猪–鱼复合养殖模式中气单胞菌I类整合子的流行情况及其耐药特征

Prevalence and characterization of class Ⅰ integron in Aeromonas from pig-fish integrated farm

为了解广东地区猪-鱼复合养殖模式下气单胞菌整合子流行情况及其耐药特征,从广东省5个不同猪-鱼复合养殖场采集分离猪粪、鱼、池塘水及池塘底泥的气单胞菌共317株,通过微量二倍稀释法测定其对20种药物的敏感性;PCR扩增Ⅰ类整合子整合酶基因intI1,并分析其基因盒阵列结构。结果表明,317株气单胞菌对20种药物耐药程度不一,氨苄西林、磺胺间甲氧嘧啶、萘啶酸的耐药率相对较高。intI1的检出率为15.77%,鱼源和猪源的整合子检出率高于环境源。整合子阳性菌对测试的12种药物的耐药率显著高于阴性菌,且表现为多重耐药;50个Ⅰ类整合子共检测到16种耐药基因盒,包括了编码氨基糖苷类耐药基因aadA1、aadA2、aac6-Ⅱ、aacA4,甲氧苄啶耐药基因drfA1、dfrA12、dfrA15、dfrA17、dfrB4,β内酰胺类耐药基因bla_(OXA-10)、bla_(OXA-21),氯霉素耐药基因catB3、catB8,利福平耐药基因arr2、arr3以及质粒介导的喹诺酮类耐药基因aac(6')-Ib-cr。携带了不同类耐药基因盒的整合子阳性菌还表现出所对应的对甲氧苄啶、链霉素、氯霉素类等的耐药表型,由此推测整合子的存在与细菌的多重耐药密切相关。研究表明,I类整合子分布于广东地区猪-鱼复合养殖模式下不同来源气单胞菌,并介导细菌对多种抗菌药物耐药。有必要开展畜禽-鱼复合养殖模式下细菌耐药性的传播机制研究,为水产养殖合理用药提供依据。

Aeromonas is regarded as an important pathogen of freshwater animals. However, little is known about genetics of antirnicrobial resistance in Aeromonas sp. in Chinese aquaculture. The aim of this study was to investigate the presence of class Ⅰ integron and characterize multidrug-resistant Aermonas sp. isolated from integrated farms. Three hundred and seventeen Aeromonas strains were isolated from pig feces, fish, pond water and sediments. These samples were collected from 5 pig-fish integrated farms in Foshan and Zhaoqing of Guangdong province. All strains were evaluated for resistance to 20 antimicrobials of 7 classes by two fold dilution method. Genomic DNA was extracted with the Omega DNA mini kit. All the isolates were screened for the presence of intI1. All the intll-positive strains were also amplified sull, qacE1 fragment and gene cassette region by PCR. Gene cassettes arrays were analyzed by BLAST. Percentages of isolates resistant to 20 antimicrobials were variable. Resistance was most prevalent for ampicillin, sulfamonomethoxine and nalidixic acid. Among 317 Aeromonas isolates, 50 （15.77%） isolates were positive for intll. The incidence of class lintegron was more prevalent in Aeromonas from fish and pig than that from environmental sources. Comparing the resistance phenotypes represented in integron-positive and integron-negative strains, the results showed that the resistance rates of trimethoprim/sulfamethoxazole, doxycycline, enrofloxacin, neomycin and florfenicol among integron-positive isolates are higher than those from integron-negative strains. All of the integron-positive strains were resistant to more than three classes of agents. There werel6 different gene cassettes encoding resistance to aminoglycosides （aadA 1, aadA2, aac6-, aacA4）, trimethoprim （drfA 1, dfrA 12, dfrA 15, dfrA 17, dfrB4）, β- lactams（blaoxA_10, blaoxA-21）, chloramphenicl （catB3, catB8）, rifampicin （art-2, arr-3） and plasmid-mediated quinolone resistance （PMQR） gene aac（6＇）-Ib-cr. Most of the integron-positive isolates carrying gene cassettes mentioned above corresponded to the resistance profile of antimicrobial agents such as trimethoprim, streptomycin, chloramphenicol. It indicated that there was a close relationship between classⅠ integron and multi-drug resistance in Aeromonas isolates. It is concluded that class Ⅰ integron mediated multi-drug resistance was wide spread in Aeromans from different origins of pig-fish integrated farms. Further study on resistance mechanism should be undertaken so as to provide a basis for rational usage of antimicrobial agents in Chinese aquaculture.