isolation rate was highest in ICUs(31.3%),followed by the geriatric department(20.0%).The male patients with the infection were dominant,and the patients aged more than 60 years old accounted for 89%.Of the 400 strains of S.maltophilia,49(12.3%)were detected positive for class I integrase gene,2(0.5%)were detected positive for class III integrase gene,49(12.3%)were detected positive for qacE△1-sul1 gene,15(3.8%)were detected positive for sul2 gene.Of 40 strains of sulfamethoxazole-trimethoprimresistant S.maltophilia,22(55.0%)were detected positive for class I integrase gene,13(26.5%)were detected positive for qacE△1-sul1 gene,and 6(15.0%)were detected positive for sul2 gene.Totally 3different sizes of bands were acquired after the variable regions of class I integrons were amplified,and aacA4,ant(3″),and aac(6′)-Ib drug resistance gene cassettes were identified after bands were sequenced.CONCLUSION The presence of class I integrons and sul,dfrAgenes in the S.maltophiliastrains may increase the drug resistance to sulfamethoxazole-trimethoprim,which is shown of a synergistic effect of a variety of factors,in addition,most of the sulfamethoxazole-trimethoprim-resistant S.maltophilia carry with quaternary ammonium salt resistance gene qacE△1,suggesting that it is urgent to monitor the bacterial resistance at any time and develop novel disinfectants.
引文
[1]Brooke JS.Stenotrophomonas maltophilia:an emerging global opportunistic pathogen[J].Clin Microbiol Rev,2012,25(1):2-41.
[2]国家卫生计生委合理用药专家委员会,全国细菌耐药监测网.2015年全国细菌耐药监测报告[J].中国执业药师,2016,13(3):3-8.
[3]Looney WJ,Narita M,Mühlemann K.Stenotrophomonas maltophilia:an emerging opportunist human pathogen[J].Lancet Infect Dis,2009,9(5):312-323.
[4]Samonis G,Karageorgopoulos DE,Maraki S,et al.Stenotrophomonas maltophiliainfections in a general hospital:patient characteristics,antimicrobial susceptibility,and treatment outcome[J].PLoS One,2012,7(5):e37375.
[5]胡立芬,叶英,沈为华,等.嗜麦芽寡养单胞菌的天然及获得性耐药机制研究进展[J].中国抗生素杂志,2011,36(9):1-5.
[6]Clinical and Laboratory Standards Institute.Performance standards for antimicrobial susceptibility testing,twenty-fourth informational supplement[S].M100-S25.CLSI,2015.
[7]梁冰.96株嗜麦芽寡养假单胞菌医院感染临床分布及耐药性分析[J].中国实用医药,2012,7(2):164-165.
[8]王玉萍,李小宁,宋骏.皖南地区74例嗜麦芽寡养单胞菌的分布及耐药分析[J].临床输血与检验,2012,14(3):221-222.
[9]Tan CK,Liaw SJ,Yu CJ,et al.Extensively drug-resistant Stenotrophomonas maltophilia in a tertiary care hospital in Taiwan:microbiologic characteristics,clinical features,and outcomes[J].Diagn Microbiol Infect Dis,2008,60(2):205-210.
[10]Hu LF,Gao LP,Ye Y,et al.Susceptibility of Stenotrophomonas maltophilia clinical strains in China to antimicrobialcombinations[J].J Chemother,2014,26(5):282-286.
[11]Neela V,Rankouhi SZ,van Belkum A,et al.Stenotrophomonas maltophiliain Malaysia:molecular epidemiology and trimethoprim-sulfamethoxazole resistance[J].Int J Infect Dis,2012,16(8):e603-607.
[12]Hu LF,Chang X,Ye Y,et al.Stenotrophomonas maltophilia resistance to trimethoprim/sulfamethoxazole mediated by acquisition of sul and dfrA genes in a plasmid-mediated class 1integron[J].Int J Antimicrob Agents,2011,37(3):230-234.
[13]Toleman MA,Bennett PM,Bennett DM,et al.Global emergence of trimethoprim/sulfamethoxazole resistance in Stenotrophomonas maltophilia mediated by acquisition of sul genes[J].Emerg Infect Dis,2007,13(4):559-565.
[14]Wu K,Wang F,Sun J,et al.Class 1integron gene cassettes in multidrug-resistant gram-negative bacteria in southern China[J].Int J Antimicrob Agents,2012,40(3):264-267.
[15]Labbate M,Case RJ,Stokes HW.The integron/gene cassette system:an active player in bacterial adaptation[J].Methods Mol Biol,2009(532):103-125.
[2]国家卫生计生委合理用药专家委员会,全国细菌耐药监测网.2015年全国细菌耐药监测报告[J].中国执业药师,2016,13(3):3-8.
[3]Looney WJ,Narita M,Mühlemann K.Stenotrophomonas maltophilia:an emerging opportunist human pathogen[J].Lancet Infect Dis,2009,9(5):312-323.
[4]Samonis G,Karageorgopoulos DE,Maraki S,et al.Stenotrophomonas maltophiliainfections in a general hospital:patient characteristics,antimicrobial susceptibility,and treatment outcome[J].PLoS One,2012,7(5):e37375.
[5]胡立芬,叶英,沈为华,等.嗜麦芽寡养单胞菌的天然及获得性耐药机制研究进展[J].中国抗生素杂志,2011,36(9):1-5.
[6]Clinical and Laboratory Standards Institute.Performance standards for antimicrobial susceptibility testing,twenty-fourth informational supplement[S].M100-S25.CLSI,2015.
[7]梁冰.96株嗜麦芽寡养假单胞菌医院感染临床分布及耐药性分析[J].中国实用医药,2012,7(2):164-165.
[8]王玉萍,李小宁,宋骏.皖南地区74例嗜麦芽寡养单胞菌的分布及耐药分析[J].临床输血与检验,2012,14(3):221-222.
[9]Tan CK,Liaw SJ,Yu CJ,et al.Extensively drug-resistant Stenotrophomonas maltophilia in a tertiary care hospital in Taiwan:microbiologic characteristics,clinical features,and outcomes[J].Diagn Microbiol Infect Dis,2008,60(2):205-210.
[10]Hu LF,Gao LP,Ye Y,et al.Susceptibility of Stenotrophomonas maltophilia clinical strains in China to antimicrobialcombinations[J].J Chemother,2014,26(5):282-286.
[11]Neela V,Rankouhi SZ,van Belkum A,et al.Stenotrophomonas maltophiliain Malaysia:molecular epidemiology and trimethoprim-sulfamethoxazole resistance[J].Int J Infect Dis,2012,16(8):e603-607.
[12]Hu LF,Chang X,Ye Y,et al.Stenotrophomonas maltophilia resistance to trimethoprim/sulfamethoxazole mediated by acquisition of sul and dfrA genes in a plasmid-mediated class 1integron[J].Int J Antimicrob Agents,2011,37(3):230-234.
[13]Toleman MA,Bennett PM,Bennett DM,et al.Global emergence of trimethoprim/sulfamethoxazole resistance in Stenotrophomonas maltophilia mediated by acquisition of sul genes[J].Emerg Infect Dis,2007,13(4):559-565.
[14]Wu K,Wang F,Sun J,et al.Class 1integron gene cassettes in multidrug-resistant gram-negative bacteria in southern China[J].Int J Antimicrob Agents,2012,40(3):264-267.
[15]Labbate M,Case RJ,Stokes HW.The integron/gene cassette system:an active player in bacterial adaptation[J].Methods Mol Biol,2009(532):103-125.