肉鸭发酵床垫料菌群结构演替、大肠杆菌耐药性和锌抗性的关系研究

发布时间：2018-08-26 15:25

【摘要】：肉鸭发酵床养殖是一种利用垫料中微生物降解动物排泄物中的有机物,进而有效控制规模化养殖带来的严重环境污染问题的新型旱养模式。抗生素因其有预防和治疗疾病、提高饲料利用率、促进生长等优点在畜牧生产中广泛使用;锌作为机体必需的元素之一,参与多种生理功能,也常作为矿物质添加剂在生产中使用。由于发酵床垫料的使用时间较长,饲料源矿物质、饲料及治疗用抗生素均会随着动物排泄物的排放而在垫料中累积,从而使垫料细菌同时面对抗生素和重金属的双重选择压力。目前,我国肉鸭发酵床旱养模式处于新兴阶段,对于肉鸭发酵床养殖过程中,垫料微生物菌群结构、垫料源大肠杆菌耐药性的变化和原因等研究较少。本论文从以上问题切入,对使用不同批次的肉鸭发酵床垫料菌群结构、垫料源大肠杆菌耐药水平和体外模型下大肠杆菌耐药性的诱导进行研究,试验分为以下四部分:一、肉鸭养殖过程中发酵床垫料菌群结构的变化本研究旨在探究发酵床使用时间和肉鸭粪便微生物对发酵床垫料菌群结构、总菌和大肠杆菌数量的影响。试验采集江苏某肉鸭发酵床养殖场内刚制作完成发酵床垫料样品,及饲养4批次、8批次肉鸭后的垫料样品,同时采集各批次34日龄肉鸭粪便样品,采用变性梯度凝胶电泳技术(PCR-DGGE)、16SrRNA基因序列分析和实时荧光定量PCR(Real-time PCR)技术对发酵床使用过程中垫料菌群结构进行定性和定量研究。结果表明:0批次(DO)与4批次(D4)、8批次(D8)垫料菌群相似性分别为68.81%、70.82%,而4批次(D4)和8批次(D8)垫料菌群的相似性则达81.93%,显著高于D4、D8与D0间相似性(P0.05)。条带6、8(最相似菌分别为Leqionella tunisiensis、Pensisddobacter bauzanensis)在三个时间点垫料菌群中均表现优势,且含量较为稳定;条带10(最相似菌为Rummeliibacillus suwonesis)仅在二个重复使用垫·料菌群中表现优势;条带12、13(最相似菌分别是Psychrobacter sp.PRwf-1、Iamia majanohamensis)共同存在于垫料样和粪便样。肉鸭粪便中大肠杆菌的数量显著高于4批次、8批次垫料中的数量(P0.05),与0批次垫料间差异不显著(P0.05)。综上所述,发酵床垫料的使用时间和肉鸭粪便微生物共同影响了垫料菌群结构和数量,菌群结构随使用时间的延长而趋于稳定。二、发酵床使用过程中垫料源大肠杆菌耐药性和锌抗性的分析本试验旨在研究肉鸭发酵床使用过程中垫料源大肠杆菌抗生素耐药性和锌抗性水平的变化,并探讨两者间的关系,为肉鸭生产中药物合理使用和发酵床垫料管理提供指导。试验以从江苏徐州某肉鸭发酵床养殖场的0(D0)、4(D4)、8(D8)批次垫料中分离的152株大肠杆菌菌株为对象,采用美国临床和实验室标准协会(CLSI)推荐的微量肉汤稀释法和琼脂稀释法进行抗生素和锌的最低抑菌浓度测定。结果表明:菌株对不同抗生素的耐药程度不同,其中对氟苯尼考、四环素和强力霉素的耐药率均在95%以上,对恩诺沙星、氧氟沙星的耐药率在76%-81%之间,对庆大霉素耐药率最低,为21.05%;不同批次垫料源大肠杆菌的耐药率也有所不同,8批次垫料源的大肠杆菌对头孢类和喹诺酮类药物的耐药率显著高于4批次垫料源的菌株(P0.05)。多重耐药现象在本研究菌株中普遍存在,多耐菌株占98.03%(149/152株),其中以5耐菌株居多。对分离菌株进行锌耐受测定,结果显示耐锌现象较为严重,耐锌率达100%,且随垫料使用时间的延长,菌株锌MIC值有所升高。本研究中并未发现菌株锌的耐受程度和抗生素耐药性之间存在相关性。本研究从垫料中分离的菌株中抗生素耐药性和锌耐受情况均较为严重,故今后生产中应合理选择有效药物用于肉鸭疾病治疗。三、发酵床垫料源大肠杆菌质粒介导恩诺沙星耐药和耐锌分析本试验通过分析大肠杆菌质粒介导恩诺沙星耐药和锌抗性的基因携带情况,并结合其表型变化,探讨发酵床垫料大肠杆菌恩诺沙星耐药和锌抗性之间的关系。通过PCR法检测肉鸭发酵床垫料中分离的66株恩诺沙星高耐菌(MIC≥32 μg/mL)和11株敏感菌株(MIC≤0.25μg/mL)中4种质粒介导喹诺酮类耐药基因(qnrS、qepA、oqxAB、aac(6')-lb-cr)和耐锌基因(ZntM),并结合耐受表型进行分析。结果表明:质粒介导的3种恩诺沙星耐药基因qnrS、oqxAB、aac(6')-lb-cr和耐锌基因ZntA在被检菌株中普遍存在。77.92%菌株至少携带一种质粒介导的耐药基因,携带最普遍的是与外排泵相关的oqxAB基因(57.14%),其次是与氨基糖苷类乙酰转移酶相关的aac(6')-lb-cr基因(38.96%)和与拓扑异构酶有关的qnnrS基因(33.77%),而qepA基因在被检菌株中并未检测到。结合相应菌株的恩诺沙星和锌耐受表型发现,9株恩诺沙星表型敏感菌,检测到至少携带一种耐药基因;15株恩诺沙星表型高耐菌,未检测到这4种耐药基因;在98.70%(76/77株)耐锌菌株中均检测到ZntA基因。本研究中菌株的耐药表型与耐药基因携带情况并不完全一致,提示存在质粒介导以外的机制影响大肠杆菌对恩诺沙星的耐受;被检菌株普遍耐锌且基本存在ZntA基因,垫料中锌的累积和质粒介导锌耐受的重要性可能是导致这一现象的原因。四、低浓度恩诺沙星和锌对体外模型中大肠杆菌敏感性的影响研究本研究选取4株恩诺沙星敏感菌株(MIC=0.5 μg/mL)分别在含1/2 MIC(0.25μg/mL)恩诺沙星的MH肉汤(E组)、含1/3 MIC(1.33 μg/mL)氯化锌的M-H肉汤(Zn组)和含1/2 MIC恩诺沙星和1/3 MIC氯化锌的M-H肉汤(E+Zn组)中进行体外诱导培养,并对诱导的第3、6、9、12、15、18代菌株测定恩诺沙星和锌MIC值;检测0、18代的菌株抗性基因携带情况。结果表明:低浓度恩诺沙星单独诱导可显著提高菌株的耐药性(MIC提高至亲本的4倍);Zn组和E+Zn组也能提高菌株耐药性,但差异不显著;对抗性基因检测可知,亲本菌株中仅能检测到qnrS基因,而诱导菌株中均能检测到oqxAB、aac(6')-lb-cr、qnrS基因;不同诱导模式均可在一定程度上提高菌株锌MIC值,Znt4基因普遍存在于所有菌株中。提示,诱导前后菌株恩诺沙星MIC值的提高可能与oqx4B、aac(6')-lb-cr基因的获得存在一定的联系,抗锌基因ZntA基因普遍存在于所有菌株中,锌的耐受机理、锌和抗生素之间的协同作用还需深入研究。
[Abstract]:Meat duck culture in fermentation bed is a new dry-farming model which utilizes microorganisms in bedding to degrade organic matter in animal excrement and effectively control the serious environmental pollution caused by large-scale farming. As one of the essential elements of the body, it participates in many physiological functions and is often used as a mineral additive in production. Because of the long use time of the fermented mattress material, the minerals of feed source, the antibiotics of feed and treatment will accumulate in the mattress along with the discharge of animal excrement, so that the bacteria in the mattress will face antibiotics and antibiotics at the same time. Dual selective pressure of heavy metals. At present, the dry-fed model of meat duck fermentation bed in China is in a new stage. In the process of duck fermentation bed culture, there are few studies on the structure of bacterial flora in mattress, the change of drug resistance of Escherichia coli from mattress source and the reasons. The study was divided into the following four parts: 1. Changes in the bacterial community structure of the fermentation mattress during duck culture. The purpose of this study was to explore the duration of the fermentation bed and the microbial community structure, total bacteria and total bacteria in duck manure. The effects of E. coli on the quantity of E. coli were studied in a meat duck fermentation bed farm in Jiangsu Province. The mattress samples were collected from 4 batches and 8 batches of meat ducks. At the same time, the stool samples of 34-day-old ducks were collected from each batch. Denaturing gradient gel electrophoresis (PCR-DGGE), 16S rRNA gene sequence analysis and real-time fluorescence were used. Quantitative and qualitative analysis of bacterial flora in the process of fermentation bed using Real-time PCR showed that the similarities of bacterial flora in 0 batches (DO) and 4 batches (D4), 8 batches (D8) were 68.81% and 70.82%, respectively, while those in 4 batches (D4) and 8 batches (D8) were 81.93%, significantly higher than those in D4, D8 and D0 (P 0). Strip 6,8 (the most similar bacteria were Leqionella tunisiensis, Pensisddobacter bauzanensis) showed superiority and relatively stable content in three time-point bedding flora; Strip 10 (the most similar bacteria were Rummeliibacillus suwonesis) showed superiority only in two reusable bedding flora; Strip 12,13 (the most similar bacteria were Psy, respectively). Chroobacter sp. PRwf-1, Iamia majanohamensis, co-existed in bedding and fecal samples. The number of E. coli in duck feces was significantly higher than that in four batches, and the number in eight batches of bedding (P 0.05) was not significantly different from that in zero batches (P 0.05). In summary, the use time of fermented bed mattress and the microorganisms in duck feces jointly affected the bedding. The structure and quantity of bacteria and the structure of bacteria tended to be stable with the prolongation of using time. 2. The purpose of this experiment was to study the changes of antibiotic resistance and zinc resistance of Escherichia coli from the bedding source during the use of the fermentation bed in ducks. To provide guidance for the rational use of medicines and the management of fermentation mattress in duck production, 152 strains of Escherichia coli were isolated from 0 (D0), 4 (D4) and 8 (D8) batches of mattress in a meat duck fermentation bed farm in Xuzhou, Jiangsu Province. The minimum inhibitory concentration of antibiotics and zinc was determined. The results showed that the resistance of the strains to different antibiotics was different. The resistance rates to florfenicol, tetracycline and doxycycline were above 95%, to enrofloxacin, ofloxacin were between 76% and 81%, and to gentamicin was the lowest (21.05%). The resistance rates of Escherichia coli to cephalosporins and quinolones in 8 batches of bedding sources were significantly higher than those in 4 batches of bedding sources (P 0.05). The results of zinc tolerance test showed that the zinc tolerance was serious, the zinc tolerance rate was 100%, and the MIC value of zinc increased with the prolongation of the use time of cushion. 3. Enrofloxacin resistance and zinc tolerance mediated by E. coli plasmid from fermented mattress material. This experiment analyzed the gene carrying of Enrofloxacin resistance and zinc resistance mediated by E. coli plasmid, and discussed fermentation by phenotypic changes. The relationship between enrofloxacin resistance and zinc resistance of Escherichia coli in mattress materials was studied. The four plasmid-mediated quinolone resistance genes (qnrS, qepA, oqxAB, AAC (6') -lb-cr and zinc resistance genes (ZntM) in 66 Enrofloxacin-resistant strains (MIC < 32 ug/mL) and 11 susceptible strains (MIC < 0.25 ug/mL) isolated from fermented mattress materials of ducks were detected by PCR. The results showed that plasmid-mediated qnrS, oqxAB, AAC (6') -lb-cr and zinc-tolerant gene ZntA were common in the tested strains. 77.92% of the strains carried at least one plasmid-mediated resistance gene, and the oqxAB gene associated with efflux pump (57.14%) was the most common. The AAC (6') - lb-cr gene associated with glycoside acetyltransferase (38.96%) and the qnnrS gene associated with topoisomerase (33.77%) were not detected in the tested strains. Combining the enrofloxacin and zinc tolerance phenotypes of the corresponding strains, 9 Enrofloxacin-sensitive strains were found to carry at least one resistance gene. In this study, the resistance phenotype of the strain was not completely consistent with the carrying of the resistance gene, suggesting that there were mechanisms other than plasmid-mediated affecting the tolerance of E. coli to enrofloxacin. ZntA gene exists basically. The accumulation of zinc in cushion and the importance of plasmid-mediated zinc tolerance may be responsible for this phenomenon. Fourth, the effect of low concentration of enrofloxacin and zinc on the susceptibility of E. coli in vitro model. In this study, four Enrofloxacin-sensitive strains (MIC = 0.5 ug/mL) were selected in Enrofloxacin containing 1/2 MIC (0.25 ug/mL) respectively. MH broth containing 1/3 MIC (1.33 ug/mL) of zinc chloride (Zn group) and M-H broth containing 1/2 MIC of enrofloxacin and 1/3 MIC of zinc chloride (E+Zn group) were induced and cultured in vitro, and the MIC values of enrofloxacin and zinc were determined for the 3rd, 6th, 9th, 12th, 15th and 18th generations of the induced strains. Low concentration of enrofloxacin alone could significantly increase the resistance of strains (MIC increased to 4 times of parents); Zn group and E + Zn group could also increase the resistance of strains, but the difference was not significant; resistance gene detection showed that only qnrS gene could be detected in parents, but oqxAB, AAC (6') - lb-cr, qnrS gene could be detected in induced strains; Znt4 gene was found in all strains, suggesting that the increase of Enrofloxacin MIC might be related to the acquisition of oqx4B, AAC (6') - lb-cr gene. ZntA gene was ubiquitous in all strains. The mechanism of zinc tolerance, zinc tolerance and antibiotic resistance were also discussed. The synergy between elements needs further study.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S834

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