大熊猫粪源大肠杆菌耐药性及整合子研究
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摘要
为了解大熊猫粪源大肠杆菌的耐药性、整合子-基因盒的分布特性,分析整合子对细菌耐药性的影响,采用Kirby-Bauer(K-B)纸片法进行了50株大肠杆菌对13种抗菌药物的药物敏感性试验;PCR-测序法检测了1、2、3型整合酶基因,进一步对阳性菌株可变区的基因盒序列鉴定分析。结果显示,菌株对13种抗菌药物表现出不同的耐药性,其中对氨苄西林、头孢唑林、四环素和复方新诺明表现出较高耐药性(耐药率为30%~68%),对其余药物耐药性较低(耐药率低于14%);50株菌中有15株(30%)含有1型整合子,未发现2型和3型整合子;15株1型整合子阳性菌中,有6株(40%)扩增出1200~2000 bp的基因盒,主要介导氨基糖苷类和磺胺-甲氧苄啶耐药的aad A和dfr A基因家族。以dfr A27+aad A2为主(检出率83.33%),1株为aac A4+aad A1+cat B2。以上说明本次检测的大熊猫粪源大肠杆菌对多种抗菌药物呈低水平耐药;1型整合子在大肠杆菌中广泛分布,整合子-基因盒是造成整合子阳性菌株耐氨基糖苷类、磺胺-甲氧苄啶类、氯霉素的主要原因。
To investigate the antimicrobial resistance,distribution of integron-gene cassettes of Escherichia coli that isolated from the feces of giant pandas,as well as determine the correlation between resistance and integron-gene cassette,KirbyBauer method was used to test the susceptibility of 50 E. coli isolates against 13 antimicrobial agents. PCR-sequencing was established to detect the integrons( class 1,2,3) and gene cassettes that were harbored in E. coli isolates. The results showed that the isolates had different resistant levels to the 13 tested antimicrobials: high resistant level was observed in ampicillin,cefazolin,tetracycline and SMZ-TMP( resistant rates ranged from 30% to 68%); 30% of the isolates were found to harbor the class 1 integron,and no class 2 or class 3 integron was detected; gene cassettes with length of 1200- 2000 bp were amplified from 40% of the 15 int I1-positive isolates,and the main resistant gens were identified as encoding aminoglycoside adenyltransferase( aad A) and dihydrofolate reductase( dfr A). The main arrays of gene cassettes were dfr A27 + aad A2( detection rate was 83. 33%),moreover,1 isolate was found to harbor the gene cassettes aac A4 + aad A1 + cat B2 discovered. In conclusion,the E. coli isolates from the feces of giant pandas were sensitive to majority of the tested antimicrobial agents. Class l integron was widely distributed in E. coli strains from giant pandas,and integron-gene cassettes were responsible for resistant to aminoglycosides,sulfonamides-TMP and chloramphenicol.
To investigate the antimicrobial resistance,distribution of integron-gene cassettes of Escherichia coli that isolated from the feces of giant pandas,as well as determine the correlation between resistance and integron-gene cassette,KirbyBauer method was used to test the susceptibility of 50 E. coli isolates against 13 antimicrobial agents. PCR-sequencing was established to detect the integrons( class 1,2,3) and gene cassettes that were harbored in E. coli isolates. The results showed that the isolates had different resistant levels to the 13 tested antimicrobials: high resistant level was observed in ampicillin,cefazolin,tetracycline and SMZ-TMP( resistant rates ranged from 30% to 68%); 30% of the isolates were found to harbor the class 1 integron,and no class 2 or class 3 integron was detected; gene cassettes with length of 1200- 2000 bp were amplified from 40% of the 15 int I1-positive isolates,and the main resistant gens were identified as encoding aminoglycoside adenyltransferase( aad A) and dihydrofolate reductase( dfr A). The main arrays of gene cassettes were dfr A27 + aad A2( detection rate was 83. 33%),moreover,1 isolate was found to harbor the gene cassettes aac A4 + aad A1 + cat B2 discovered. In conclusion,the E. coli isolates from the feces of giant pandas were sensitive to majority of the tested antimicrobial agents. Class l integron was widely distributed in E. coli strains from giant pandas,and integron-gene cassettes were responsible for resistant to aminoglycosides,sulfonamides-TMP and chloramphenicol.
引文
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林居纯,陈雅莉,曹三杰,等.2011.食品动物源大肠杆菌多重耐药菌株整合子-基因盒的分子特征[J].畜牧兽医学报,42(1):77-81.
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岳磊,陈雪影,庄娜,等.2012.动物源qnr基因阳性肠杆菌I类整合子研究[J].中国农业科学,45(2):385-390.
张安云,王红宁,周万蓉,等.2006.多重PCR对大熊猫和野生动物肠道分离菌氨基糖苷类抗生素耐药基因检测研究[C]//中国畜牧医学会家畜传染病学分会.第六届理事会第二次会议暨教学专业委员会第六届代表大会论文集.北京:中国农业出版社:620-623.
张发苏,李涛,徐无宏,等.2006.大肠埃希菌I类整合子携带率及可转移耐药率研究[J].临床输血与检验,8(3):164-166.
邹立扣,蒲妍君,杨莉,等.2012.四川省猪肉源大肠杆菌和沙门氏菌的分离与耐药性分析[J].食品科学,33(13):202-206.
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Clinical and Laboratory Standards Institute.2011.Performance standards for antimicrobial susceptibility testing[S].CLSI:21th Informational Supple-ment M100-S21.
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Wang X,Yan QG,Xia XD,et al.2013.Serotypes,virulence factors factors,and antimicrobial susceptibilities of vaginal and fecal isolates o Escherichia coli from giant pandas[J].Appl Environ Microbiol,7(17):5146-5150.
Zhang AY,Wang HN,Tian GB,et al.2009.Phenotypic and genotypic characterisation of antimicrobial resistance in faecal bacteria from 30 giant panda[J].International Journal of Antimicrobial Agents,33(2):456-460.
李蓓,李旭林,郭丽娟,等.2012.大熊猫大肠杆菌的分离鉴定和耐药性分析[J].西南农业学报,25(3):1109-1113.
林居纯,陈雅莉,曹三杰,等.2011.食品动物源大肠杆菌多重耐药菌株整合子-基因盒的分子特征[J].畜牧兽医学报,42(1):77-81.
林居纯,舒刚,张辉建,等.2014.健康畜禽肠道大肠杆菌耐药性及整合子-基因盒检测[J].中国兽医学报,34(1):56-62.
刘书亮,张晓利,韩新锋,等.2011.动物性食品源大肠杆菌耐药性研究[J].中国食品学报,11(7):163-168.
陆思静,管希周,王睿,等.2010.59株大肠杆菌中整合子的研究[J].中国药房,21(10):901-904.
魏秀丽,高迎春,魏秀青,等.2013.多重耐药大肠埃希菌rmtB基因检测与整合子初步研究[J].中国抗生素杂志,38(4):289-292.
伍清林,金兰梅,周正凯,等.2011.动物同珍禽舍内大肠杆菌的分离鉴定与耐药性的试验[J].中国兽医杂志,47(9):24-27.
熊焰,李德生,王印,等.2000.卧龙自然保护区大熊猫粪样菌群的分离鉴定与分布研究[J].畜牧兽医学报,31(2):165-170.
岳磊,陈雪影,庄娜,等.2012.动物源qnr基因阳性肠杆菌I类整合子研究[J].中国农业科学,45(2):385-390.
张安云,王红宁,周万蓉,等.2006.多重PCR对大熊猫和野生动物肠道分离菌氨基糖苷类抗生素耐药基因检测研究[C]//中国畜牧医学会家畜传染病学分会.第六届理事会第二次会议暨教学专业委员会第六届代表大会论文集.北京:中国农业出版社:620-623.
张发苏,李涛,徐无宏,等.2006.大肠埃希菌I类整合子携带率及可转移耐药率研究[J].临床输血与检验,8(3):164-166.
邹立扣,蒲妍君,杨莉,等.2012.四川省猪肉源大肠杆菌和沙门氏菌的分离与耐药性分析[J].食品科学,33(13):202-206.
朱靖.1974.关于大熊猫分类地位的探讨[J].动物学报,20(2):174-187.
Clinical and Laboratory Standards Institute.2011.Performance standards for antimicrobial susceptibility testing[S].CLSI:21th Informational Supple-ment M100-S21.
Sacristán C,Esperón F,Herrera-León S,et al.2014.Virulence genes,antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain[J].Avian Pathol,43(2):172-175.
Villa L,Mammina C,Miriagou V,et al.2002.Multidrug and broadspectrum cephalosporin resistance among Salmonella enterica serotype enteritidis clinical isolates in southern Italy[J].J Clin Microbiol,40(7):2662-2665.
Wang X,Yan QG,Xia XD,et al.2013.Serotypes,virulence factors factors,and antimicrobial susceptibilities of vaginal and fecal isolates o Escherichia coli from giant pandas[J].Appl Environ Microbiol,7(17):5146-5150.
Zhang AY,Wang HN,Tian GB,et al.2009.Phenotypic and genotypic characterisation of antimicrobial resistance in faecal bacteria from 30 giant panda[J].International Journal of Antimicrobial Agents,33(2):456-460.