【摘要】：肉鴨發(fā)酵床養(yǎng)殖是一種利用墊料中微生物降解動物排泄物中的有機物,進而有效控制規(guī)模化養(yǎng)殖帶來的嚴重環(huán)境污染問題的新型旱養(yǎng)模式�？股匾蚱溆蓄A防和治療疾病、提高飼料利用率、促進生長等優(yōu)點在畜牧生產(chǎn)中廣泛使用;鋅作為機體必需的元素之一,參與多種生理功能,也常作為礦物質(zhì)添加劑在生產(chǎn)中使用。由于發(fā)酵床墊料的使用時間較長,飼料源礦物質(zhì)、飼料及治療用抗生素均會隨著動物排泄物的排放而在墊料中累積,從而使墊料細菌同時面對抗生素和重金屬的雙重選擇壓力。目前,我國肉鴨發(fā)酵床旱養(yǎng)模式處于新興階段,對于肉鴨發(fā)酵床養(yǎng)殖過程中,墊料微生物菌群結(jié)構(gòu)、墊料源大腸桿菌耐藥性的變化和原因等研究較少。本論文從以上問題切入,對使用不同批次的肉鴨發(fā)酵床墊料菌群結(jié)構(gòu)、墊料源大腸桿菌耐藥水平和體外模型下大腸桿菌耐藥性的誘導進行研究,試驗分為以下四部分:一、肉鴨養(yǎng)殖過程中發(fā)酵床墊料菌群結(jié)構(gòu)的變化本研究旨在探究發(fā)酵床使用時間和肉鴨糞便微生物對發(fā)酵床墊料菌群結(jié)構(gòu)、總菌和大腸桿菌數(shù)量的影響。試驗采集江蘇某肉鴨發(fā)酵床養(yǎng)殖場內(nèi)剛制作完成發(fā)酵床墊料樣品,及飼養(yǎng)4批次、8批次肉鴨后的墊料樣品,同時采集各批次34日齡肉鴨糞便樣品,采用變性梯度凝膠電泳技術(shù)(PCR-DGGE)、16SrRNA基因序列分析和實時熒光定量PCR(Real-time PCR)技術(shù)對發(fā)酵床使用過程中墊料菌群結(jié)構(gòu)進行定性和定量研究。結(jié)果表明:0批次(DO)與4批次(D4)、8批次(D8)墊料菌群相似性分別為68.81%、70.82%,而4批次(D4)和8批次(D8)墊料菌群的相似性則達81.93%,顯著高于D4、D8與D0間相似性(P0.05)。條帶6、8(最相似菌分別為Leqionella tunisiensis、Pensisddobacter bauzanensis)在三個時間點墊料菌群中均表現(xiàn)優(yōu)勢,且含量較為穩(wěn)定;條帶10(最相似菌為Rummeliibacillus suwonesis)僅在二個重復使用墊·料菌群中表現(xiàn)優(yōu)勢;條帶12、13(最相似菌分別是Psychrobacter sp.PRwf-1、Iamia majanohamensis)共同存在于墊料樣和糞便樣。肉鴨糞便中大腸桿菌的數(shù)量顯著高于4批次、8批次墊料中的數(shù)量(P0.05),與0批次墊料間差異不顯著(P0.05)。綜上所述,發(fā)酵床墊料的使用時間和肉鴨糞便微生物共同影響了墊料菌群結(jié)構(gòu)和數(shù)量,菌群結(jié)構(gòu)隨使用時間的延長而趨于穩(wěn)定。二、發(fā)酵床使用過程中墊料源大腸桿菌耐藥性和鋅抗性的分析本試驗旨在研究肉鴨發(fā)酵床使用過程中墊料源大腸桿菌抗生素耐藥性和鋅抗性水平的變化,并探討兩者間的關(guān)系,為肉鴨生產(chǎn)中藥物合理使用和發(fā)酵床墊料管理提供指導。試驗以從江蘇徐州某肉鴨發(fā)酵床養(yǎng)殖場的0(D0)、4(D4)、8(D8)批次墊料中分離的152株大腸桿菌菌株為對象,采用美國臨床和實驗室標準協(xié)會(CLSI)推薦的微量肉湯稀釋法和瓊脂稀釋法進行抗生素和鋅的最低抑菌濃度測定。結(jié)果表明:菌株對不同抗生素的耐藥程度不同,其中對氟苯尼考、四環(huán)素和強力霉素的耐藥率均在95%以上,對恩諾沙星、氧氟沙星的耐藥率在76%-81%之間,對慶大霉素耐藥率最低,為21.05%;不同批次墊料源大腸桿菌的耐藥率也有所不同,8批次墊料源的大腸桿菌對頭孢類和喹諾酮類藥物的耐藥率顯著高于4批次墊料源的菌株(P0.05)。多重耐藥現(xiàn)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。對分離菌株進行鋅耐受測定,結(jié)果顯示耐鋅現(xiàn)象較為嚴重,耐鋅率達100%,且隨墊料使用時間的延長,菌株鋅MIC值有所升高。本研究中并未發(fā)現(xiàn)菌株鋅的耐受程度和抗生素耐藥性之間存在相關(guān)性。本研究從墊料中分離的菌株中抗生素耐藥性和鋅耐受情況均較為嚴重,故今后生產(chǎn)中應合理選擇有效藥物用于肉鴨疾病治療。三、發(fā)酵床墊料源大腸桿菌質(zhì)粒介導恩諾沙星耐藥和耐鋅分析本試驗通過分析大腸桿菌質(zhì)粒介導恩諾沙星耐藥和鋅抗性的基因攜帶情況,并結(jié)合其表型變化,探討發(fā)酵床墊料大腸桿菌恩諾沙星耐藥和鋅抗性之間的關(guān)系。通過PCR法檢測肉鴨發(fā)酵床墊料中分離的66株恩諾沙星高耐菌(MIC≥32 μg/mL)和11株敏感菌株(MIC≤0.25μg/mL)中4種質(zhì)粒介導喹諾酮類耐藥基因(qnrS、qepA、oqxAB、aac(6')-lb-cr)和耐鋅基因(ZntM),并結(jié)合耐受表型進行分析。結(jié)果表明:質(zhì)粒介導的3種恩諾沙星耐藥基因qnrS、oqxAB、aac(6')-lb-cr和耐鋅基因ZntA在被檢菌株中普遍存在。77.92%菌株至少攜帶一種質(zhì)粒介導的耐藥基因,攜帶最普遍的是與外排泵相關(guān)的oqxAB基因(57.14%),其次是與氨基糖苷類乙酰轉(zhuǎn)移酶相關(guān)的aac(6')-lb-cr基因(38.96%)和與拓撲異構(gòu)酶有關(guān)的qnnrS基因(33.77%),而qepA基因在被檢菌株中并未檢測到。結(jié)合相應菌株的恩諾沙星和鋅耐受表型發(fā)現(xiàn),9株恩諾沙星表型敏感菌,檢測到至少攜帶一種耐藥基因;15株恩諾沙星表型高耐菌,未檢測到這4種耐藥基因;在98.70%(76/77株)耐鋅菌株中均檢測到ZntA基因。本研究中菌株的耐藥表型與耐藥基因攜帶情況并不完全一致,提示存在質(zhì)粒介導以外的機制影響大腸桿菌對恩諾沙星的耐受;被檢菌株普遍耐鋅且基本存在ZntA基因,墊料中鋅的累積和質(zhì)粒介導鋅耐受的重要性可能是導致這一現(xiàn)象的原因。四、低濃度恩諾沙星和鋅對體外模型中大腸桿菌敏感性的影響研究本研究選取4株恩諾沙星敏感菌株(MIC=0.5 μg/mL)分別在含1/2 MIC(0.25μg/mL)恩諾沙星的MH肉湯(E組)、含1/3 MIC(1.33 μg/mL)氯化鋅的M-H肉湯(Zn組)和含1/2 MIC恩諾沙星和1/3 MIC氯化鋅的M-H肉湯(E+Zn組)中進行體外誘導培養(yǎng),并對誘導的第3、6、9、12、15、18代菌株測定恩諾沙星和鋅MIC值;檢測0、18代的菌株抗性基因攜帶情況。結(jié)果表明:低濃度恩諾沙星單獨誘導可顯著提高菌株的耐藥性(MIC提高至親本的4倍);Zn組和E+Zn組也能提高菌株耐藥性,但差異不顯著;對抗性基因檢測可知,親本菌株中僅能檢測到qnrS基因,而誘導菌株中均能檢測到oqxAB、aac(6')-lb-cr、qnrS基因;不同誘導模式均可在一定程度上提高菌株鋅MIC值,Znt4基因普遍存在于所有菌株中。提示,誘導前后菌株恩諾沙星MIC值的提高可能與oqx4B、aac(6')-lb-cr基因的獲得存在一定的聯(lián)系,抗鋅基因ZntA基因普遍存在于所有菌株中,鋅的耐受機理、鋅和抗生素之間的協(xié)同作用還需深入研究。
[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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S834

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