【作者】 吴聪明；

【导师】 陈杖榴；

【作者基本信息】 华南农业大学 ， 基础兽医学， 2003， 博士

【摘要】 开展动物源细菌耐药性调查,了解养殖场抗菌药物选择压力,监测动物源细菌尤其是动物源致病菌耐药性的发生发展动态,跟踪动物源耐药菌的扩散并评估其危险性具有重要的理论及实际意义。目前我国这方面的研究尚待开展。本研究分两部分: (一)采用WHO推荐的纸片扩散法,按NCCLS(1999)判定标准,检测了1524株分离自规模化猪场(522株)、鸡场(220株)、奶牛场(200株)、猪屠场(120株)、各类市售鲜肉(148株)及健康学生志愿者(85株)的大肠杆菌对21种常用抗菌药物的耐药性,分析比较各来源大肠杆菌对各种抗菌药物的耐药率及多重耐药情况,结果如下: 各类养殖场动物大肠杆菌对常用抗菌药物的耐药率以青霉素类、四环素类、磺胺类抗菌药物最高,以头孢菌素类、丁胺卡那霉素、多粘菌素B抗菌药物最低,对链霉索、卡那霉素、庆大霉素、氯霉素等抗菌药物的耐药性较普遍,各类动物分离菌对FQs耐药率虽有差异,但耐药菌也较普遍;来源于各类动物的大肠杆菌对多种抗菌药物耐药性以猪源及鸡源较严重,牛源较轻,其中对FQs的耐药性,鸡源大肠杆菌约40%,猪源大肠杆菌约25%,牛源仅3.5%,三者之间存在显著差异;不同年龄动物分离大肠杆菌对多数测试药物(尤其是FQs)的耐药性,以幼龄动物分离菌较严重,成年动物分离菌较轻,预防性用药动物群最严重。分离大肠杆菌的耐药性与各养殖场的用药模式即使用抗菌药物种类、数量、频度等高度相关。 养殖场内动物、环境及饲养员分离大肠杆菌对21种抗菌药物的耐药率存有差异,但整体耐药趋势分布相似,尤其是动物与环境分离菌的耐药趋势分布更接近;同场动物、环境及饲养员分离大肠杆菌中还有一定比例的同耐药谱多耐药菌株,包括FREC,表明养殖动物、养殖环境及饲养员之间可能存在耐药菌相互传播,或者处于相同的药物选择压力,筛选出相似的耐药菌。分离自饲养员、屠宰人员及健康学生人群的大肠杆菌对21种抗菌药物的耐药性存在差异,除几种敏感药物(头孢菌素类、丁胺卡那霉素、多粘菌素B等)外,对养殖业常用抗菌药物如青霉素类、四环素类、磺胺药等的耐药率以饲养员分离菌最高,屠宰加工人员次之,普通人群最低;三类人群分离菌的多重耐药情况也一样,表明长期从事动物养殖或动物屠宰等工作的人群,体内肠道菌耐药性将因动物耐药菌的扩散而加重。 屠宰场候宰猪、屠宰环境分离大肠杆菌与宰后猪胴体分离大肠杆菌对21种抗菌更多还原

【Abstract】 The development and spread of resistant organisms is a major global problem and many countries have become increasingly concerned about the potential dangers to public health. Because of this, the use of antimicrobial agents in veterinary services is widely debated. In order to assess the risk of transfer of antimicrobial resistance from animals, standardized and continuous data must be collected from the veterinary, agricultural and medical departments. In this dissertation, an investigation of the use of antimicrobials in food animals and the susceptibility of bacteria in food animals and in food of animal origin was done. Besides, the epidemiology of integrons/gene cassettes in Escherichia coli isolates collected from pig farm was carried out.1. Antimicrobial susceptibility testing of 21 antimicrobial agents was performed on a total of 1524 commensal Escherichia coli isolates collected from pigs industry (522), poultry industry (220) ,dairy cattle farm (200), pig slaughterhouse (120) ,retail meats (148), and healthy student volunteers (85) in Guangdong province between Nov.2001 to Aug. 2002. Antibiograms and their interpretation were made using the disk diffusion method following the NCCLS(1999) standards.Among the 21 antimicrobial agents tested, resistance of E.coli isolated from all sources was the most frequent for sulfonamides, tetracyclines, and penicillins. Isolates from swine farm and poultry farm presented significantly more resistant than those from dairy cattle farm. These results were correlative with the use of antimicrobial agents in livestock production. The resistance of E.coli isolated from young animals (including porket, chickling, and calf) were higher than those from adults animals, from infected animals were higher than those from healthy animals, and from medicating animals were higher than those from non-medicating animals.These may be due to younger animals were at a greater risk of contracting disease, and had a higher frequent prophylactic medication.The susceptibility of E.coli isolates collected from farmers and environment was consistent with those of collected from animals, and the same multi-resistant patterns of E.coli isolates from different sources were identified. It suggested that the spread of resistant bacteria among animals, environment and humans occurred on the farm. The susceptibility of E.coli strains isolated from pig carcasses consistent with those of pre-slaughtered pig, the susceptibility of E.coli isolates collected from retail beef,pork and chicken were corresponded to those of market-age cattle, pig and poultry,and a large proportion of resistant E.coli strains were isolated from cooked food. The results suggested that the resistant bacteria have access to enter human body through contaminated food.2. One-hundred and five (54.7%) strains of Escherichia coli which carry class I integrons were identified by PCR with primers designed to hybridize to integrase genes from 192 E.coli isolates collected from one pig farm. To further characterize the integrons of E.coli isolates, using primers specific for the 5 ’ -CS and 3 ’ -CS, touch-down PCR was used to amplify and sequence the gene cassettes associated with the identified class I integrons, and six different sized amplicons were identified. The DNA sequence of each amplicon was compared with published gene cassettes from genbank and confirmed the presence of the aminoglycoside adenyltransferase (aadA), dihydrofolate reductase (dhfr), beta-lactamase (bla), streptothricin acetyl-transferase (sat), and erythromycin esterase (ereA) genes, which confers resistance to strepto-mycin/spectinomycin, trimethoprim, β -lactams, streptothricin, and erythromycin, respectively.Six different class I integrons were prevalent in pig farm, which carried different genecassette (type and number). The most prevalent class I integron represented 36.6%, which carried dhfr and aadA2 two types of gene cassettes. One E.coli strain could carry one or two integrons, and almost all the integrons were located on the plasmids. Isolates collected from one pig material or from the same pigpen material had the similar integron profiles, but isolates with the same resistant patterns not always carried the same integrons. All isolates carried class I integrons were multiresistant, especially resistant to Sulfonamides (100%), streptomycin (90%), and spectinomycin (75%).Class I integrons carried dhfr and aadA cassettes confered resistance to trimethoprim and streptomycin represented 78% and 100% of cassettes detected, respectively. It was likely that selection for cassettes carrying dhfr genes and aadA genes had occurred in pig farm because trimethoprim was used for antibacterial growth promoter, and the extensive and long-term use of streptomycin, this could therefore account for their high prevalence, other cassettes identified in class I integrons were bla,sat,and ereA.The presence of these integrons and gene cassettes did not account for the total phenotypic resistance of all the isolates and did not exclude the presence of other mobile DNA elements in the resistant E.coli isolates.更多还原