【作者】 刘连杰；

【导师】 刘晓强；

【作者基本信息】 西北农林科技大学 ， 基础兽医学， 2023， 硕士

【摘要】 金黄色葡萄球菌是奶山羊乳房炎最主要致病菌之一,也是最易生成生物被膜的细菌之一。当前,兽医临床对于奶山羊乳房炎的治疗主要以抗菌药物为主,但长期使用抗菌药物会导致药物残留和细菌耐药性等问题,严重阻碍奶山羊养殖业的健康持续发展。现代药理学研究证明短穗兔耳草具有良好的抗菌活性,并且具备多靶点、毒性和副作用小且不易产生耐药性的天然优势。将短穗兔耳草应用于金黄色葡萄球菌感染性乳房炎的治疗,是抗菌药物替代品研发的新思路。本试验通过药敏试验研究短穗兔耳草提取物对金黄色葡萄球菌的体外抗菌活性。从功能、形态和分子三个层面讨论短穗兔耳草提取物对奶山羊乳房炎金黄色葡萄球菌生物被膜的作用及机制。通过酶解试验分析金黄色葡萄球菌生物被膜胞外聚合物基质中主要组分的含量以及短穗兔耳草提取物对生物被膜胞外蛋白、胞外多糖和胞外eDNA的影响;采用结晶紫染色法分析短穗兔耳草提取物对金黄色葡萄球菌生物被膜的抑制和清除作用;结合扫描电镜和激光共聚焦显微镜观察短穗兔耳草提取物对金黄色葡萄球菌生物被膜形态的作用效果;利用qRT-PCR技术探讨短穗兔耳草提取物对金黄色葡萄球菌生物被膜调控基因icaA和icaD表达的影响。主要研究内容及结果总结如下:1.奶山羊乳房炎金黄色葡萄球菌的分离鉴定和药物敏感性分析本试验结合革兰染色、甘露醇高盐琼脂培养基、金黄色葡萄球菌耐热核酸酶编码基因(nuc)PCR扩增和16S rDNA基因片段测序等手段,从70份患乳房炎奶山羊乳样中共计分离鉴定出28株金黄色葡萄球菌,占致病菌总数的20.44%。分离所得28株金黄色葡萄球菌,对10类共计16种兽医临床常用抗菌药物均呈现不同程度的耐药性,其耐药率介于7.14%～75.00%之间,并且呈现出多重耐药性,多重耐药菌株占比57.14%。其中,7重耐药菌株占比7.14%,5重耐药菌株占比21.43%,4重耐药菌株占比17.86%,3重耐药菌株占比10.71%。结合刚果红平板法和结晶紫染色法发现,分离所得28株金黄色葡萄球菌中有25株具备生物被膜形成能力,其中5株生物被膜形成能力中等,20株生物被膜形成能力较弱。2.短穗兔耳草提取物对金黄色葡萄球菌的体外抗菌活性药敏试验显示,短穗兔耳草提取物对28株金黄色葡萄球菌的MIC介于15.625～62.5 mg/m L之间,与抗菌药物联用对多数金黄色葡萄球菌表现出相加或无关作用,其中与氨苄西林、泰万菌素和头孢噻肟联用对少数金黄色葡萄球菌表现出协同作用,与甲砜霉素和头孢曲松联用对极少数金黄色葡萄球菌表现出拮抗作用。3.短穗兔耳草提取物对金黄色葡萄球菌生物被膜的作用及机制研究酶解试验显示,金黄色葡萄球菌生物被膜胞外聚合物基质中胞外蛋白含量最多,胞外多糖次之,胞外eDNA含量最少,短穗兔耳草提取物对胞外多糖含量的影响最为显著。通过结晶紫染色法显示,4MIC、2MIC、MIC、0.5MIC和0.25MIC等5个梯度浓度的短穗兔耳草提取物对金黄色葡萄球菌生物被膜均具有抑制和清除作用,抑制率介于9.974%～83.256%之间,清除率介于5.461%～66.826%之间。结合扫描电镜和激光共聚焦显微镜观察发现,不同浓度的短穗兔耳草提取物均能显著破坏金黄色葡萄球菌生物被膜的形态结构,有效杀灭膜内细菌,并且呈现浓度依赖性,其中抑制效果明显优于清除效果。qRT-PCR试验结果表明,不同浓度的短穗兔耳草提取物能够显著下调金黄色葡萄球菌生物被膜icaA和icaD基因的相对表达量,icaA基因相对表达量的下调幅度为5.40%～94.59%,icaD基因相对表达量的下调幅度为2.69%～80.84%,其中icaA基因相对表达量的下降幅度更为明显。综上所述,短穗兔耳草提取物对奶山羊乳房炎金黄色葡萄球菌具有良好的抗菌和抗生物被膜活性。能够下调金黄色葡萄球菌生物被膜调控基因icaA和icaD的表达,降低生物被膜胞外聚合物基质的含量,减弱细菌细胞表面疏水性,干预生物被膜形成初始阶段细菌的黏附过程,破坏生物被膜结构和功能的稳定性,从而发挥抗菌和抗生物被膜作用。更多还原

【Abstract】 Staphylococcus aureus(S.aureus)is one of the main pathogens of dairy goat mastitis,and also one of the leading bacteria causing biofilm associated infections.Currently,the clinical treatment of related infections caused by S.aureus is mainly treated by antibacterials which may lead to drug residues and drug resistance,thus seriously hindering the development of dairy goat farming.Modern pharmacological studies showed that Lagotis brachystachya Maxim.(L.brachystachya)has antibacterial effect and rich active components,and it is less liable to produce drug resistance.Natural medicine therapies seem to be an alternative to antibiotic treatment for control of S.aureus infected mastitis.In this study,the antibacterial activity of L.brachystachya on S.aureus was studied by drug sensitivity test in vitro.The antibiofilm activity and the mechanisms of L.brachystachya on the biofilm formation of S.aureus from goat mastitis was investigated from the function,morphology,and molecule levels.Analysis of extracellular polymer components of S.aureus biofilm by enzymatic digestion assay.The effect of L.brachystachya extract on the production of extracellular protein,extracellular polysaccharides and extracellular DNA(eDNA)of S.aureus were evaluated.The crystal violet staining method was used to evaluate the effect of L.brachystachya extract on biofilm inhibition and clearance of S.aureus isolates.CLSM and SEM were used to observe the inhibition and clearance effect of L.brachystachya on S.aureus biofilm.Quantitative real time PCR(qRT-PCR)assay was performed to evaluate the effect of L.brachystachya extract on the expression levels of the biofilm-related genes(icaA and icaD)in S.aureus was evaluated using.The main study contents and the corresponding results are summarized as follows.1.Isolation,identification and antimicrobial susceptibility analysis of S.aureus in dairy goats mastitis.All bacteria were identified by Gram staining,Manitol Salt Agar(MSA),S.aureus thermostable nuclease coding gene(nuc)PCR amplification and 16S rDNA gene sequencing.A total of 28 S.aureus isolates were isolated and identified from 70 milk samples of dairy goat mastitis,accounting for 20.44% of the total pathogenic bacteria.Besides,the isolates S.aureus showed different degrees of resistance to sixteen antimicrobial of ten categories agents with resistance rates ranging from 7.14% to 75.00%,and the multi-drug resistance accounted for 57.15%.Among them,seven-resistant isolates accounted for 7.14%,five-resistant isolates accounted for 21.43%,four-resistant isolates accounted for 17.86%,and three-resistant isolates accounted for 10.71%.The results of Congo red and crystal violet staining revealed that five isolates showed moderate biofilm production ability and twenty isolates showed weak biofilm-forming ability.2.Antimicrobial activity of L.brachystachya extract against S.aureus.The results of the drug sensitivity tests showed that L.brachystachya extract had significant antimicrobial activity with the MIC ranging from 15.625 to 62.5 mg/m L,and the combination with antimicrobials showed additive or irrelevant effects for most of isolates.Combining L.brachystachya extract with ampicillin,oxacillin,ciprofloxacin,and norfloxacin exhibited synergistic effects against several S.aureus isolates,whereas the combination of L.brachystachya extract with thiamphenicol or ceftriaxone showed antagonistic effects.3.Study on the effect and mechanism of extracts from L.brachystachya on the biofilm of S.aureus.The enzymatic digestion assays indicated that extracellular protein was the most of the extracellular polymer content of S.aureus biofilm,followed by extracellular polysaccharides and the least was eDNA.Moreover,L.brachystachya extract showed the most inhibition effect to the polysaccharide.The crystal violet staining results indicated that L.brachystachya extracat had various degrees of biofilm inhibitory and clearance effects at five concentrations,including 4MIC,2MIC,MIC,0.5MIC and 0.25 MIC,with the inhibition and clearance rates ranging from 9.974% to 83.256% and 5.461% to 66.826%,respectively.Similarly,the CLSM and SEM images further revealed that the extract could effectively destroy the structure of biofilm and kill the internal bacteria with a concentration-dependent manner,and the inhibition effect was significantly better than the clearance effect.The results of qRT-PCR showed that different concentrations of L.brachystachya extracts could significantly down-regulated the expression of icaA and icaD genes ranging from 5.40% to 94.59% and 2.69% to 80.84%,respectively.Furthermore,the extracts showed a more supressing effect on the transcription of icaA.In conclusion,L.brachystachya extract effective antibacterial and antibiofilm activity against the S.aureus from dairy goat mastitis.And L.brachystachya extract could down-regulating the expression of S.aureus biofilm-related genes(icaA and icaD),reducing the production of EPS,intervening the inter-adhesive process of isolates in the early stage of biofilm formation,and even clearing the mature biofilms.更多还原