發(fā)布時間：2018-06-07 16:48

  本文選題：奶牛乳腺炎 + 葡萄球菌�。� 參考：《揚州大學》2016年博士論文

【摘要】：乳腺炎是制約世界奶牛養(yǎng)殖業(yè)發(fā)展的最重要疾病之一。奶牛乳腺炎的病原微生物種類繁多,但以葡萄球菌、鏈球菌和大腸桿菌為主,其中金黃色葡萄球菌是接觸傳染性病原菌,而且具有公共衛(wèi)生意義；凝固酶陰性葡萄球菌(coagulase-negative staphylococci, CNS)有20多種,已成為許多國家奶牛乳腺炎的主要病原菌,但不同國家流行的CNS種類不同。葡萄球菌毒力基因多達數(shù)十種,但具體的致病作用知之甚少。奶牛乳腺炎主要靠加強飼養(yǎng)管理和使用抗菌藥物進行控制,但隨著抗生素的長期、盲目使用,細菌耐藥現(xiàn)象日益嚴重。國內(nèi)對奶牛乳腺炎葡萄球菌的系統(tǒng)研究較少,本研究對乳腺炎葡萄球菌的種類、毒力基因、抗性基因及其與耐藥表型的關系進行了系統(tǒng)的調(diào)查分析,旨在為奶牛乳腺炎葡萄球菌致病與耐藥機制的深入研究奠定基礎。一、奶牛乳腺炎葡萄球菌的分離與種類鑒定根據(jù)臨床檢查和乳汁體細胞檢測結(jié)果,在2012年6月到2014年6月期間采集乳腺炎陽性奶樣209份,接種血瓊脂平板培養(yǎng)后挑取疑似菌落,根據(jù)菌落形態(tài)和革蘭氏染色鏡檢結(jié)果,篩選到104份葡萄球菌感染陽性奶樣。在分離的104株葡萄球菌中,所有菌株均為過氧化氫酶試驗陽性,29株(27.9%)為耐熱核酸酶試驗陽性,27株(26.0%)為凝固酶試驗陽性,24株(23.1%)為耐熱核酸酶基因檢測陽性,28株(26.9%)為凝固酶基因檢測陽性。綜合上述結(jié)果,將28株(26.9%)初步鑒定為金黃色葡萄球菌,76株(73.1%)鑒定為CNS。用PCR擴增葡萄球菌蛋白翻譯Tu延伸因子(elongation Tu factor, Tuf)和16SrRNA基因片段并進行序列測定,以同源性≥98%為種的區(qū)分標準,將28株確認為金黃色葡萄球菌,76株CNS分為13個種,其中阿爾萊特葡萄球菌(S. arlettae)、松鼠葡萄球菌(S. sciuri)、木糖葡萄球菌(S. xylosus)和產(chǎn)色葡萄球菌(S. chromogenes)為優(yōu)勢CNS,分別占15.8%；表皮葡萄球菌(S. epidermidis)、模仿葡萄球菌(S. simulans)、馬胃葡萄球菌(S. equorum)、溶血性葡萄球菌(S. haemolyticus)和沃氏葡萄球菌(S. warneri)次之,豬葡萄球菌(S. hyicus)、腐生葡萄球菌(S. saprophyticus)、琥珀葡萄球菌(S. succinus)和蒼蠅葡萄球菌(S. muscae)為次要CNS,各占1.3%。這些研究結(jié)果表明,葡萄球菌是該場奶牛乳腺炎的主要病原菌,并以CNS為主,但優(yōu)勢CNS種類不同于國外報道；Tuf基因序列分析可用于葡萄球菌種的鑒定,但少數(shù)菌株需用16S rRNA基因序列予以確認。二、奶牛乳腺炎葡萄球菌毒力基因的檢測與分析將37個葡萄球菌毒力基因分為普通毒力基因、外毒素基因和生物被膜形成基因。以葡萄球菌基因組DNA為模板進行單一、雙重或多重PCR檢測,結(jié)果在28株金黃色葡萄球菌中,14個普通毒力基因的檢出率依次為凝固酶基因coa和免疫球蛋白結(jié)合蛋白基因坦(100%)、溶血素A基因hla(96.4%)、溶血素B基因hlb和纖維蛋白原結(jié)合蛋白基因∥b(92.9%)、凝集因子A基因clfA(89.3%)、凝集因子B基因clfB和耐熱核酸酶基因聆uc(85.7%)、MHC Ⅱ類分子類似物蛋白基因map(78.6%)、莢膜多糖5基因cap5(46.4%)、莢膜多糖8基因cap8和彈性蛋白結(jié)合蛋白基因e助(39.3%)以及膠原結(jié)合蛋白基因cnα(10.7%)；12個外毒素基因的檢出率依次為腸毒素G基因seg(14.3%)、腸毒素I基因sei(10.7%)和腸毒素A基因sea(7.1%)；11個生物被膜形成基因的檢出率依次為層粘連蛋白結(jié)合蛋白基因etno(100%)、葡萄球菌蛋白A基因spa(96.4%)、纖連蛋白結(jié)合蛋白B基因fnbB(75.0%)、細胞粘附D基因icaD(71.4%)、附屬調(diào)節(jié)1基因agr-1(64.3%)和附屬調(diào)節(jié)2基因agr-2(17.9%)。這些毒力基因以19個不同的基因組合存在,但僅1個為優(yōu)勢毒力基因組合(32.1%)。在76株CNS中,普通毒力基因的檢出率依次為國(18.4%)、map(11.8%)和cap8(1.3%),外毒素基因的檢出率依次為sei(5.3%)和seb(2.6%),生物被膜形成基因的檢出率依次為eno(53.9%)、bap(10.5%)、agr-2(3.9%)、fnbA(2.6%)和fnbB(2.6%),其毒力基因組合不僅數(shù)量少,而且非常簡單。多數(shù)金黃色葡萄球菌均攜帶溶血素A基因,提示對奶牛乳腺的致病性較強。所有葡萄球菌的外毒素和生物被膜基因檢出率均顯著低于國外報道,提示其相關致病機制的復雜性。三、奶牛乳腺炎葡萄球菌抗性基因檢測及其與耐藥性的關系分析以葡萄球菌基因組DNA為模板,用單一、雙重或多重PCR檢測甲氧西林抗性基因mecA,氨基糖苷類抗性基因aacA-aphD.aac(6')laph(2")、aph(3')-Ⅲa和ant(4')-Ia,鏈陽霉素A抗性基因vatA、vatB和vatC,紅霉素與克林霉素抗性基因ermA.ermB和ermC,四環(huán)素抗性基因tetK和tetM,大環(huán)內(nèi)酯類抗性基因msrA和msrB,林可酰胺類抗性基因linA,以及青霉素抗性基因blaZ。結(jié)果在28株金黃色葡萄球菌中,17種抗性基因的檢出率依次為blaZ(82.1%)、 mecA(35.7%)、aacA-aphD(32.1%)、aac(6')/aph(2")(28.6%)、tetK(10.7%)、ermC(7.1%)和linA(7.1%),多數(shù)菌株攜帶多個抗性基因組合且含blaZ基因。在76株CNS中,抗性基因的檢出率依次為linA(38.2%)、tetK(34.2%)、blaZ(30.3%)、aacA-aphD(21.1%)、msrB (19.7%)、mecA和msrA(17.1%)、ermC(13.2%)、aac(6')/aph(2")(10.5%)、ermB(9.2%)和tetM(2.6%),mecA基因主要見于松鼠葡萄球菌、表皮葡萄球菌和溶血葡萄球菌,多數(shù)菌株攜帶多個抗性基因組合,其中表皮葡萄球菌的抗性基因組合最多。藥敏試驗分析結(jié)果顯示在28株金黃色葡萄球菌中,100%對兩種或以上抗生素耐藥；82.1%對青霉素耐藥,與blaZ因密切相關；46.4%對鏈霉素耐藥,與aacA-aphD、aac(6')/aph(2")或未知基因相關；35.7%對卡那霉素和妥布霉素耐藥,32.1%對慶大霉素耐藥,與aacA-aphD或aac(6')/aph(2")基因相關；32.1%對頭孢西丁耐藥,主要與blaZ和mecA基因相關；14.3%對紅霉素耐藥,與ermC.或未知基因相關；10.7%對四環(huán)素耐藥,與tetK或未知基因相關。在76株CNS中,79.4%對兩種或以上抗生素耐藥；86.8%對青霉素耐藥,與blaZ或未知基因相關；48.7%對紅霉素耐藥,與erm、msr或未知基因相關：46.1%對鏈霉素耐藥,27.6%對卡那霉素耐藥,13.2%對慶大霉素耐藥,11.8%對妥布霉素耐藥,與aacA-aphD、aac(6')/aph(2")或未知基因相關；39.5%對四環(huán)素耐藥,與tetK基因密切相關；30.3%對克林霉素耐藥,與ermB、ermC、linA或未知基因相關；27.6%對頭孢西丁耐藥,與mecA、blaZ或未知基因相關。這些研究結(jié)果表明,本研究的葡萄球菌blaZ和mecA基因檢出率均顯著高于國內(nèi)外報道,可能與該奶牛場長期使用青霉素進行乳腺炎防治和場內(nèi)耐藥菌株循環(huán)傳播有關。部分抗生素抗性與未知基因相關,提示葡萄球菌耐藥機制的復雜性。
[Abstract]:Mastitis is one of the most important diseases that restrict the development of dairy farming in the world. There are a wide variety of pathogenic microbes in cow mastitis, but mainly Staphylococcus, Streptococcus and Escherichia coli, and Staphylococcus aureus is in contact with infectious pathogens and is of public health significance; coagulase negative staphylococcus (coagulase-negativ E staphylococci, CNS) has more than 20 kinds, and has become the main pathogen of cow mastitis in many countries, but the prevalence of CNS in different countries is different. The virulence genes of Staphylococcus are as many as dozens, but the specific pathogenicity is very little. Long term, blind use, the phenomenon of bacterial resistance to bacteria is becoming more and more serious. There are few systematic studies on the Staphylococcus of cow mastitis in China. This study systematically investigated the types, virulence genes, resistance genes and the relationship between the resistance genes and the resistance phenotype of Staphylococcus mastitis, aiming at the pathogenesis and drug resistance mechanism of Staphylococcus mastitis. The isolation and identification of Staphylococcus from dairy cow mastitis, 209 samples of mastitis positive milk samples were collected from June 2012 to June 2014, and suspected bacterial colonies were picked up after inoculation of blood agar plate, according to colony morphology and Gram stain microscopic examination results. 104 staphylococcal infection positive milk samples were screened. Among the 104 strains of Staphylococcus isolates, all strains were tested positive for catalase, 29 (27.9%) were heat resistant nuclease test positive, 27 (26%) was coagulase test positive, 24 (23.1%) was positive for heat resistant nuclease gene, 28 (26.9%) was positive for coagulase gene. According to the above results, 28 strains (26.9%) were identified as Staphylococcus aureus, 76 (73.1%) were identified as CNS. PCR amplification of staphylococcal protein translation Tu extension factor (elongation Tu factor, Tuf) and 16SrRNA gene fragment and sequence determination. The 28 strains were identified as Staphylococcus aureus, 76 with homology more than 98% as the standard. Strain CNS is divided into 13 species, including S. arlettae, Staphylococcus squirrel (S. sciuri), Staphylococcus xylose (S. xylosus) and Staphylococcus aureus (S. chromogenes) as the dominant CNS, accounting for 15.8% respectively; Staphylococcus epidermidis (S. epidermidis), mimic Staphylococcus aureus, hemolysis Staphylococcus (S. haemolyticus) and Staphylococcus veviae (S. warneri) are secondary, and Staphylococcus (S. hyicus), Staphylococcus saprophys (S. saprophyticus), Staphylococcus amber (S. succinus) and Staphylococcus aureus (S. muscae) are secondary. The results show that Staphylococcus is the main pathogen of mastitis in this field. CNS, but the dominant CNS species are different from foreign reports, Tuf gene sequence analysis can be used for the identification of Staphylococcus species, but a few strains need to be confirmed by the 16S rRNA gene sequence. Two, the detection and analysis of the virulence genes of Staphylococcus mastitis of cow mastitis divide 37 Vitis Vitis virulence genes into common virulence gene, exotoxin base A single, double or multiple PCR test was carried out with the staphylococcal genome DNA as a template. The results of 14 common virulence genes in 28 strains of Staphylococcus aureus were the coagulase gene COA and the immunoglobulin binding protein gene (100%), the HLA (96.4%) of the hemolysin A gene, and the HLB of the hemolysin B gene HLB. And fibrinogen binding protein gene B (92.9%), agglutinating factor A gene clfA (89.3%), agglutinating factor B gene clfB and heat resistant nuclease gene hearing UC (85.7%), MHC class II molecular analogs protein gene map (78.6%), capsule polysaccharide 5 gene cap5 (46.4%), capsule polysaccharide 8 gene cap8 and elastin binding protein gene e assistance (39.3%) and collagen Binding protein gene CN alpha (10.7%); 12 exotoxin genes were detected in sequence of enterotoxin G gene SEG (14.3%), enterotoxin I gene SEI (10.7%) and enterotoxin A gene sea (7.1%); the detection rate of 11 biofilm formation genes in turn was laminin binding protein based etno (100%), staphylococcal protein A Spa (96.4%), fibronectin. Binding to the protein B gene fnbB (75%), the cells adhered to the D gene icaD (71.4%), the accessory regulation 1 gene agr-1 (64.3%) and the accessory regulation 2 gene agr-2 (17.9%). These virulence genes existed in 19 different gene combinations, but only 1 were the dominant virulence gene combinations (32.1%). In 76 CNS, the detection rates of common virulence genes were in China (18.4%), map (11.). 8%) and cap8 (1.3%), the detection rates of exotoxin genes were SEI (5.3%) and SEB (2.6%). The detection rates of biofilm formation genes were eno (53.9%), BAP (10.5%), agr-2 (3.9%), fnbA (2.6%) and fnbB (2.6%), and their virulence gene combinations were not only small, but also very simple. The pathogenicity of bovine mammary gland was stronger. All staphylococcal exotoxin and biofilm gene detection rate were significantly lower than that of foreign reports, suggesting the complexity of the related pathogenic mechanism. Three, the relationship between the resistance gene detection of Staphylococcus aureus and its resistance to the drug resistance of cow mastitis was based on the genome DNA of Vitis Vitis, single, double or more. Heavy PCR detection of methoxiin resistance gene mecA, aminoglycoside resistance gene aacA-aphD.aac (6') laph (2 "), APH (3') - III A and ant (4') -Ia, chain Yang mycin A resistance gene, erythromycin and clindamycin resistance gene The resistance gene linA and the results of penicillin resistance gene blaZ. in the 28 strains of Staphylococcus aureus, the detection rates of the 17 resistant genes were blaZ (82.1%), mecA (35.7%), aacA-aphD (32.1%), AAC (6') /aph (2 ") (28.6%), tetK (10.7%), ermC (7.1%) and linA (7.1%). Most of the strains carried multiple resistant gene combinations and contained blaZ genes. In 76 strains In S, the detection rates of resistance genes were linA (38.2%), tetK (34.2%), blaZ (30.3%), aacA-aphD (21.1%), msrB (19.7%), mecA and msrA (17.1%), ermC (13.2%), AAC (6') /aph (2 ") (10.5%), 9.2% and 2.6%), which were mainly found in Staphylococcus epidermidis, Staphylococcus epidermidis and hemolytic Staphylococcus, and most of the strains carried multiple resistance genes. The combination, among which Staphylococcus epidermidis had the most resistance genes, showed that 100% of 28 strains of Staphylococcus aureus were resistant to two or more antibiotics; 82.1% to penicillin, closely related to blaZ, 46.4% to streptomycin, associated with aacA-aphD, AAC (6') /aph (2 ") or unknown genes; 35.7% pairs of cards. Drug resistance of mycophencin and tobramycin, 32.1% resistant to gentamicin, associated with aacA-aphD or AAC (6') /aph (2 ") genes; 32.1% resistant to cefoxitin, mainly associated with blaZ and mecA genes; 14.3% to erythromycin, associated with ermC. or unknown genes; 10.7% against tetracycline, associated with tetK or unknown genes. 79.4% pairs in 76 strains of CNS Resistance to two or more antibiotics; 86.8% to penicillin resistant to blaZ or unknown genes; 48.7% to erythromycin, associated with ERM, MSR or unknown genes: 46.1% to streptomycin, 27.6% to kanamycin, 13.2% to gentamicin, 11.8% to todamin, and aacA-aphD, AAC (6') /aph (2 ") or unknown gene 39.5% resistance to tetracycline, closely related to the tetK gene; 30.3% to clindamycin resistance, related to ermB, ermC, linA or unknown genes; 27.6% to cefoxitin, related to mecA, blaZ or unknown genes. These results showed that the detection rates of staphylococcal blaZ and mecA genes in this study were significantly higher than those reported at home and abroad. It is related to the long-term use of penicillin in the dairy farm for the prevention of mastitis and the circulatory transmission of field resistant strains. Some antibiotic resistance is related to the unknown genes, suggesting the complexity of the mechanism of staphylococcal resistance.
【學位授予單位】：揚州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S858.23

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