本文關(guān)鍵詞： 金黃色葡萄球菌 耐藥 分子標(biāo)志 基因分型　出處：《鄭州大學(xué)》2015年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：金黃色葡萄球菌(Staphylococcus aureus)是目前全球范圍內(nèi)引起人類(lèi)感染性疾病的主要病原微生物之一,簡(jiǎn)稱(chēng)金葡菌。耐甲氧西林金黃色葡萄球菌(Methicillin-resistant Staphylococeus aureus,MRSA)的出現(xiàn)是由于甲氧西林敏感金葡菌(Methicillin-sensitive Staphylococcus aureus,MSSA)獲取了葡萄球菌盒式染色體(Staphyloccoccal Cassette Chromosome mec,SCCmec)元件。成簇的規(guī)律間隔的短回文重復(fù)序列(Clustered Regularly Interspaced Short Palindromic Repeat,CRISPR)是最近發(fā)現(xiàn)的針對(duì)噬菌體等外源基因的細(xì)菌免疫系統(tǒng)。目的1.了解分離的金葡菌耐藥性、耐藥基因分布及分子特征。2.了解鄭州地區(qū)分離的金葡菌spa和MLST主要流行型別,揭示該地區(qū)金葡菌分子流行病學(xué)特征。3.了解CRISPR/Cas系統(tǒng)的cas核心基因cas1和cas2的分布情況,初步探索金葡菌cas1和cas2與金葡菌耐藥關(guān)系。方法1.采用最低抑菌濃度(MIC)對(duì)分離的金葡菌進(jìn)行25種藥物敏感試驗(yàn)。2.PCR方法擴(kuò)增介導(dǎo)β-內(nèi)酰胺類(lèi)、氨基糖苷類(lèi)、紅霉素、四環(huán)素等藥物耐藥的11種耐藥基因及cas1,cas2基因,并測(cè)序分析。3.多重PCR方法對(duì)MRSA進(jìn)行SCCmec分型。4.PCR方法進(jìn)行金葡菌spa分型,PCR產(chǎn)物經(jīng)測(cè)序確定spa型別。PCR擴(kuò)增MRSA7個(gè)管家基因,PCR產(chǎn)物經(jīng)測(cè)序后通過(guò)查找數(shù)據(jù)庫(kù)確定ST型別。5.不同菌株耐藥表型、基因型以及cas1,cas2基因分布以百分率描述。采用SPSS21.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。MRSA和MSSA敏感率、耐藥基因的陽(yáng)性率比較以及菌株耐藥表型、耐藥基因型、SCCmec分型與cas1,cas2基因關(guān)聯(lián)性采用χ2檢驗(yàn)或Fisher確切檢驗(yàn)。6.利用DNAstar7.0軟件的Meg Align進(jìn)行序列比對(duì)同源性分析并構(gòu)建系統(tǒng)發(fā)育樹(shù)。結(jié)果1.分離的86株金葡菌中,MRSA共28株,MSSA共58株。青霉素耐藥率最高,為95.35%,耐藥率大于50%的有11種。MRSA菌株對(duì)19種抗菌藥物的耐藥率均高于MSSA菌株。2.對(duì)86株金葡菌菌耐藥基因檢測(cè),acc(6')/aph(2'')、aph(3')-III、ant(4',4")陽(yáng)性率分別為100.00%、72.09%、86.05%;erm A、erm B、erm C、msr A陽(yáng)性率分別為95.35%、79.07%、93.02%、58.14%;tet M、tet K陽(yáng)性率為97.67%、81.40%;未檢測(cè)出blaTEM;3.86株金葡菌spa分型可分為11個(gè)型別,28株MRSA的SCCmec分型共分為3個(gè)型別,MLST分為4個(gè)型別。4.86株金葡菌中有44株cas1陽(yáng)性,30株cas2基因陽(yáng)性。cas1基因在MRSA和MSSA組間以及SCCmec基因型組間差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。15種藥物的耐藥性與cas1基因的陽(yáng)性率有關(guān),2種藥物耐藥性與cas2基因的陽(yáng)性率有關(guān)。cas1基因的陽(yáng)性率與erm C、msr A以及tet K陽(yáng)性率有關(guān)。結(jié)論1.金葡菌對(duì)多種抗生素耐藥率均較高,且耐藥基因陽(yáng)性率高。MRSA耐藥率普遍高于MSSA。2.分離的MRSA主要有SCCmecⅠ型、SCCmecⅢ型和SCCmecⅣE型。SCCmec盒式染色體與MRSA的多重耐藥相關(guān)。3.分離的金葡菌spa基因分型有11個(gè)型別,MSRA MLST分為4個(gè)型別。4.cas1基因以及cas2基因差異與金葡菌耐藥可能有關(guān),cas1基因與MRSA耐藥表型、耐藥基因以及SCCmec分型可能有關(guān)。
[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，

本文編號(hào)：1500422