牛源金黄色葡萄球菌凝固酶分型与致病因子检测

为了解青海和甘肃地区奶牛养殖场的金黄色葡萄球菌(S.aureus)基因型分布状况,本研究对来自该两个地区的210份奶样进行了S.aureus的分离与鉴定,利用凝固酶基因(Coa)扩增和限制性内切酶AluⅠ酶切方法对所有分离株进行了凝固酶多态性分型。并对不同凝固酶基因型的S.aureus进行了白细胞毒素F (LukF)、白细胞毒素S (LukS)、α溶血素(α-hemolysin)、β溶血素(β-hemolysin)等主要致病因子检测。结果显示,共分离鉴定出35株S.aureus (青海13株,甘肃22株),所有分离株均能够扩增出Coa基因片段,并有5种PCR分型结果(本文简称PCR1～PCR5型),其中PCR 1、4和5型只有1种亚型,而PCR 2型有3种亚型,PCR 3型有2种亚型。PCR3型是青海(6/13)和甘肃地区(8/22)主要流行的基因型。致病因子检测结果显示,所有分离株均携带致病因子LukS和α溶血素;致病因子LukF和β溶血素检出率也很高,分别是30株(85.7%)和31株(88.6%)。β溶血素基因在甘肃地区检出率为95.5%(21/22),青海地区检出率为76.9%(10/13),致病因子LukF在甘肃地区检出率为91.0%(20/22),青海地区检出率为76.9%(10/13),且在各基因型中均有分布,甘肃地区S.aureus致病因子LukF和β溶血素检出率高于青海地区。本研究首次对青海和甘肃地区致奶牛乳房炎S.aureus进行了鉴定分析,为这两个地区奶牛乳房炎的防治提供了参考依据。     

奶牛乳房炎金黄色葡萄球菌对四环素耐药性分析

为分析奶牛乳房炎中分离的金黄色葡萄球菌(S.aureus)四环素耐药机制,本研究以K-B法和常量肉汤法测定最低抑菌浓度(MIC)值,并对26株奶牛乳房炎S.aureus进行四环素耐药表型检测及加入利血平后的MIC值检测;以PCR检测四环素耐药基因tetM、tetO、tetL和tetK,对扩增产物进行序列分析。检测结果表明:26株菌株中,7株对四环素耐药,19株敏感;利血平能显著降低部分耐药株对四环素的MIC值;在7株耐药菌株中,1株检测出tetM基因,6株检测出tetK基因,没有检测到tetL和tetO基因;tetK基因与S.aureus质粒pT181同源性为100%,tetM基因与转座子Tn916序列同源性为99.9%。实验研究表明,26株奶牛乳房炎S.aureus四环素耐药表型与耐药基因相符,耐药机制以外排蛋白介导为主,并在国内牛源S.aureus中首次检测到tetK基因。     

兔源金黄色葡萄球菌的耐药性及耐药基因分析

为了解四川地区规模化兔场金黄色葡萄球菌(S.aureus)的耐药情况,本研究从四川地区部分规模化兔场共分离鉴定出41株S.aureus,采用纸片扩散法对分离株进行了耐药表型鉴定,同时采用PCR方法对相关耐药基因进行了检测。药敏试验结果显示:分离株对β-内酰胺类、氨基糖苷类、四环素类和大环内脂类药物的耐药率分别为87.80%、97.56%、78.05%和56.10%,对多肽类、喹诺酮类及磺胺类耐药率较低。耐药基因检测结果显示:所有分离株均检测到耐药基因,并且29株分离株具有多重耐药性,其中耐药基因mec A、tet、erm、aac(6')/aph(2')、ant(4',4')及aph(3')-III的检出率分别为75.61%、70.73%、46.34%、92.68%、80.49%及53.66%。结合耐药表型与耐药基因结果分析显示兔源S.aureus的耐药表型与耐药基因检出率基本呈正相关。本研究通过检测41株兔源S.aureus对临床常用抗生素的耐药性及相关耐药基因,初步掌握了四川地区规模化兔场S.aureus的耐药情况,为该地区规模化兔场预防及治疗葡萄球菌病临床合理用药提供了依据。     

奶牛乳房炎金黄色葡萄球菌荚膜多糖主要血清型的鉴定

为了解内蒙古地区奶牛乳房炎金黄色葡萄球菌荚膜多糖的主要血清型,试验从内蒙古地区9个牧场共采集236份奶牛乳房炎患牛的奶样,采用常规微生物方法、生物化学反应方法、动物试验和PCR方法对金黄色葡萄球菌荚膜多糖的血清型进行鉴定。结果表明:经常规微生物学检验分离得到162株葡萄球菌,其中金黄色葡萄球菌(S.aureus)124株、表皮葡萄球菌(S.epidermidis)29株、腐生葡萄球菌(S.saparophytics)9株。动物试验得到116株致病性S.aureus。PCR方法鉴定出荚膜多糖5型S.aureus23株,占致病性S.aureus的19.83%;荚膜多糖8型S.aureus61株,占致病性S.aureus的52.59%;荚膜多糖5型和8型S.aureus占致病性S.aureus的72.41%。说明5型和8型S.aureus是内蒙古地区奶牛乳房炎金黄色葡萄球菌荚膜多糖的主要血清型。     

猪源金黄色葡萄球菌分离鉴定及16srRNA基因遗传进化分析

从湖南省养猪场采集猪拭子和肺脏300份进行金黄色葡萄球菌(S.aureus)分离.用生化试验和PCR进行鉴定;选择5株分离菌进行遗传进化分析.结果显示,所有分离株触酶试验、糖发酵试验、VP试验、溶血试验和耐热核酸酶试验均为阳性.92.3%的分离株血浆凝固酶试验为阳性.PCR鉴定均为阳性.分离株16s rRNA和nuc部分基因片段与Genbank公开的相关序列相似性分别为98.0%以上和99.0%以上.5株分离株与S.aureus GHA10等处于同一进化分支.S.aureus分离率21.67%.肺脏、拭子、母猪和仔猪分离率分别是21.52%、21.79%、19.71%和23.41%.本研究结果对湖南地区S.aure-us致病性、耐药性和耐药机制的研究提供了素材.     

我国部分地区牛源金黄色葡萄球菌基因多态性分型研究

为了解我国牛源金黄色葡萄球菌(S.aureus)的基因多态性,本研究利用随机引物多态性扩增(RAPD)体系对174株分离自贵州、内蒙古、四川、上海、甘肃等地奶牛乳房炎的S.aureus及一株标准菌CVCC2246进行基因分型。结果表明,175株S.aureus均得到清晰的RAPD指纹图谱,扩增产物为1~9个片段,产物大小为240 bp~4 500 bp。所有菌株共分为8个基因型,其中1型16株;2型和3型各37株;4型15株;5型18株;6型14株;7型13株;8型8株。2型和3型菌株占总菌株42%以上,在贵州、内蒙古、四川、上海、甘肃分布广泛,为流行优势基因型;但5型是内蒙古地区的流行优势基因型。各地区菌株基因型比例有明显差异,可能与奶牛饲养水平和环境差异有关。     

抑制奶牛乳房炎源金黄色葡萄球菌的乳酸菌的筛选

为获得抑制奶牛乳房炎源金黄色葡萄球菌(S. aureus)的乳酸菌(LAB),本研究从新疆巴音布鲁克牧区鲜牛乳和哈萨克族乳制品奶疙瘩样品中分离培养LAB,通过传统的分离鉴定与16S rDNA基因序列测序相结合的方法鉴定LAB种类,同时以临床奶牛乳房炎源金黄色葡萄球菌S. aureus N2为指示菌,采用双层琼脂扩散法检测分离株的抑菌能力。通过测定生长曲线确定分离株的生长稳定期,进而利用硫酸铵沉淀法透析提取稳定期内分离株的细菌素,并检测其细菌素抑菌效价。结果显示:从样品中筛选获得5株能够抑制指示菌生长的LAB,分别为希氏乳杆菌、干酪乳杆菌、粪肠球菌、戊糖片球菌和乳酸乳球菌亚种。生长规律曲线表明20 h^30 h为5株LAB的稳定期,此期培养液pH值维持在3.8~4.5。从培养20 h的5株LAB上清液中提取到了细菌素,经检测其具有抑菌活性,抑菌效价分别为457 IU/mL、1 023 IU/mL、676 IU/mL、1 862 IU/mL和1 023 IU/mL。本研究结果表明5株LAB通过在生长稳定期内维持较低酸性环境(pH<4.5),代谢产生细菌素对乳房炎源S. aureus发挥抑制生长作用。本研究为S. aureus性奶牛乳房炎的生物防治提供了实验依据。     

奶牛乳房炎金黄色葡萄球菌FnbpB-D基因的表达及其抗血清活性

纤维素结合蛋白B(FnbpB)是金黄色葡萄球菌(S.aureus)最主要的黏附因子之一,它能够介导S.aureus结合于细胞表面的纤维连结素(Fn)和纤维蛋白原(Fg)。FnbpB基因中D区为主要活性部位。本实验利用pET-32a表达载体表达了S.aureus FnbpB-D重组蛋白(34.7ku),并通过动物实验对重组FnbpB-D蛋白的抗血清活性进行了初步研究。采用ELISA对抗血清进行抗粘附性检测,结果显示实验组与对照组差异极显著(p0.01),抗血清具有较强的抑制S.aureus黏附纤维连结素的作用。此外,调理吞噬实验结果显示,免疫兔全血对S.aureus有较强的调理作用。这些实验结果将为制备奶牛乳房炎S.aureus黏附素疫苗的研制提供参考。     

耐甲氧西林金黄色葡萄球菌分离鉴定及耐药基因检测

为了解动物源性金黄色葡萄球菌对市售常见的各种兽用抗生素药物的耐药情况,掌握并分析其耐药性,指导临床合理使用抗菌药物。本研究通过常规细菌分离鉴定患病动物炎性分泌物,对分离菌株进行生化试验、耐药性试验、耐甲氧西林金黄色葡萄球菌(Methicillin-resistant S.aureus,MRSA)鉴定和耐药基因鉴定,获得4株耐药金黄色葡萄球菌,2株为MRSA,2株为甲氧西林敏感的金黄色葡萄球菌(Methicillinsusceptible S.aureus,MSSA);4株金黄色葡萄球菌均检测出tetA、aac(6')/aph(2')、sul1耐药基因,其中2株MRSA检测出mecA。MRSA与MSSA均表现出了多重耐药现象,其中MRSA比MSSA的耐药现象更为广泛。因此,要更加关注MRSA的流行情况,制定规范的用药行为,严控抗生素药物的滥用。     

生鲜乳中金黄色葡萄球菌LAMP检测方法的建立

针对金黄色葡萄球菌的TRAP基因设计LAMP引物,建立了金黄色葡萄球菌环介导等温扩增(LAMP)方法,进行了特异性和灵敏性试验,并对临床乳样进行初步检测。结果显示,建立的LAMP检测方法特异性良好,与生鲜乳中常见的大肠埃希菌、肺炎克雷伯菌、无乳链球菌、粪肠球菌无交叉反应,灵敏度为10CFU/mL。分别用传统分离鉴定法与LAMP法对50个乳腺炎乳样进行检测,传统方法分离得到S.aureus 14株,LAMP法检测得到S.aureus 14株,符合率100%,阳性检出率均为28%。结果表明,成功建立了生鲜乳中金黄色葡萄球菌的快速检测方法,该方法无需特殊的仪器,有利于在临床和基层进行推广。     

Milk whey induction of agglutination in ovine and bovine mastitis Staphylococcus aureus

A total of 59 mastitis staphylococcic strains were tested for growth agglutination upon supplementation of growth media with ovine and bovine milk whey and mammary secretions from dry cows. Differences were observed when comparing bacterial species or origins (ovine vs. bovine) of bacteria and whey. All of the ovine and bovine S. aureus strains tested, but only 4 among 22 other ovine mastitis staphylococcic strains, showed growth agglutination in Todd Hewitt broth (THB) supplemented with greater than or equal to 30% (v/v) ovine milk whey. None of the strains agglutinated during growth in regular THB medium. Ovine whey had an agglutination induction capacity higher than bovine whey (P less than 0.005), concerning the number of responsive ovine and bovine S. aureus strains. There were no differences between whey samples from different ewes with regard to their capacity to induce agglutination. Ovine S. aureus strains were more responsive than bovine strains of this bacterial species, concerning the number of responsive strains (P less than 0.001) to bovine whey (greater than or equal to 30% in THB), the proportion of responsive strains at low (10%) ovine whey concentration (P less than 0.001), and the strength of reaction (precipitation timing and clump size). Secretions from dry cows systematically induced agglutination in all of the bovine and ovine S. aureus strains tested.     

Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase

Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and GapC proteins share considerable homology to the GapB and GapC products of human strains of S. aureus. These two proteins could be distinguished by their different GAPDH activities and binding to bovine transferrin properties. Both gapB and gapC genes were conserved in 11 strains tested, and the GapC protein was present on the surface of all S. aureus strains.     

Microarray based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle

Staphylococcus aureus is a common pathogen which can colonise and infect not only man, but also domestic animals. Especially, infection of cattle is of high economic relevance as S. aureus is an important causal agent of bovine mastitis. In the present contribution, a DNA microarray was applied for the study of 144 different gene targets, including resistance genes and genes encoding exotoxins, in S. aureus isolated from cows. One hundred and twenty-eight isolates from Germany and Switzerland were tested. These isolates were assigned to 20 different strains and nine clonal complexes. The majority of isolates belonged either to apparently closely related clonal complexes 8, 25, and 97 (together 34.4%) or were related to the sequenced bovine strain RF122 (48.4%). Notable characteristics of S. aureus of bovine origin are the carriage of intact haemolysin beta (in 82% of isolates tested), the absence of staphylokinase (in 89.1%), the presence of allelic variants of several exotoxins such as toxic shock syndrome toxin and enterotoxin N, and the occurrence of the leukocidin lukF-P83/lukM (in 53.1%). Two isolates were methicillin-resistant S. aureus (MRSA). One of them was a clonal complex 8 MRSA related to the epidemic MRSA strain Irish 01. The other one belonged to ST398/spa-type 34 resembling a newly emerging MRSA strain which has been described to occur in humans as well as in domestic animals. The presence of these two strains highlights the possibility of transfers of S. aureus strains between different host species.     

The dynamics of Staphylococcus aureus intramammary infection in nine Danish dairy herds

The aim of the present study was to examine the diversity of Staphylococcus aureus isolates from bovine intramammary infections (IMI) in nine dairy herds, and compare these with isolates from other sites on the cows by phage- and ribotyping. Whether colonisation of milkers with S. aureus could be a source of infection for bovine IMI was investigated. In addition, 100 epidemiologically unrelated S. aureus isolates from asymptomatic human carriers were also phage- and ribotyped to compare the human and bovine reservoir of S. aureus in Denmark. A total of 625 S. aureus isolates from bovine IMI, bovine skin lesions, milking personnel, and non-farm-related human carriers were included in the study. Certain types predominated in one or several herds during the study period of one-and-a-half to two years, whereas the presence of other types was of a more sporadic nature. Within the individual herds, there was a close correspondence between ribo- and phage types of S. aureus isolated from bovine intramammary infections and skin lesions. Isolates from milking personnel, however, were not identical to any of the predominant intramammary strains. Furthermore, several of the isolates from milking personnel showed ribo- and phage patterns identical to S. aureus isolates from human carriers. The findings of the present study underline the importance of strict milking hygiene and improvement of current mastitis therapy. The results support the hypothesis that some S. aureus mastitis strains are more contagious, virulent or persistent than others. The human reservoir of S. aureus does not play a major role as a source of bovine intramammary infections.     

Induction of anti-phagocytic surface properties of Staphylococcus aureus from bovine mastitis by growth in milk whey

The respiratory burst activity of bovine polymorphonuclear (PMN) cells in response to milk whey- and TSB-grown S. aureus strains isolated from bovine mastitis was studied in whole blood chemiluminescence (CL) and in a CL system with purified bovine neutrophils. In both cases milk whey-grown S. aureus strains elicited significantly less CL than homologous strains grown in TSB. Ingestion of milk whey-grown S. aureus strains by bovine neutrophils was also considerably lower than that of the corresponding homologous organisms grown in TSB. Binding of complement factor C3 to serum-opsonized milk whey-grown S. aureus strains was lower compared with TSB-grown homologous organisms. Moreover, 5 of 6 S. aureus strains grown in milk whey were significantly more resistant to in vivo clearance from the peritoneal cavity of mice compared with homologous bacteria grown in TSB. S. aureus strains grown in TSB exhibited hydrophobic surface properties, whereas homologous strains grown in milk whey were hydrophilic.     

Species identification and some characteristics of coagulase-negative staphylococci isolated from bovine udders.

The species of 203 strains of coagulase-negative staphylococci (CNS), isolated from bovine udder quarters was determined; all were tested for hydrophobicity and encapsulation, attributes that may relate to virulence. Twelve species were identified, of which Staphylococcus simulans, (34.5%), S. chromogenes (16.7%), S. epidermidis (13.8%) and S. xylosus (8.9%) were the most frequent. The majority of strains possessed a hydrophilic cell surface. However, strains from two species (S. chromogenes and S. epidermidis) were more hydrophobic than the others. Only five strains were encapsulated (S. xylosus, 3; S. saprophyticus, 1; and S. sciuri, 1). Judging from the low frequencies of hydrophobic and encapsulated strains, and comparing with strains isolated from clinical cases, it is suggested that these properties are not major virulence determinants of CNS.     

甘肃地区牛源金黄色葡萄球菌分子鉴定及RAPD分型

本研究目的是分离鉴定引起甘肃地区奶牛乳房炎的金黄色葡萄球菌,掌握其基因型情况。利用16S、23SrRNA保守序列PCR扩增对乳房炎奶样中的金黄色葡萄球菌进行鉴定,并进行RAPD基因分型。结果表明,310份奶样中共分离出金黄色葡萄球菌100株,RAPD结果显示这100株金黄色葡萄球菌均可得到清晰的RAPD指纹图谱,扩增产物在2～7条带之间,具有多种带型组成。通过聚类分析100株菌产生11个基因型,其中Ⅰ型4株,Ⅱ型4株,Ⅲ型10株,Ⅳ型13株,Ⅴ型7株,Ⅵ型24株,Ⅶ型16株,Ⅷ型6株,Ⅸ型4株,Ⅹ型10株,Ⅺ型2株。Ⅵ型为该地区的流行优势菌群,不同牛场各基因型菌株分布有明显差异。本研究说明牛场的环境与养殖条件对病原菌流行传播有明显的影响,这一结果对地区性奶牛乳房炎的防治提供了可靠的理论依据。     

Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis

In the present study, 188 coagulase-negative Staphylococcus (CNS) strains were isolated from bovine mastitis cases from 56 different Brazilian dairy herds, located in the Southeast region of the country, and were tested for antimicrobial substance production. Twelve CNS strains (6.4%) exhibited antagonistic activity against a Corynebacterium fimi indicator strain. Most antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). Amongst the CNS producers, six were identified as S. epidermidis, two as S. simulans, two as S. saprophyticus, one as S. hominis and one as S. arlettae. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. The Bac(+) strains presented either no or few antibiotic resistance phenotypes. Three strains were shown to produce a bacteriocin either identical or similar to aureocin A70, a bacteriocin previously isolated from an S. aureus strain isolated from food. The remaining Bac(+) strains produce antimicrobial peptides that seem to be distinct from the best characterised staphylococcal bacteriocins described so far. Some of them were able to inhibit Listeria monocytogenes, an important food-borne pathogen, and several strains of Streptococcus agalactiae associated with bovine mastitis, suggesting a potential use of these bacteriocins either in the prevention or in the treatment of streptococcal mastitis.     

牛源耐甲氧西林金黄色葡萄球菌的检测

本研究旨在了解甘肃地区奶牛乳房炎金黄色葡萄球菌的耐药性和耐甲氧西林金黄色葡萄球菌的感染情况,为奶牛乳房炎的防制提供理论依据。采用KB纸片扩散法,检测17株金黄色葡萄球菌对8种不同抗菌药物的敏感性;再用琼脂稀释法检测了苯唑西林、万古霉素对金黄色葡萄球菌的最小抑菌浓度(MICs);头孢西丁纸片扩散法和PCR扩增特异性mecA耐药基因对所有受试菌株进行全面的耐甲氧西林金黄色葡萄球菌检测。结果表明,菌株对青霉素、磺胺异恶唑具有较强抗性,而对环丙沙星、头孢唑啉、万古霉素和苯唑西林全敏感;头孢西丁纸片扩散法未能检测出表型为MRSA的阳性菌株,而PCR方法却检测出8株mecA基因阳性菌株,且这些菌株的苯唑西林MIC均小于2 μg/mL。菌株的耐药情况较严重,对甲氧西林敏感而携带mecA基因的菌株高频存在于被调查地区的奶牛场中。     

Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences

Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens.