【作者】 唐一鸣；

【导师】 姜中其；

【作者基本信息】 浙江大学 ， 基础兽医学， 2010， 硕士

【摘要】 近十几年来,为防治疾病及促进生长,大量抗菌药物应用于集约化养猪生产中。抗菌药物长期、广泛和不合理使用以及抗菌药物的选择压力等原因使大肠杆菌和沙门氏菌耐药菌株不断增多、耐药谱不断扩大,有的菌株甚至对尚未用于临床的新型抗菌药物也表现出了耐药性。因此,积极开展猪源大肠杆菌和沙门氏菌耐药性监测,阐明耐药表型与基因型特征,将有利于遏制大肠杆菌和沙门氏菌耐药性发生发展,优化抗菌药物的合理应用。本文采集浙江省规模化猪场患病猪只的病料,经细菌分离纯化、致病性试验、生化试验和PCR鉴定等,共获得79株病原性大肠杆菌和35株沙门氏菌。通过WHO推荐的Kirby-Bauer (K-B)法测定其对21种抗菌药物的耐药性。结果表明,大肠杆菌仅对头孢噻肟／克拉维酸、头孢噻肟和多粘菌素B敏感性较高,而对大部分抗菌药物表现出较高的耐药性,其中对苯唑西林、利福平、复方新诺明、强力霉素、羧苄西林、氨苄西林、阿莫西林、链霉素及氯霉素的耐药率均超过90%,其次是对庆大霉素、环丙沙星、新霉素、恩诺沙星、诺氟沙星、阿莫西林／克拉维酸及氟苯尼考的耐药率均超过50%。进一步的耐药谱分析表明,79株大肠杆菌分离株全部为耐药菌,其中以对16～18种抗菌药物多重耐药最为突出(60.8%,48/79)；相对于大肠杆菌,沙门氏菌除了对苯唑西林和利福平耐药率为100%外,对其余抗菌药物的耐药率均低于50%。其中对头孢噻肟／克拉维酸、头孢噻肟、阿米卡星、诺氟沙星及多黏菌素B的敏感率均为100%,对庆大霉素、环丙沙星、阿莫西林／克拉维酸、壮观霉素、氟苯尼考、氨苄西林、新霉素和复方新诺明的敏感率超过80%,对恩诺沙星和氯霉素的敏感率超过60%。35株沙门氏菌虽然同样对1种或多种抗生素耐药,但多重耐药频数显著低于大肠杆菌,主要表现为对2～4种抗菌药物多重耐药。Touch-down PCR扩增随机挑选的26株病原性大肠杆菌和35株沙门氏菌分离株,检测超广谱β-内酰胺酶基因(extended-spectrum beta-lactamases, ESBLs) (TEM, SHV, OXA)、氨基糖苷类药物耐药基因(aadA1、aadA2、aadB、aadD、aph(3’)-Ⅱa、aacC2、aacC4、aac(3)-Ⅰa、aac(3)-Ⅱa、磺胺类耐药基因(sulⅠ、sulⅡ、sulⅢ)、氯霉素类耐药基因(Cat1、Cat2、CmlA、CmlB、Flor)、四环素类耐药基因(tetA、tetB、tetC、tetD、tetE、tetG),结果表明,26株大肠杆菌中耐药基因sulⅡ、TEM、aph(3’)-Ⅱa、aacC2、Cat1、Cat2、aac(3)-Ⅱa、tetB、tetE、aadA2和sul I较为普遍,检出率率分别为96.2%、88.5%、80.8%、69.2%、69.2%、69.2%、65.4%、65.4%、61.5%、57.7%和50%,耐药基因Flor、CmlA、aadA1、tetA、sulⅢ和aadB的检出率分别为34.6%、26.9%、19.2%、19.2%、11.5%和3.9%。未能检测到SHV、OXA、aadD、aacC4、aac(3)-Ⅰa、tetC、tetD、tetG和CmlB基因。沙门氏菌含有的耐药基因类型没有大肠杆菌多,且耐药基因的检出率较低,tetA、TEM、sulⅡ、aacC2、Cat1、Cat2、tetB、aph(3’)-Ⅱa、sulⅠ、aadA2、aac(3)-Ⅱa和tetG的检出率分别为28.6%、20%、20%、17.1%、17.1%、17.1%、11.4%、8.6%、8.6%、2.9%、2.9%和2.9%。未能检测到SHV、OXA、aadA1、aadB、aadD、aacC4、aac(3)-Ⅰa、sulⅢ、tetC、tetD、tetE、CmlA、CmlB、Flor基因。分别采用微量肉汤稀释法和琼脂二倍稀释法测定恩诺沙星和盐酸环丙沙星对由K-B法筛选出的猪源沙门氏菌敏感株的最低抑菌浓度(minimum inhibitory concentration,MIC)和防突变浓度(mutant prevention concentration,MPC),并计算耐药选择指数(selection index, SI=MPC/MIC)。试验结果表明,恩诺沙星和盐酸环丙沙星对沙门氏菌的MIC分别为0.031～0.5μg/mL和0.031～0.13μg/mL,敏感率均为100%,与K-B法试验结果一致。恩诺沙星和盐酸环丙沙星对敏感菌的MPC分别分布于1～4μg/mL和0 .25～1μg/mL,盐酸环丙沙星对沙门氏菌的SI值(8～16)低于恩诺沙星(16～32),因此认为盐酸环丙沙星不仅抗菌活性较高,其防耐药突变能力也强于恩诺沙星。本研究表明,浙江省规模化猪场猪源病原性大肠杆菌的耐药性已相当严重,耐药基因种类多、检出率高；而猪源沙门氏菌的耐药程度相对较低,对很多抗菌药物的敏感性高,耐药基因种类较少,检出率低。恩诺沙星和盐酸环丙沙星对猪源沙门氏菌的防突变能力有一定差异,因此,为减少和控制耐药性的发生发展,在临床使用抗菌药物时不仅要参考抗菌药物对病原菌的MIC,还要重视菌株的MPC及突变选择窗(mutant selection window, MSW),以防止耐药突变菌选择及优势生长。更多还原

【Abstract】 Since recent decade ago,many antimicrobials have been extensively used in pig production to prevent infectious diseases and improve growth. The wide and irrational application of the antimicrobials resulted in heavy antibiotic selection pressure, and increasingly severe bacterial resistance and multi-resistance to pathogenic Escherichia coli and Salmonella. Some clinical isolates could survive even in several new drugs which even not yet introduced in clinic.Therefore,it’s wise and favorable to monitor the drug resistance, characterize antimicrobial resistance both phenotype and genotype, so that better contain the development and dissemination of pathogenic E. coli and Salmonella, and more rationally administer regimens of antimicrobial agents.Samples from sick piglets in intensive pig farms of Zhejiang Province were collected for isolates of pathogenic E. coli and Salmonella. Through biochemical tests, PCR and virulence tests in mice, total of 79 pathogenic E. coli isolates and 35 Salmonella isolates were identified. The susceptibility testing was carried out by Kirby-Bauer(K-B) method according to CLSI(Clinical and Laboratory Standards Institute) against 21 commonly used antimicrobials, respectively. The results indicated that, apart from great sensitivity to cefotaxime/clavulanate, cefotaxime and polymyxin B,the E. coli isolates exhibited severe resistance to the majority of antimicrobials.The resistance frequencies of oxacillin, rifampicin, sulphamethoxazole and trimethoprim, doxycycline, carbenicillin, ampicillin, amoxicillin, streptomycin and chloromycetin were all above 90%, and the resistance frequencies of gentamycin, ciprofloxacin, neomycin, enrofloxacin, norfloxacin, amoxicillin/clavulanate and florfenicol were also over 50%.The resistance patterns analysis demonstrated that all the E. coli isolates were resistant to 1 and more antimicrobials, and the majority of the isolates are resistant to 16-18 antimicrobials(60.8%,48/79).Compared with E. coli, the Salmonella isolates are sensitive to most antimicrobials except for oxacillin and rifampicin. They showed totally sensitive to cefotaxime/clavulanate, cefotaxime, amikacin, norfloxacin and polymyxin B.The sensitivity frequencies of gentamycin, ciprofloxacin, amoxicillin/clavulanate, spectinomycin, florfenicol,ampicillin, neomycin and sulphamethoxazole and trimethoprim were over 80%,and the sensitivity frequencies of enrofloxacin and chloromycetin were also over 60%.Though the 35 Salmonella isolates were also resistant to 1 and more antimicrobials, the multidrug resistance was less severe than E. coli isolates, and the majority of the isolates were resistant to 2-4 antimicrobials.The ESBLs(extended-spectrum beta-lactamases) genes(TEM, SHV, OXA), aminoglycoside-resistant genes(aadA1,aadA2, aadB, aadD, aph(3’)-Ⅱa, aacC2, aacC4, aac(3)-Ⅰa, aac(3)-Ⅱa), sulfonamide-resistant genes (sulⅠ, sulⅡ, sulⅢ), chloramphenicol-resistant genes(Cat1, Cat2, CmlA, CmlB, Flor), tetracycline-resistant genes(tetA, tetB, tetC, tetD, tetE, tetG) were amplified by Touch-down PCR and detected by agarose gel electrophoresis, respectively. The results showed that the following antimicrobial resistance genes were more prevalent among the 26 E. coli isolates randomly selected:sulⅡ(96.2%), TEM(88.5%), aph(3’)-Ⅱa(80.8%), aacC2(69.2%), Cat1(69.2%), Cat2(69.2%), aac(3)-Ⅱa(65.4%), tetB(65.4%), tetE(61.5%), aadA2(57.1%), sulⅠ(50%). The positive rate of the gene Flor, CmlA, aadA1, tetA, sulⅢand aadB was 34.6%,26.9%,19.2%,19.2%,11.5% and 3.9%, respectively. However, SHV, OXA, aadD, aacC4, aac(3)-Ⅰa, tetC, tetD, tetG and CmlB genes were not detected. There were not so many resistance genes in the 35 Salmonella isolates. The positive rate of the gene was lower than that of E. coli isolates:tetA(28.6%), TEM(20%), sulⅡ(20%), aacC2(17.1%), Car1(17.1%), Cat2(17.1%), tetB(11.4%), aph(3’)-Ⅱa(8.6%), sulⅠ(8.6%), aadA2(2.9%), aac(3)-Ⅱa(2.9%), tetG(2.9%). However, SHV, OXA, aadA1, aadB, aadD, aacC4, aac(3)-Ⅰa, sulⅢ, tetC, tetD, tetE, CmlA, CmlB and Flor genes were not detected.The minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of enrofloxacin and ciprofloxacin hydrochloride against sensitive Salmonella isolates from piglets, which were confirmed by K-B method, were determined by microdilution and agar dilution methods,respectively.The MICs of enrofloxacin and ciprofloxacin hydrochlorid were 0.031-0.5μg/mL and 0.031-0.13μg/mL, and the sensitivity rates were both 100%, which were consistent with the sensitivity testing by K-B method. The MPCs of enrofloxacin and ciprofloxacin hydrochlorid to the sensitive isolates were 1-4μg/mL and 0.25-1μg/mL, and the selection index (SI) values of ciprofloxacin hydrochlorid (8-16) were lower than those of enrofloxacin (16-32). Therefore, the efficacy of ciprofloxacin hydrochlorid for containing the selection of Salmonella resistant mutants was greater than that of enrofloxacin.The results in the study indicated that the pathogenic E. coli isolates from piglet in Zhejiang province had severe resistance to antimicrobials, and they harbored more varieties of the resistance genes with high positive rates. However, the Salmonella isolates were sensitive to the majority of antimicrobials tested. Enrofloxacin and ciprofloxacin hydrochlorid presented different capacity to restrict the selection of strains resistant mutants. In order to control the development of antimicrobial resistance, so not only the MIC should be considered in clinical application, but the MPC and MSW (mutant selection window, MSW) values of isolates should also be taken into account to contain the development and dissemination of bacterial resistance.更多还原