四川省食品动物源性大肠杆菌耐药性监测及整合子—基因盒检测
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摘要
伴随着抗菌药物在兽医临床的广泛使用,耐药菌株迅速出现,多重耐药现象十分普遍,已经引起全世界的广泛关注。做好耐药性监测,从而指导兽医临床合理用药已刻不容缓。对于细菌耐药性检测,传统的方法主要是进行耐药表型监测,随着分子生物技术的发展,PCR技术在耐药基因型的检测及新耐药机制的发现方面发挥着重要的作用。
本研究首先从四川省不同养殖场收集食品动物源大肠杆菌病样本,按常规的细菌分离鉴定方法进行大肠杆菌菌株的鉴定。按照CLSI推荐方法及结果判断标准,应用微量肉汤稀释法测定细菌对于不同抗菌药物的最小抑菌浓度(MIC),从而对四川省食品动物源大肠杆菌的耐药情况进行监测。对于多重耐药菌株,采用多重PCR技术对其携带的3类整合子进行检测,同时对整合子阳性菌株进行基因盒分析。本次研究分2部分完成。
1.不同食品动物源性大肠杆菌耐药性的监测
从四川不同地区规模较大的养殖场收集食品动物源(鸡源、鸭源、牛源、猪源)大肠杆菌病样本,分离鉴定出705株大肠杆菌。采用微量肉汤稀释法进行了25种抗菌药物的药敏实验。结果显示：705株大肠杆菌对25种抗菌药物表现出程度不同的耐药性,特别是对兽医临床长期使用的甲氧苄啶、复方新诺明、利福平的耐药率接近100%；其次是对氨苄西林、四环素、氯霉素、氟喹诺酮类药物的耐药率均超过了70%；对头孢唑啉、头孢噻呋、头孢曲松也表现出一定的耐药性,耐药率分别为34.34%、41.30%、26.25%,但中介率较高,可见大肠杆菌对β-内酰胺类药物存在耐药率上升的趋势；本次监测发现所有大肠杆菌只有对阿米卡星保持有一定的敏感性,敏感率超过67%。
多重耐药性分析显示,705株菌均为多重耐药菌株,主要表现出15-24耐,在各种耐药谱型中,23耐菌株所占比率最大,其次为21耐。不同动物源菌株耐药性比较显示,禽源菌株的耐药情况较猪源菌株,牛源菌株严重。不同地区来源菌株耐药性比较可见,不同地区来源菌株对所检测的25种药物耐药性分布大体一致,对部分药物表现出一定地域差异。
2.食品动物源性大肠杆菌的整合子—基因盒检测
采用多重PCR方法同时扩增327株大肠杆菌整合酶Ⅰ型、Ⅱ型和Ⅲ型基因。多重PCR检测结果显示,在32Ⅳ株菌株中,294株细菌携带有Ⅰ型整合酶基因,其检出率为89.91%,本次检测未发现携带有Ⅱ型和Ⅲ型整合酶基因的菌株。随机抽取Ⅰ型整合酶阳性菌株81株,进行基因盒扩增,扩增结果显示14株Ⅰ型整合酶阳性菌株未扩增出基因盒片段,其余67株扩增出845bp-2731bp的基因盒片段。测序结果显示,67株菌中含有6种不同类型的基因盒,即dfrA17+aadA5、dfrA17、aadA22、dhfrAI+ aadA2、dfrA17+orfF+aadA2、aacA4+catB3+dfrA1,编码甲氧苄啶-磺胺类药物、氨基糖苷类药物和氯霉素耐药。本次研究发现在2株鸡源大肠杆菌株中,发现了通过整合子-基因盒携带的一种介导氨基糖苷类药物耐药的新基因aadA22,这是首次在四川分离菌株中发现该基因的存在,其介导的耐药性有待进一步的研究。
With the worldwide use of antimicrobial agents in veterinary medicine, the incidence of resistance and multidrug-resistance among pathogenic bacteria has been increasing, which attracts global attentions. Surveillance of antimicrobial resistance is very urgent to clinical medicine. The traditional method used in surveillance of resistance is to test resistant phenotype. With the development of molecular biological technology, PCR technology have been extensivly used in detection of resistant genotype and novel resistant mechanism.
In this study, The samples of colibacillosis were collected from food animals in different farms in Sichuan province, E.coli isolates were identified by conventional test. Antimicrobial susceptibility of these isolates was examined using the broth microdilution method and the results interpreted according to Clinical and laboratory standards institute (CLSI). Multiplex PCR was used to detect 3 different classes of integron in multi-resistant strains, and PCR-sequencing method was used to analysis gene cassettes. The study was classified into two parts as follows.
1. Surveillance of different antimicrobial resistance among E.coli isolates from food animals.
The samples of colibacillosis were collected from food animals, including chickens, ducks, cattles and pigs, in different farms in Sichuan province,705 isolates were identified in the study. Antimicrobial susceptibility of these isolates to 25 different agents indicated: The resistance of 705 isolates to 25 agents were different, more than 100% of the 705 E.coli isolates were resistant to TMP, Trimethoprim-sulfamethoxazole and Rifampin respectively; Then, above 70% of isolates were resistance to Ampicillin,Tetracyline, Chloramphenicol and Fluoroquinolone,respectively. Isolates exhibited relatively low resistant level toβ-lactams, resistane rate of Cephazolin,Ceftiofur and Ceftriaxone, was 34.34%,41.30% and 26.25%, respectively. However, the medium rates of 3 kinds ofβ-lactams were very high, which indicated that the resistant changing trends was ascendant; All of isolates showed different susceptible level to Amikacin, susceptible rates exceeded 67%.
Multidrug-resistance was very serious among isolates, All of 705 isolates exhibited multiple resistance to 15-24 antimicrobial agents. The rate of resistant strains to 23 agents was at most, then to 21 agents. Resistant rate of Avian strains was the most serious among different animal isolates; Although the resistant levels of the partial agents were different in isolates from different locals, however, resistant patterns among E.coli isolates from different geographical origins was similar.
2. Detection of integron-gene cassettes among Escherichia coli isolates from food-animals
Multiplex PCR was used to detect 3 different classes of integron in 327 isolates. The results of multiplex PCR indicated 294 of 327 isolates harbored intI1 integrase genes, the detection rate of intI1 was 89.91%, intI2 and intI3 genes were not discoveried in the study. Amplification of Gene cassettes in 81 intI1-positive strains choosed randomly was carried out. The results showed that amplicons of 845bp-2731bp were yielded in 67 intI1-positive strains. The sequencing of gene cassettes indicated that 6 kinds of gene cassettes were harbored in 67 strains. The gene cassettes arrays were dfrA17+aadA5,dfrA17, aadA22, dhfrAI+aadA2, dfrA17+orfF+aadA2 and aacA4+catB3+dfrA1, which were encoding resistant to TMP+Sulfonamides, Aminoglycosides and chloramphenicol, respectively. aadA22, which was novel gene encoding resistant to Aminoglycosides, was first acquired in avian E.coli strains from Sichuan province. The study of aadA22 gene will be carried out in future.
本研究首先从四川省不同养殖场收集食品动物源大肠杆菌病样本,按常规的细菌分离鉴定方法进行大肠杆菌菌株的鉴定。按照CLSI推荐方法及结果判断标准,应用微量肉汤稀释法测定细菌对于不同抗菌药物的最小抑菌浓度(MIC),从而对四川省食品动物源大肠杆菌的耐药情况进行监测。对于多重耐药菌株,采用多重PCR技术对其携带的3类整合子进行检测,同时对整合子阳性菌株进行基因盒分析。本次研究分2部分完成。
1.不同食品动物源性大肠杆菌耐药性的监测
从四川不同地区规模较大的养殖场收集食品动物源(鸡源、鸭源、牛源、猪源)大肠杆菌病样本,分离鉴定出705株大肠杆菌。采用微量肉汤稀释法进行了25种抗菌药物的药敏实验。结果显示：705株大肠杆菌对25种抗菌药物表现出程度不同的耐药性,特别是对兽医临床长期使用的甲氧苄啶、复方新诺明、利福平的耐药率接近100%；其次是对氨苄西林、四环素、氯霉素、氟喹诺酮类药物的耐药率均超过了70%；对头孢唑啉、头孢噻呋、头孢曲松也表现出一定的耐药性,耐药率分别为34.34%、41.30%、26.25%,但中介率较高,可见大肠杆菌对β-内酰胺类药物存在耐药率上升的趋势；本次监测发现所有大肠杆菌只有对阿米卡星保持有一定的敏感性,敏感率超过67%。
多重耐药性分析显示,705株菌均为多重耐药菌株,主要表现出15-24耐,在各种耐药谱型中,23耐菌株所占比率最大,其次为21耐。不同动物源菌株耐药性比较显示,禽源菌株的耐药情况较猪源菌株,牛源菌株严重。不同地区来源菌株耐药性比较可见,不同地区来源菌株对所检测的25种药物耐药性分布大体一致,对部分药物表现出一定地域差异。
2.食品动物源性大肠杆菌的整合子—基因盒检测
采用多重PCR方法同时扩增327株大肠杆菌整合酶Ⅰ型、Ⅱ型和Ⅲ型基因。多重PCR检测结果显示,在32Ⅳ株菌株中,294株细菌携带有Ⅰ型整合酶基因,其检出率为89.91%,本次检测未发现携带有Ⅱ型和Ⅲ型整合酶基因的菌株。随机抽取Ⅰ型整合酶阳性菌株81株,进行基因盒扩增,扩增结果显示14株Ⅰ型整合酶阳性菌株未扩增出基因盒片段,其余67株扩增出845bp-2731bp的基因盒片段。测序结果显示,67株菌中含有6种不同类型的基因盒,即dfrA17+aadA5、dfrA17、aadA22、dhfrAI+ aadA2、dfrA17+orfF+aadA2、aacA4+catB3+dfrA1,编码甲氧苄啶-磺胺类药物、氨基糖苷类药物和氯霉素耐药。本次研究发现在2株鸡源大肠杆菌株中,发现了通过整合子-基因盒携带的一种介导氨基糖苷类药物耐药的新基因aadA22,这是首次在四川分离菌株中发现该基因的存在,其介导的耐药性有待进一步的研究。
With the worldwide use of antimicrobial agents in veterinary medicine, the incidence of resistance and multidrug-resistance among pathogenic bacteria has been increasing, which attracts global attentions. Surveillance of antimicrobial resistance is very urgent to clinical medicine. The traditional method used in surveillance of resistance is to test resistant phenotype. With the development of molecular biological technology, PCR technology have been extensivly used in detection of resistant genotype and novel resistant mechanism.
In this study, The samples of colibacillosis were collected from food animals in different farms in Sichuan province, E.coli isolates were identified by conventional test. Antimicrobial susceptibility of these isolates was examined using the broth microdilution method and the results interpreted according to Clinical and laboratory standards institute (CLSI). Multiplex PCR was used to detect 3 different classes of integron in multi-resistant strains, and PCR-sequencing method was used to analysis gene cassettes. The study was classified into two parts as follows.
1. Surveillance of different antimicrobial resistance among E.coli isolates from food animals.
The samples of colibacillosis were collected from food animals, including chickens, ducks, cattles and pigs, in different farms in Sichuan province,705 isolates were identified in the study. Antimicrobial susceptibility of these isolates to 25 different agents indicated: The resistance of 705 isolates to 25 agents were different, more than 100% of the 705 E.coli isolates were resistant to TMP, Trimethoprim-sulfamethoxazole and Rifampin respectively; Then, above 70% of isolates were resistance to Ampicillin,Tetracyline, Chloramphenicol and Fluoroquinolone,respectively. Isolates exhibited relatively low resistant level toβ-lactams, resistane rate of Cephazolin,Ceftiofur and Ceftriaxone, was 34.34%,41.30% and 26.25%, respectively. However, the medium rates of 3 kinds ofβ-lactams were very high, which indicated that the resistant changing trends was ascendant; All of isolates showed different susceptible level to Amikacin, susceptible rates exceeded 67%.
Multidrug-resistance was very serious among isolates, All of 705 isolates exhibited multiple resistance to 15-24 antimicrobial agents. The rate of resistant strains to 23 agents was at most, then to 21 agents. Resistant rate of Avian strains was the most serious among different animal isolates; Although the resistant levels of the partial agents were different in isolates from different locals, however, resistant patterns among E.coli isolates from different geographical origins was similar.
2. Detection of integron-gene cassettes among Escherichia coli isolates from food-animals
Multiplex PCR was used to detect 3 different classes of integron in 327 isolates. The results of multiplex PCR indicated 294 of 327 isolates harbored intI1 integrase genes, the detection rate of intI1 was 89.91%, intI2 and intI3 genes were not discoveried in the study. Amplification of Gene cassettes in 81 intI1-positive strains choosed randomly was carried out. The results showed that amplicons of 845bp-2731bp were yielded in 67 intI1-positive strains. The sequencing of gene cassettes indicated that 6 kinds of gene cassettes were harbored in 67 strains. The gene cassettes arrays were dfrA17+aadA5,dfrA17, aadA22, dhfrAI+aadA2, dfrA17+orfF+aadA2 and aacA4+catB3+dfrA1, which were encoding resistant to TMP+Sulfonamides, Aminoglycosides and chloramphenicol, respectively. aadA22, which was novel gene encoding resistant to Aminoglycosides, was first acquired in avian E.coli strains from Sichuan province. The study of aadA22 gene will be carried out in future.
引文
[1]陆承平.兽医微生物学[M].北京,中国农业出版社，2001.215-223
[2]林维庆.鸡大肠杆菌病[J].中国兽医杂志,1991,17(6)：49-51.
[3]李玲,汪铭书,程安春等.雏鸭致病性大肠杆菌的分离鉴定药敏试验及耐药性分析[J].中国兽医杂志，2007,43(12)：34-36.
[4]周新民.鸭大肠杆菌病[J].饲料博览,2005,3：57-57.
[5]李永明.猪大肠杆菌病病原学研究概况[J].贵州农业科学,1990,4：12-13.
[6]邱春媚,何家康,毕玉海.猪源大肠杆菌的耐药性调查与对策[J].猪业科学,2008,8：38-41.
[7]赵永唐.犊牛大肠杆菌病的防治报告[J].中国畜牧兽医,2008,35(4)：119-120.
[8]王世荣,任俊源.犊牛大肠杆菌病的病原及防治[J].山东农业科学,1989,1：44-45.
[9]沈建忠.动物性食品的安全性及其监控[J].中国兽医杂志,2001,37(1)：38-39
[10]陈代杰.抗菌药物与细菌耐药性[M].上海,华东理工大学出版社,2001.120-121
[11]Orman B, Pineiro S, Arduino S et.al. Evolution of Multiresistance in Nontyphoid Salmonella Serovars from 1984 to 1998 in Argentina[J].Antimicrobial agents and chemotherapy,2002,46(12): 3963-3970.
[12]刘文字,廖晓萍.兽用抗菌药物耐药性产生机制及其防治对策探讨[J].养禽与禽病防治,2007,8：6-8.
[13]朱力军.动物大肠杆菌耐药性的变化趋势[J].中国兽药杂志,2001,35(2)：16-18.
[14]雷连成,江文正,韩文瑜等.致病性大肠杆菌的耐药性监测[J].中国兽医杂志,2001,37(1)：12-13.
[15]雷连成,韩文瑜,郑丹.大肠杆菌流行株耐药特性对其免疫效果的影响[J].吉林农业大学学报,2005,27(5)：554-558.
[16]徐小艳,王国相,印继华等.1975-2002年大肠杆菌耐药性变化情况调查[J].畜牧与兽医,2003,35(3)：16-17.
[17]宋立,宁宜宝,沈建忠等.中国不同年代食品动物大肠杆菌耐药性调查研究[J].中国科学,2009,39(7)：692-698.
[18]陈杖榴.兽医药理学[M].北京,中国农业出版社,2002,195-240.
[19]陈民钧.细菌对β-内酰胺药的耐药性及检测方法[J].中华检验医学杂志,2001,24(4)：197-200.
[20]马传新,唐海英.β-内酰胺抗生素的耐药机制及对策[J].中国医院药学杂志,2000,20(12)：743-745.
[21]徐修礼,刘家云.细菌对抗生素耐药机制研究的某些进展[J].国外医学：临床生物化学与检验学分册,2002,23(2)：83-84.
[22]王波云,李立安,顾亚夫.细菌耐药性研究进展与对策[J].解放军药学学报,2001,17(5)：259-261.
[23]Schwarz S, Chaslus-Dancla E. Use of Antimicrobials in Veterinary Medicine and Mechanisms of Resistance[J].Veterinary research,2001,32:201-225.
[24]Rosamond J, Allsop A. Harnessing the Power of the Genome in the Search for New Antibiotics[J].Science,2000,287(5460):1973-1976.
[25]顾兵,童明庆.整合子与细菌耐药[J].临床检验杂志,2005,23(3)：226-229.
[26]Bonomo R, Szabo D. Mechanisms of Multidrug Resistance in Acinetobacter Species and Pseudomonas Aeruginosa[J].Clinical infectious diseases,2006.43:S49-S56.
[27]刘芳萍,刘立新,曲鹏等.细菌耐药机制研究进展[J].畜牧兽医科技信息；2006,5：12-13.
[28]Rowe-Magnus D, Mazel D. The Role of Integrons in Antibiotic Resistance Gene Capture[J].International Journal of Medical Microbiology,2002,292(2):115-125.
[29]Hall R, Collis C. Mobile Gene Cassettes and Integrons:Capture and Spread of Genes by Site-Specific Recombination[J].Molecular Microbiology,1995,15(4):593-600.
[30]Stokes H W, Hall R M. A Novel Family of Potentially Mobile DNA Elements Encoding Site-Specific Gene-Integration Functions:lntegrons[J]. Molecular Microbiology,1989,3(12): 1669-1683.
[31]Hall R, Brookes D, Stokes H. Site-Specific Insertion of Genes into Integrons:Role of the 59-Base Element and Determination of the Recombination Cross-over Point[J].Molecular Microbiology, 1991,5(8):1941-1959.
[32]李超,周铁丽,刘庆中等.细菌整合子及其介导的耐药研究进展[J].中华检验医学杂志,2007,30(2)：227-230.
[33]陆思静,刘又宁,管希周等.细菌整合子系统研究新进展[J].国外医学：呼吸系统分册,2005,25(3)：228-231.
[34]Boonkerd N, Pibalpakdi P, Tiloklurs M et.al. Class 1 Integron Containing Metallo β-Lactamase Gene BlaIMP-1 in Carbapenem-Resistant Pseudomonas Aeruginosa in Thailand[J].Journal of Infection and Chemotherapy,2009,15(4):257-261.
[35]赵春燕,李凡.整合子与细菌耐药性[J].国外医学：微生物学分册,2003,26(3)：15-17.
[36]Collis C, Kim M, Partridge S et.al. Characterization of the Class 3 Integron and the Site-Specific Recombination System It Determines[J].Journal of bacteriology,2002,184(11):3017-3026.
[37]Mazel D, Dychinco B, Webb V et.al. A Distinctive Class of Integron in the Vibrio Cholerae Genome[J].Science,1998,280:605-608.
[38]张婷,石磊.细菌演化的天然工具——超级整合子[J].中国抗生素杂志,2006,31(12)：709-713.
[39]卓超.细菌基因盒-整合子系统研究进展[J].国外医药：抗生素分册,2002,23(2)：57-61.
[40]杜艳,陈端.整合子中耐药基因盒的研究进展[J].医学综述,2005,11(2)：116-118.
[41]王会娟.细菌耐药机制-整合子系统研究进展[J].山西医药杂志,2007,36(2)：143-144.
[42]韩立中,周敏,王勇等.整合子在细菌耐药性中的作用[J].检验医学,2004,19(1)：80-83.
[43]万珊,莫非，蒋朝晖.临床铜绿假单胞菌β-内酰胺类耐药相关基因实验观察[J].中国保健,2006,14：24-25.
[44]段玉平,徐兴然,曾忠良等.猪源大肠杆菌整合子与耐药性的相关性研究[J].中国兽药杂志,2009,43(5)：24-26.
[45]张安云,王红宁,周万蓉等.野生动物分离菌耐药基因多重PCR检测及序列分析[J].畜牧与兽医,2007,39(3)：1-4.
[46]张险朋,兰婷妹,赖笑娴等.实时荧光PCR技术在动物疫病诊断中的应用[J].动物医学进展,2006,27(3)：102-105.
[47]金大智,谢明杰,曹际娟.食品中单增李氏菌实时荧光PCR检测鉴定方法的建立[J].辽宁师范大学学报：自然科学版,2003,26(1)：73-76.
[48]樊慧珍,于化鹏,黄文杰等.应用DNA探钊检测甲氧西林耐药葡萄球菌[J].临床检验杂志,2006,24(5)：351-352.
[49]林居纯,曾振灵,蒋红霞等.耐氟喹诺酮类病原菌Gyra基因突变的寡核苷酸芯片检测[J].中国兽医科学,2007,37(9)：811-814.
[50]毛红菊,林东防,徐晓刚等.利用基因芯片快速检测多种临床常见致病菌及其耐药性基因[J].中华传染病杂志,2006,24(6)：372-377.
[51]John P H, Renate R, Takashi A et al. Methods for Broth Hilution Susceptibility Testing of Bacteria Isolated from Aquatic Animals; Approved Guideline [S].Pennsylvania:Clinical and Laboratory Standards Institute,2005.
[52]倪语星,洪秀华.细菌耐药性监测与抗感染治疗[M].北京,人民军医出版社,2002,314-315.
[53]孙海虹,马同锁,于珊等.鸡大肠杆菌的菌株鉴定及药物敏感试验[J].河北师范大学学报：自然科学版,2005,29(6)：623-625.
[54]金凌艳,顾欣,蔡金华等.2008年上海市动物源大肠杆菌耐药性监测[J].上海畜牧兽医通讯,2009，3：31-32.
[55]李灶平,刘衡川,刘渠.食品中大肠埃希菌耐药性与整合子的关系研究[J].四川大学学报：医学版,2005,36(3)：400-403.
[56]宋立,宁宜宝,张秀英等.中国不同地区家禽大肠杆菌血清型分布和耐药性比较研究[J].中国农业科学,2005,38(7)：1466-1473.
[57]林红燕，沈怀亮,魏衍超.临床检出肠球菌耐药谱4年的变迁[J].中华医院感染学杂志,2005,15(1)：115-117.
[58]林居纯.氟喹诺酮类药物耐药性检测基因芯片的研究.华南农业大学博士学位论文.广州,华南农业大学,2005.
[59]王红宁,刘书亮.规模化猪场致病性大肠杆菌,沙门氏菌药敏区系调查[J].西南农业学报,2000,13(C00)：84-90.
[60]赵红霞,李培锋,吴聪明等.奶牛子宫内膜炎大肠杆菌耐药性及其与整合子关系的研究[J].畜牧兽医学报,2009,40(1)：142-144.
[61]陈希文,王雄清,代敏等.绵阳地区猪源致病性大肠杆菌的耐药性监测[J].江苏农业科学,2007,1：131-134.
[62]刘书亮,王红宁.84株仔猪黄,白痢E. Coli的体外药敏试验[J].畜禽业,2000,9：16-17.
[63]刘梦元,吴斌,刘建杰等.规模化猪场大肠杆菌的耐药性监测及血清流行病学调查[J].中国兽医学报,2004,24(1)：16-18.
[64]姜涛,孙智勇,邓旭明.动物源性大肠杆菌的耐药性分析[J].吉林畜牧兽医,2008,29(5)：10-11.
[65]苏健裕,石磊,杨连生.临床大肠埃希菌第1类整合子检测及耐药基因盒分析[J].检验医学与临床,2008,5(7)：385-387
[66]Leverstein-van Hall M, Blok H, Donders A et.al. Multidrug Resistance among Enterobacteriaceae Is Strongly Associated with the Presence of Integrons and Is Independent of Species or Isolate Origin[J].The Journal of infectious diseases,2003,187:251-259.
[67]Goldstein C, Lee M D, Sanchez S et.al. Incidence of Class 1 and 2 Integrases in Clinical and Commensal Bacteria from Livestock. Companion Animals, and Exotics[J].Antimicrob Agents Chemother,2001,45(3):723-726.
[68]Sambrook J, Fritsch EF, Maniatis T et.al.分子克隆实验指南(第三版)[M].北京,科学出版社,2002，1564-1604.
[69]石磊，陈洵,肖增璜.多重耐药临床菌株中整合子结构的检测与分析[J].中国抗生素杂志,2007,32(1)：43-48.
[70]White P, Mclver C, Deng Y et.al. Characterisation of Two New Gene Cassettes, aadA5 and dfrA17[J].FEMS microbiology letters,2006,182(2):265-269.
[71]吴聪明.广东大肠杆菌耐药性及整合子/基因盒分子流行病学研究.华南农业大学博士学位论文.广州,华南农业大学,2003.
[72]Chamberlain J, Gibbs R, Rainer J et.al. Deletion Screening of the Duchcnne Muscular Dystrophy Locus Via Multiplex DNA Amplification[J].Nucleic Acids Research,1988,16(23):11141-11156.
[73]赵红庆，苑锡铜,黄留玉.多重PCR技术在病原检测中的应用[J].生物技术通讯,2007,18(5)：863-865.
[74]丁良君,张秀英,付锐等.大肠埃希菌Ⅰ类整合子与多重耐药的相关性分析[J].东北农业大学学 报,2007,6：771-774.
[75]张发苏,李涛,徐无宏等.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,2006,8(3)：164-]66.
[76]Van Essen-Zandbergen A, Smith H, Veldman K et.al. Occurrence and Characteristics of Class 1,2 and 3 Integrons in Escherichia Coli, Salmonella and Campylobacter Spp. In the Netherlands[J].Journal of Antimicrobial Chemotherapy,2007,59(4):746-750.
[77]王桂琴,吴聪明,曹兴元等.奶牛乳房炎大肠杆菌整合子与耐药表型的相关性研究[J].畜牧兽医学报,2007,38(12)：1345-1350.
[78]Wang G, Wu C, Du X et.al. Characterization of lntegrons-Mediated Antimicrobial Resistance among Escherichia Coli Strains Isolated from Bovine Mastitis[J].Veterinary microbiology,2008, 127(1-2):73-78.
[79]刘衡川,兰全学.多重耐药大肠埃希菌中Ⅰ类整合子的研究[J].中国感染与化疗杂志,2006,6(3)：189-192.
[80]Lee M, Chen Y, Peng C. Molecular Characterisation of Class 1 Integrons in Salmonella Enterica Serovar Choleraesuis Isolates from Southern Taiwan[J].International journal of antimicrobial agents,2009,33(3):216-222.
[81]李景云,崔生辉,张力等.鸡来源产超广谱β-内酰胺酶大肠埃希菌携带整合子基因的研究[J].中华流行病学杂志,2008,29(10)：1020-1023.
[82]Hammad A, Ishida Y, Shimamoto T. Prevalence and Molecular Characterization of Ampicillin-Resistant Enterobacteriaceae Isolated from Traditional Egyptian Domiati Cheese[J].Journal of Food Protection,2009,72(3):624-630.
[2]林维庆.鸡大肠杆菌病[J].中国兽医杂志,1991,17(6)：49-51.
[3]李玲,汪铭书,程安春等.雏鸭致病性大肠杆菌的分离鉴定药敏试验及耐药性分析[J].中国兽医杂志，2007,43(12)：34-36.
[4]周新民.鸭大肠杆菌病[J].饲料博览,2005,3：57-57.
[5]李永明.猪大肠杆菌病病原学研究概况[J].贵州农业科学,1990,4：12-13.
[6]邱春媚,何家康,毕玉海.猪源大肠杆菌的耐药性调查与对策[J].猪业科学,2008,8：38-41.
[7]赵永唐.犊牛大肠杆菌病的防治报告[J].中国畜牧兽医,2008,35(4)：119-120.
[8]王世荣,任俊源.犊牛大肠杆菌病的病原及防治[J].山东农业科学,1989,1：44-45.
[9]沈建忠.动物性食品的安全性及其监控[J].中国兽医杂志,2001,37(1)：38-39
[10]陈代杰.抗菌药物与细菌耐药性[M].上海,华东理工大学出版社,2001.120-121
[11]Orman B, Pineiro S, Arduino S et.al. Evolution of Multiresistance in Nontyphoid Salmonella Serovars from 1984 to 1998 in Argentina[J].Antimicrobial agents and chemotherapy,2002,46(12): 3963-3970.
[12]刘文字,廖晓萍.兽用抗菌药物耐药性产生机制及其防治对策探讨[J].养禽与禽病防治,2007,8：6-8.
[13]朱力军.动物大肠杆菌耐药性的变化趋势[J].中国兽药杂志,2001,35(2)：16-18.
[14]雷连成,江文正,韩文瑜等.致病性大肠杆菌的耐药性监测[J].中国兽医杂志,2001,37(1)：12-13.
[15]雷连成,韩文瑜,郑丹.大肠杆菌流行株耐药特性对其免疫效果的影响[J].吉林农业大学学报,2005,27(5)：554-558.
[16]徐小艳,王国相,印继华等.1975-2002年大肠杆菌耐药性变化情况调查[J].畜牧与兽医,2003,35(3)：16-17.
[17]宋立,宁宜宝,沈建忠等.中国不同年代食品动物大肠杆菌耐药性调查研究[J].中国科学,2009,39(7)：692-698.
[18]陈杖榴.兽医药理学[M].北京,中国农业出版社,2002,195-240.
[19]陈民钧.细菌对β-内酰胺药的耐药性及检测方法[J].中华检验医学杂志,2001,24(4)：197-200.
[20]马传新,唐海英.β-内酰胺抗生素的耐药机制及对策[J].中国医院药学杂志,2000,20(12)：743-745.
[21]徐修礼,刘家云.细菌对抗生素耐药机制研究的某些进展[J].国外医学：临床生物化学与检验学分册,2002,23(2)：83-84.
[22]王波云,李立安,顾亚夫.细菌耐药性研究进展与对策[J].解放军药学学报,2001,17(5)：259-261.
[23]Schwarz S, Chaslus-Dancla E. Use of Antimicrobials in Veterinary Medicine and Mechanisms of Resistance[J].Veterinary research,2001,32:201-225.
[24]Rosamond J, Allsop A. Harnessing the Power of the Genome in the Search for New Antibiotics[J].Science,2000,287(5460):1973-1976.
[25]顾兵,童明庆.整合子与细菌耐药[J].临床检验杂志,2005,23(3)：226-229.
[26]Bonomo R, Szabo D. Mechanisms of Multidrug Resistance in Acinetobacter Species and Pseudomonas Aeruginosa[J].Clinical infectious diseases,2006.43:S49-S56.
[27]刘芳萍,刘立新,曲鹏等.细菌耐药机制研究进展[J].畜牧兽医科技信息；2006,5：12-13.
[28]Rowe-Magnus D, Mazel D. The Role of Integrons in Antibiotic Resistance Gene Capture[J].International Journal of Medical Microbiology,2002,292(2):115-125.
[29]Hall R, Collis C. Mobile Gene Cassettes and Integrons:Capture and Spread of Genes by Site-Specific Recombination[J].Molecular Microbiology,1995,15(4):593-600.
[30]Stokes H W, Hall R M. A Novel Family of Potentially Mobile DNA Elements Encoding Site-Specific Gene-Integration Functions:lntegrons[J]. Molecular Microbiology,1989,3(12): 1669-1683.
[31]Hall R, Brookes D, Stokes H. Site-Specific Insertion of Genes into Integrons:Role of the 59-Base Element and Determination of the Recombination Cross-over Point[J].Molecular Microbiology, 1991,5(8):1941-1959.
[32]李超,周铁丽,刘庆中等.细菌整合子及其介导的耐药研究进展[J].中华检验医学杂志,2007,30(2)：227-230.
[33]陆思静,刘又宁,管希周等.细菌整合子系统研究新进展[J].国外医学：呼吸系统分册,2005,25(3)：228-231.
[34]Boonkerd N, Pibalpakdi P, Tiloklurs M et.al. Class 1 Integron Containing Metallo β-Lactamase Gene BlaIMP-1 in Carbapenem-Resistant Pseudomonas Aeruginosa in Thailand[J].Journal of Infection and Chemotherapy,2009,15(4):257-261.
[35]赵春燕,李凡.整合子与细菌耐药性[J].国外医学：微生物学分册,2003,26(3)：15-17.
[36]Collis C, Kim M, Partridge S et.al. Characterization of the Class 3 Integron and the Site-Specific Recombination System It Determines[J].Journal of bacteriology,2002,184(11):3017-3026.
[37]Mazel D, Dychinco B, Webb V et.al. A Distinctive Class of Integron in the Vibrio Cholerae Genome[J].Science,1998,280:605-608.
[38]张婷,石磊.细菌演化的天然工具——超级整合子[J].中国抗生素杂志,2006,31(12)：709-713.
[39]卓超.细菌基因盒-整合子系统研究进展[J].国外医药：抗生素分册,2002,23(2)：57-61.
[40]杜艳,陈端.整合子中耐药基因盒的研究进展[J].医学综述,2005,11(2)：116-118.
[41]王会娟.细菌耐药机制-整合子系统研究进展[J].山西医药杂志,2007,36(2)：143-144.
[42]韩立中,周敏,王勇等.整合子在细菌耐药性中的作用[J].检验医学,2004,19(1)：80-83.
[43]万珊,莫非，蒋朝晖.临床铜绿假单胞菌β-内酰胺类耐药相关基因实验观察[J].中国保健,2006,14：24-25.
[44]段玉平,徐兴然,曾忠良等.猪源大肠杆菌整合子与耐药性的相关性研究[J].中国兽药杂志,2009,43(5)：24-26.
[45]张安云,王红宁,周万蓉等.野生动物分离菌耐药基因多重PCR检测及序列分析[J].畜牧与兽医,2007,39(3)：1-4.
[46]张险朋,兰婷妹,赖笑娴等.实时荧光PCR技术在动物疫病诊断中的应用[J].动物医学进展,2006,27(3)：102-105.
[47]金大智,谢明杰,曹际娟.食品中单增李氏菌实时荧光PCR检测鉴定方法的建立[J].辽宁师范大学学报：自然科学版,2003,26(1)：73-76.
[48]樊慧珍,于化鹏,黄文杰等.应用DNA探钊检测甲氧西林耐药葡萄球菌[J].临床检验杂志,2006,24(5)：351-352.
[49]林居纯,曾振灵,蒋红霞等.耐氟喹诺酮类病原菌Gyra基因突变的寡核苷酸芯片检测[J].中国兽医科学,2007,37(9)：811-814.
[50]毛红菊,林东防,徐晓刚等.利用基因芯片快速检测多种临床常见致病菌及其耐药性基因[J].中华传染病杂志,2006,24(6)：372-377.
[51]John P H, Renate R, Takashi A et al. Methods for Broth Hilution Susceptibility Testing of Bacteria Isolated from Aquatic Animals; Approved Guideline [S].Pennsylvania:Clinical and Laboratory Standards Institute,2005.
[52]倪语星,洪秀华.细菌耐药性监测与抗感染治疗[M].北京,人民军医出版社,2002,314-315.
[53]孙海虹,马同锁,于珊等.鸡大肠杆菌的菌株鉴定及药物敏感试验[J].河北师范大学学报：自然科学版,2005,29(6)：623-625.
[54]金凌艳,顾欣,蔡金华等.2008年上海市动物源大肠杆菌耐药性监测[J].上海畜牧兽医通讯,2009，3：31-32.
[55]李灶平,刘衡川,刘渠.食品中大肠埃希菌耐药性与整合子的关系研究[J].四川大学学报：医学版,2005,36(3)：400-403.
[56]宋立,宁宜宝,张秀英等.中国不同地区家禽大肠杆菌血清型分布和耐药性比较研究[J].中国农业科学,2005,38(7)：1466-1473.
[57]林红燕，沈怀亮,魏衍超.临床检出肠球菌耐药谱4年的变迁[J].中华医院感染学杂志,2005,15(1)：115-117.
[58]林居纯.氟喹诺酮类药物耐药性检测基因芯片的研究.华南农业大学博士学位论文.广州,华南农业大学,2005.
[59]王红宁,刘书亮.规模化猪场致病性大肠杆菌,沙门氏菌药敏区系调查[J].西南农业学报,2000,13(C00)：84-90.
[60]赵红霞,李培锋,吴聪明等.奶牛子宫内膜炎大肠杆菌耐药性及其与整合子关系的研究[J].畜牧兽医学报,2009,40(1)：142-144.
[61]陈希文,王雄清,代敏等.绵阳地区猪源致病性大肠杆菌的耐药性监测[J].江苏农业科学,2007,1：131-134.
[62]刘书亮,王红宁.84株仔猪黄,白痢E. Coli的体外药敏试验[J].畜禽业,2000,9：16-17.
[63]刘梦元,吴斌,刘建杰等.规模化猪场大肠杆菌的耐药性监测及血清流行病学调查[J].中国兽医学报,2004,24(1)：16-18.
[64]姜涛,孙智勇,邓旭明.动物源性大肠杆菌的耐药性分析[J].吉林畜牧兽医,2008,29(5)：10-11.
[65]苏健裕,石磊,杨连生.临床大肠埃希菌第1类整合子检测及耐药基因盒分析[J].检验医学与临床,2008,5(7)：385-387
[66]Leverstein-van Hall M, Blok H, Donders A et.al. Multidrug Resistance among Enterobacteriaceae Is Strongly Associated with the Presence of Integrons and Is Independent of Species or Isolate Origin[J].The Journal of infectious diseases,2003,187:251-259.
[67]Goldstein C, Lee M D, Sanchez S et.al. Incidence of Class 1 and 2 Integrases in Clinical and Commensal Bacteria from Livestock. Companion Animals, and Exotics[J].Antimicrob Agents Chemother,2001,45(3):723-726.
[68]Sambrook J, Fritsch EF, Maniatis T et.al.分子克隆实验指南(第三版)[M].北京,科学出版社,2002，1564-1604.
[69]石磊，陈洵,肖增璜.多重耐药临床菌株中整合子结构的检测与分析[J].中国抗生素杂志,2007,32(1)：43-48.
[70]White P, Mclver C, Deng Y et.al. Characterisation of Two New Gene Cassettes, aadA5 and dfrA17[J].FEMS microbiology letters,2006,182(2):265-269.
[71]吴聪明.广东大肠杆菌耐药性及整合子/基因盒分子流行病学研究.华南农业大学博士学位论文.广州,华南农业大学,2003.
[72]Chamberlain J, Gibbs R, Rainer J et.al. Deletion Screening of the Duchcnne Muscular Dystrophy Locus Via Multiplex DNA Amplification[J].Nucleic Acids Research,1988,16(23):11141-11156.
[73]赵红庆，苑锡铜,黄留玉.多重PCR技术在病原检测中的应用[J].生物技术通讯,2007,18(5)：863-865.
[74]丁良君,张秀英,付锐等.大肠埃希菌Ⅰ类整合子与多重耐药的相关性分析[J].东北农业大学学 报,2007,6：771-774.
[75]张发苏,李涛,徐无宏等.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,2006,8(3)：164-]66.
[76]Van Essen-Zandbergen A, Smith H, Veldman K et.al. Occurrence and Characteristics of Class 1,2 and 3 Integrons in Escherichia Coli, Salmonella and Campylobacter Spp. In the Netherlands[J].Journal of Antimicrobial Chemotherapy,2007,59(4):746-750.
[77]王桂琴,吴聪明,曹兴元等.奶牛乳房炎大肠杆菌整合子与耐药表型的相关性研究[J].畜牧兽医学报,2007,38(12)：1345-1350.
[78]Wang G, Wu C, Du X et.al. Characterization of lntegrons-Mediated Antimicrobial Resistance among Escherichia Coli Strains Isolated from Bovine Mastitis[J].Veterinary microbiology,2008, 127(1-2):73-78.
[79]刘衡川,兰全学.多重耐药大肠埃希菌中Ⅰ类整合子的研究[J].中国感染与化疗杂志,2006,6(3)：189-192.
[80]Lee M, Chen Y, Peng C. Molecular Characterisation of Class 1 Integrons in Salmonella Enterica Serovar Choleraesuis Isolates from Southern Taiwan[J].International journal of antimicrobial agents,2009,33(3):216-222.
[81]李景云,崔生辉,张力等.鸡来源产超广谱β-内酰胺酶大肠埃希菌携带整合子基因的研究[J].中华流行病学杂志,2008,29(10)：1020-1023.
[82]Hammad A, Ishida Y, Shimamoto T. Prevalence and Molecular Characterization of Ampicillin-Resistant Enterobacteriaceae Isolated from Traditional Egyptian Domiati Cheese[J].Journal of Food Protection,2009,72(3):624-630.