金黄色葡萄球菌分子特征及耐药相关分子标志

发布时间：2018-02-10 11:34

  本文关键词： 金黄色葡萄球菌 耐药 分子标志 基因分型　出处：《郑州大学》2015年硕士论文　论文类型：学位论文

【摘要】：金黄色葡萄球菌(Staphylococcus aureus)是目前全球范围内引起人类感染性疾病的主要病原微生物之一,简称金葡菌。耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococeus aureus,MRSA)的出现是由于甲氧西林敏感金葡菌(Methicillin-sensitive Staphylococcus aureus,MSSA)获取了葡萄球菌盒式染色体(Staphyloccoccal Cassette Chromosome mec,SCCmec)元件。成簇的规律间隔的短回文重复序列(Clustered Regularly Interspaced Short Palindromic Repeat,CRISPR)是最近发现的针对噬菌体等外源基因的细菌免疫系统。目的1.了解分离的金葡菌耐药性、耐药基因分布及分子特征。2.了解郑州地区分离的金葡菌spa和MLST主要流行型别,揭示该地区金葡菌分子流行病学特征。3.了解CRISPR/Cas系统的cas核心基因cas1和cas2的分布情况,初步探索金葡菌cas1和cas2与金葡菌耐药关系。方法1.采用最低抑菌浓度(MIC)对分离的金葡菌进行25种药物敏感试验。2.PCR方法扩增介导β-内酰胺类、氨基糖苷类、红霉素、四环素等药物耐药的11种耐药基因及cas1,cas2基因,并测序分析。3.多重PCR方法对MRSA进行SCCmec分型。4.PCR方法进行金葡菌spa分型,PCR产物经测序确定spa型别。PCR扩增MRSA7个管家基因,PCR产物经测序后通过查找数据库确定ST型别。5.不同菌株耐药表型、基因型以及cas1,cas2基因分布以百分率描述。采用SPSS21.0软件进行统计学分析。MRSA和MSSA敏感率、耐药基因的阳性率比较以及菌株耐药表型、耐药基因型、SCCmec分型与cas1,cas2基因关联性采用χ2检验或Fisher确切检验。6.利用DNAstar7.0软件的Meg Align进行序列比对同源性分析并构建系统发育树。结果1.分离的86株金葡菌中,MRSA共28株,MSSA共58株。青霉素耐药率最高,为95.35%,耐药率大于50%的有11种。MRSA菌株对19种抗菌药物的耐药率均高于MSSA菌株。2.对86株金葡菌菌耐药基因检测,acc(6')/aph(2'')、aph(3')-III、ant(4',4")阳性率分别为100.00%、72.09%、86.05%;erm A、erm B、erm C、msr A阳性率分别为95.35%、79.07%、93.02%、58.14%;tet M、tet K阳性率为97.67%、81.40%;未检测出blaTEM;3.86株金葡菌spa分型可分为11个型别,28株MRSA的SCCmec分型共分为3个型别,MLST分为4个型别。4.86株金葡菌中有44株cas1阳性,30株cas2基因阳性。cas1基因在MRSA和MSSA组间以及SCCmec基因型组间差异具有统计学意义(P0.05)。15种药物的耐药性与cas1基因的阳性率有关,2种药物耐药性与cas2基因的阳性率有关。cas1基因的阳性率与erm C、msr A以及tet K阳性率有关。结论1.金葡菌对多种抗生素耐药率均较高,且耐药基因阳性率高。MRSA耐药率普遍高于MSSA。2.分离的MRSA主要有SCCmecⅠ型、SCCmecⅢ型和SCCmecⅣE型。SCCmec盒式染色体与MRSA的多重耐药相关。3.分离的金葡菌spa基因分型有11个型别,MSRA MLST分为4个型别。4.cas1基因以及cas2基因差异与金葡菌耐药可能有关,cas1基因与MRSA耐药表型、耐药基因以及SCCmec分型可能有关。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus) is one of the major pathogenic microorganisms that cause human infectious diseases worldwide. Staphylococcus aureus. Methicillin-resistant Staphylococeus aureus (Staphylococeus aureus) was found because Methicillin-sensitive Staphylococcus aureus (MSSAs) obtained Staphyloccoccal Cassette Chromosome meccscmec. short palindromes with regular intervals in clusters. Complex Regularly Interspaced Short Palindromic Repeatr is a recently discovered bacterial immune system targeting foreign genes such as phage. Objective 1. To understand the drug resistance of isolated Staphylococcus aureus. The distribution and molecular characteristics of drug resistance genes. 2. To understand the main epidemic types of spa and MLST isolated in Zhengzhou area, and to reveal the molecular epidemiological characteristics of Staphylococcus aureus in this area. 3. To understand the distribution of cas core genes cas1 and cas2 in CRISPR/Cas system. To explore the relationship between cas1 and cas2 and drug resistance of Staphylococcus aureus methods 1. 25 drug sensitive tests were carried out on isolated Staphylococcus aureus with minimal inhibitory concentration (MIC). 2. PCR was used to amplify 尾-lactams, aminoglycosides and erythromycin. Tetracycline and other drug resistance genes of 11 kinds of drug resistance and cas1 cas2 gene, Multiplex PCR method for SCCmec typing of MRSA .4.PCR method for spa typing of Staphylococcus aureus by sequencing to determine the spa type. PCR amplification of MRSA7 housekeeping gene products after sequencing, after searching the database to determine the St type. 5. Drug resistance phenotype of the same strain, SPSS21.0 software was used to analyze the susceptibility rate of. MRSA and MSSA, the positive rate of drug resistance gene and the phenotype of drug resistance of the strain were compared. The correlation between SCCmec genotyping and cas1 cas2 gene was analyzed by 蠂 2 test or Fisher exact test .6.The homology of sequence alignment was analyzed by Meg Align of DNAstar7.0 software and phylogenetic tree was constructed. 1. 86 isolates of Staphylococcus aureus were isolated from Staphylococcus aureus. There were 58 strains of MSSA. Penicillin resistance rate was the highest. The resistance rate of 11 strains with resistance rate greater than 50% to 19 antimicrobial agents was higher than that of MSSA strain .2.The positive rates of 86 strains of Staphylococcus aureus resistance gene were 100.0072.090.09 and 86.05erm Berm Berm CMSR A, respectively. The SCCmec genotyping, which can be divided into 11 types and 28 strains of MRSA, can be divided into three types. The SCCmec typing is divided into 4 types. 44 of the 44 cas1 positive strains of Staphylococcus aureus are cas1 positive and 30 of cas2 gene positive. Cas1 gene is between MRSA and MSSA groups. And the difference of SCCmec genotypes was statistically significant. The drug resistance of 15 drugs was related to the positive rate of cas1 gene. The drug resistance of two drugs was related to the positive rate of cas2 gene, the positive rate of .cas1 gene and the positive rate of erm CSR A and tet K were related to the drug resistance of two drugs and the positive rate of cas2 gene. Conclusion: 1. Staphylococcus aureus is resistant to many kinds of antibiotics. The positive rate of drug resistance gene was higher. MRSA resistance rate was generally higher than that of MSSA.2.The isolated MRSA mainly consisted of SCCmec type 鈪，

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