奶牛乳腺炎葡萄球菌种类、毒力基因及抗性基因与耐药性的调查分析

发布时间：2018-06-07 16:48

本文选题：奶牛乳腺炎 + 葡萄球菌　；参考：《扬州大学》2016年博士论文

【摘要】：乳腺炎是制约世界奶牛养殖业发展的最重要疾病之一。奶牛乳腺炎的病原微生物种类繁多,但以葡萄球菌、链球菌和大肠杆菌为主,其中金黄色葡萄球菌是接触传染性病原菌,而且具有公共卫生意义；凝固酶阴性葡萄球菌(coagulase-negative staphylococci, CNS)有20多种,已成为许多国家奶牛乳腺炎的主要病原菌,但不同国家流行的CNS种类不同。葡萄球菌毒力基因多达数十种,但具体的致病作用知之甚少。奶牛乳腺炎主要靠加强饲养管理和使用抗菌药物进行控制,但随着抗生素的长期、盲目使用,细菌耐药现象日益严重。国内对奶牛乳腺炎葡萄球菌的系统研究较少,本研究对乳腺炎葡萄球菌的种类、毒力基因、抗性基因及其与耐药表型的关系进行了系统的调查分析,旨在为奶牛乳腺炎葡萄球菌致病与耐药机制的深入研究奠定基础。一、奶牛乳腺炎葡萄球菌的分离与种类鉴定根据临床检查和乳汁体细胞检测结果,在2012年6月到2014年6月期间采集乳腺炎阳性奶样209份,接种血琼脂平板培养后挑取疑似菌落,根据菌落形态和革兰氏染色镜检结果,筛选到104份葡萄球菌感染阳性奶样。在分离的104株葡萄球菌中,所有菌株均为过氧化氢酶试验阳性,29株(27.9%)为耐热核酸酶试验阳性,27株(26.0%)为凝固酶试验阳性,24株(23.1%)为耐热核酸酶基因检测阳性,28株(26.9%)为凝固酶基因检测阳性。综合上述结果,将28株(26.9%)初步鉴定为金黄色葡萄球菌,76株(73.1%)鉴定为CNS。用PCR扩增葡萄球菌蛋白翻译Tu延伸因子(elongation Tu factor, Tuf)和16SrRNA基因片段并进行序列测定,以同源性≥98%为种的区分标准,将28株确认为金黄色葡萄球菌,76株CNS分为13个种,其中阿尔莱特葡萄球菌(S. arlettae)、松鼠葡萄球菌(S. sciuri)、木糖葡萄球菌(S. xylosus)和产色葡萄球菌(S. chromogenes)为优势CNS,分别占15.8%；表皮葡萄球菌(S. epidermidis)、模仿葡萄球菌(S. simulans)、马胃葡萄球菌(S. equorum)、溶血性葡萄球菌(S. haemolyticus)和沃氏葡萄球菌(S. warneri)次之,猪葡萄球菌(S. hyicus)、腐生葡萄球菌(S. saprophyticus)、琥珀葡萄球菌(S. succinus)和苍蝇葡萄球菌(S. muscae)为次要CNS,各占1.3%。这些研究结果表明,葡萄球菌是该场奶牛乳腺炎的主要病原菌,并以CNS为主,但优势CNS种类不同于国外报道；Tuf基因序列分析可用于葡萄球菌种的鉴定,但少数菌株需用16S rRNA基因序列予以确认。二、奶牛乳腺炎葡萄球菌毒力基因的检测与分析将37个葡萄球菌毒力基因分为普通毒力基因、外毒素基因和生物被膜形成基因。以葡萄球菌基因组DNA为模板进行单一、双重或多重PCR检测,结果在28株金黄色葡萄球菌中,14个普通毒力基因的检出率依次为凝固酶基因coa和免疫球蛋白结合蛋白基因坦(100%)、溶血素A基因hla(96.4%)、溶血素B基因hlb和纤维蛋白原结合蛋白基因∥b(92.9%)、凝集因子A基因clfA(89.3%)、凝集因子B基因clfB和耐热核酸酶基因聆uc(85.7%)、MHC Ⅱ类分子类似物蛋白基因map(78.6%)、荚膜多糖5基因cap5(46.4%)、荚膜多糖8基因cap8和弹性蛋白结合蛋白基因e助(39.3%)以及胶原结合蛋白基因cnα(10.7%)；12个外毒素基因的检出率依次为肠毒素G基因seg(14.3%)、肠毒素I基因sei(10.7%)和肠毒素A基因sea(7.1%)；11个生物被膜形成基因的检出率依次为层粘连蛋白结合蛋白基因etno(100%)、葡萄球菌蛋白A基因spa(96.4%)、纤连蛋白结合蛋白B基因fnbB(75.0%)、细胞粘附D基因icaD(71.4%)、附属调节1基因agr-1(64.3%)和附属调节2基因agr-2(17.9%)。这些毒力基因以19个不同的基因组合存在,但仅1个为优势毒力基因组合(32.1%)。在76株CNS中,普通毒力基因的检出率依次为国(18.4%)、map(11.8%)和cap8(1.3%),外毒素基因的检出率依次为sei(5.3%)和seb(2.6%),生物被膜形成基因的检出率依次为eno(53.9%)、bap(10.5%)、agr-2(3.9%)、fnbA(2.6%)和fnbB(2.6%),其毒力基因组合不仅数量少,而且非常简单。多数金黄色葡萄球菌均携带溶血素A基因,提示对奶牛乳腺的致病性较强。所有葡萄球菌的外毒素和生物被膜基因检出率均显著低于国外报道,提示其相关致病机制的复杂性。三、奶牛乳腺炎葡萄球菌抗性基因检测及其与耐药性的关系分析以葡萄球菌基因组DNA为模板,用单一、双重或多重PCR检测甲氧西林抗性基因mecA,氨基糖苷类抗性基因aacA-aphD.aac(6')laph(2")、aph(3')-Ⅲa和ant(4')-Ia,链阳霉素A抗性基因vatA、vatB和vatC,红霉素与克林霉素抗性基因ermA.ermB和ermC,四环素抗性基因tetK和tetM,大环内酯类抗性基因msrA和msrB,林可酰胺类抗性基因linA,以及青霉素抗性基因blaZ。结果在28株金黄色葡萄球菌中,17种抗性基因的检出率依次为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%),多数菌株携带多个抗性基因组合且含blaZ基因。在76株CNS中,抗性基因的检出率依次为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基因主要见于松鼠葡萄球菌、表皮葡萄球菌和溶血葡萄球菌,多数菌株携带多个抗性基因组合,其中表皮葡萄球菌的抗性基因组合最多。药敏试验分析结果显示在28株金黄色葡萄球菌中,100%对两种或以上抗生素耐药；82.1%对青霉素耐药,与blaZ因密切相关；46.4%对链霉素耐药,与aacA-aphD、aac(6')/aph(2")或未知基因相关；35.7%对卡那霉素和妥布霉素耐药,32.1%对庆大霉素耐药,与aacA-aphD或aac(6')/aph(2")基因相关；32.1%对头孢西丁耐药,主要与blaZ和mecA基因相关；14.3%对红霉素耐药,与ermC.或未知基因相关；10.7%对四环素耐药,与tetK或未知基因相关。在76株CNS中,79.4%对两种或以上抗生素耐药；86.8%对青霉素耐药,与blaZ或未知基因相关；48.7%对红霉素耐药,与erm、msr或未知基因相关：46.1%对链霉素耐药,27.6%对卡那霉素耐药,13.2%对庆大霉素耐药,11.8%对妥布霉素耐药,与aacA-aphD、aac(6')/aph(2")或未知基因相关；39.5%对四环素耐药,与tetK基因密切相关；30.3%对克林霉素耐药,与ermB、ermC、linA或未知基因相关；27.6%对头孢西丁耐药,与mecA、blaZ或未知基因相关。这些研究结果表明,本研究的葡萄球菌blaZ和mecA基因检出率均显著高于国内外报道,可能与该奶牛场长期使用青霉素进行乳腺炎防治和场内耐药菌株循环传播有关。部分抗生素抗性与未知基因相关,提示葡萄球菌耐药机制的复杂性。
[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.
【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.23

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