本文選題：人葡萄球菌 + 生物被膜　； 參考：《四川醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的：人葡萄球菌是引起血流感染的重要病原菌,分離率逐年增高,耐藥性強(qiáng)、易產(chǎn)生物被膜,臨床治療十分棘手,引起醫(yī)學(xué)界廣泛關(guān)注。本文旨在通過研究血液來源人葡萄球菌生物被膜形成能力、耐藥性特點(diǎn),分析psm-mec基因與生物被膜形成能力和耐藥性之間的相關(guān)性,為人葡萄球菌感染的防治提供依據(jù)。方法：1.收集血液來源經(jīng)全自動微生物分析系統(tǒng)鑒定的人葡萄球菌。2.采用瓊脂稀釋法檢測血液來源人葡萄球菌對16種抗菌藥物的最低抑菌濃度(Minimal inhibitory concentration,MIC),通過PCR擴(kuò)增mecA基因準(zhǔn)確驗證和區(qū)分耐甲氧西林人葡萄球菌(methicillin-resistant Staphylococcus hominis,MRSHo)和甲氧西林敏感人葡萄球菌(methicillin-susceptive Staphylococcus hominis,MSSHo),比較二者在耐藥譜上的差異。3.半定量粘附實(shí)驗(Microtite Plate Assay,TCP)檢測血液來源人葡萄球菌生物被膜形成能力,分析生物被膜組與非生物被膜組菌株在耐藥譜上的差異。4.PCR初篩攜帶psm-mec基因菌株,擴(kuò)增fudoh基因和DNA測序驗證psm-mec檢測結(jié)果,分析psm-mec基因與生物被膜形成能力和耐藥性之間的相關(guān)性。5.PCR擴(kuò)增mecRl psm-mec與psm-mec/xylR基因間隔序列,探究psm-mec基因在SCCmec上的定位特點(diǎn)。6多重PCR法對攜帶psm-mec基因菌株SCCmec分型,了解其SCCmec的分型情況。7.采用多重PCR分別檢測攜帶psm-mec基因菌株的mec與ccr型別,分析其SCCmec的構(gòu)成特點(diǎn)。結(jié)果：1.收集血液來源人葡萄球菌共55株。2.藥敏試驗顯示,55株血液來源人葡萄球菌對利奈唑胺、呋喃妥因、普丁/達(dá)福、萬古霉素和替加環(huán)素五種抗菌藥物全部敏感,對苯唑西林、青霉素、紅霉素、復(fù)方新諾明、克林霉素、環(huán)丙沙星、左氧氟沙星、莫西沙星、四環(huán)素、利福平和慶大霉素的耐藥率分別為85.45%、92.73%、87.27%、65.45%、67.27%、47.27%、47.27%、34.55%、41.82%、16.36%和10.91%。3.55株血液來源人葡萄球菌中,46(83.64%)株為MRSHo,其中45株mecA基因陽性,1株陰性,其余9(16.36%)株為MSSHo,均未檢出mecA基因。MRSHo對環(huán)丙沙星、左旋氧氟沙星、莫西沙星、青霉素、四環(huán)素和復(fù)方新諾明六種抗菌藥物的耐藥率高于MSSHo,差異具有統(tǒng)計學(xué)意義(P0.05)。4.TCP實(shí)驗顯示,55株血液來源人葡萄球菌中,生物被膜陽性35株,占63.6%,陰性20株,占36.4%。5.生物被膜組細(xì)菌對16種抗菌藥物的耐藥率與非生物被膜組間不存在差異(P0.05)。6. psm-mec基因在MRSHo中的檢出率為39.13%(18/46),在MSSHo中未檢出。18株psm-mec基因陽性的菌株均擴(kuò)增出fudoh,測序結(jié)果經(jīng)Blast比對顯示,均與psm-mec基因編碼序列完全匹配。7.攜帶psm-mec基因的人葡萄球菌產(chǎn)生物被膜率為83.33%(15/18),高于未攜帶psm-mec基因人葡萄球菌54.1%(20/37)的產(chǎn)生物被膜率,差異具有統(tǒng)計學(xué)意義(P0.05)。分析fudoh基因測序結(jié)果發(fā)現(xiàn),3株攜帶psm-mec未產(chǎn)生物被膜的菌株中,2株在psm-mec上游-12處單堿基突變(GA)。攜帶psm-mec基因菌株較未攜帶psm-mec菌株更易對p-內(nèi)酰胺類、四環(huán)素和復(fù)方新諾明耐藥,差異具有統(tǒng)計學(xué)意義(P0.05)。8.所有18株psm-mec陽性菌株,mecRl/psm-mec和psm-mec/xylR兩段基因間隔序列擴(kuò)增均陽性。9.18株攜帶psm-mec基因的SCCmec型別檢測顯示,2(11.1%)株為典型SCCmec Ⅲ型(ⅢA, ⅢB各一株),16(88.9%)株無法分型,其中,4株為類ⅢA型ⅢA-Tn554/orfX),3株為類ⅢB型(2株ⅢB+pls,1株ⅢB-IS431-Pub110),9株為SCCmec新型別。10.攜帶psm-mec基因菌株的mec與ccr型別檢測顯示,18株菌株均為Class A mec; 11株擴(kuò)增出ccr,其中6株為ccr type 1,1株為ccr type 1+2,2株為ccr type 1+3,其余7株未擴(kuò)增出ccr。結(jié)論：1.血液來源人葡萄球菌甲氧西林耐藥率高,耐甲氧西林菌株易同時對環(huán)丙沙星、左旋氧氟沙星、莫西沙星、青霉素、四環(huán)素和復(fù)方新諾明耐藥。2.血液來源人葡萄球菌生物被膜能力形成強(qiáng),生物被膜組細(xì)菌耐藥譜與非生物被膜組間無差異。3.定位于mecRl與xylR間的psm-mec基因廣泛存在血液來源的SCCmecⅢ亞型、變異型和新型別人葡萄球菌中。4.血液來源人葡萄球菌中,攜帶psm-mec基因的SCCmec元件具有多樣性,其組成中,mec保守,均為Class A類,而ccr高度變異。5.在血液來源人葡萄球菌中,攜帶psm-mec基因較未攜帶pSm-mec基因的菌株生物被膜形成能力更強(qiáng),psm-mec基因與生物被膜的形成存在相關(guān)性,同時攜帶psm-mec基因的菌株更易對p-內(nèi)酰胺類、四環(huán)素和復(fù)方新諾明耐藥。
[Abstract]:Objective: Staphylococcus aureus is an important pathogen causing blood flow infection, the separation rate is increasing year by year, the drug resistance is strong and the biofilm is easy to produce. The clinical treatment is very difficult, which has aroused widespread concern in the medical field. The aim of this paper is to study the energy and drug resistance characteristics of the biofilm of human Staphylococcus from the blood, and analyze the psm-mec gene and the biofilm. The correlation between formation ability and drug resistance provides the basis for the prevention and control of staphylococcal infection. Methods: 1. the human staphylococcal.2., identified by the automatic microbiological analysis system, was collected by the agar dilution method to detect the minimum inhibitory concentration of Staphylococcus human Staphylococcus from the blood (Minimal inhibitory concen) by the agar dilution method (Minimal inhibitory concen) Tration, MIC), the mecA gene was amplified by PCR to accurately verify and distinguish between methicillin resistant Staphylococcus (methicillin-resistant Staphylococcus hominis, MRSHo) and methicillin sensitive Staphylococcus (methicillin-susceptive Staphylococcus hominis, MSSHo). Te Plate Assay, TCP) detection of human staphylococcal biofilm formation ability, analysis of the difference between the biofilm group and the non biological membrane group on the resistance spectrum,.4.PCR initially screened by the psm-mec gene strain, amplified fudoh gene and DNA sequencing to verify the psm-mec detection results, and analyzed the psm-mec gene and biofilm formation ability and drug resistance. The correlation between sex and.5.PCR amplification of the mecRl psm-mec and psm-mec/xylR gene interval sequence, and explore the location characteristics of the psm-mec gene on SCCmec,.6 multiplex PCR method for the SCCmec genotyping of the strains of the psm-mec gene. Results: 1. the results were as follows: 1. a total of 55 strains of Staphylococcus from blood derived from Staphylococcus aureus showed that 55 strains of Staphylococcus were all sensitive to linezolid, furanoin, Putin / Dafoe, vancomycin and tegacycline, and benzoxacillin, penicillin, erythromycin, compound novamine, clindamycin, cyclopropane, and cyclosporin. The resistance rates of star, levofloxacin, moxifloxacin, tetracycline, rifampicin, and gentamicin were 85.45%, 92.73%, 87.27%, 65.45%, 67.27%, 47.27%, 47.27%, 34.55%, 41.82%, 16.36% and 10.91%.3.55, 46 (83.64%) strains were MRSHo, among which the 45 strain mecA gene was positive, and the other strains were MSSHo. The resistance rate of the mecA gene.MRSHo to ciprofloxacin, levofloxacin, moxifloxacin, penicillin, tetracycline, and compound neminoxin was higher than that of MSSHo, and the difference was statistically significant (P0.05).4.TCP experiment showed that among the 55 strains of human Staphylococcus, 35 strains of biofilm were positive, 63.6% and 20 negative, accounting for 36.4%.5. birth. There was no difference between the resistance rate of the 16 kinds of antibacterial drugs and the non biological membrane group (P0.05), the detection rate of.6. psm-mec gene in MRSHo was 39.13% (18/46). The strains that did not detect the.18 strain of psm-mec gene in MSSHo all amplified fudoh. The sequencing results showed that the sequences of the psm-mec gene were all matched with the psm-mec gene coding sequence. The biofilm rate of Staphylococcus with.7. carrying psm-mec gene was 83.33% (15/18), which was higher than that of Staphylococcus 54.1% (20/37) without psm-mec gene. The difference was statistically significant (P0.05). The analysis of fudoh gene sequencing results showed that 3 strains with psm-mec did not produce biofilm, 2 were in psm-mec upstream -12. Single base mutation (GA). The strain carrying psm-mec gene was more susceptible to p- lactam, tetracycline and compound Novamin than without psm-mec strain. The difference was statistically significant (P0.05).8. all 18 strains of psm-mec positive strains, mecRl/psm-mec and psm-mec/xylR two segment sequence amplification of both positive.9.18 strains carrying psm-mec gene SCC MEC type detection showed that 2 (11.1%) strains were typical SCCmec III (III A, III B each), 16 (88.9%) could not be classified, of which 4 were class III A type III A-Tn554/orfX), 3 were class III B (2 III B+pls, 1 III B-IS431-Pub110), 9 strains were SCCmec NEW.10. carrying psm-mec gene strains and 18 strains were detected. SS A MEC; 11 strains amplified CCR, of which 6 strains were CCR type 1,1 strains for CCR type 1+2,2 strain for CCR type, and the other 7 strains did not amplify the conclusion: 1. blood derived from Staphylococcus methoxicillin resistance rate is high, methicillin resistant strains are easily simultaneous to ciprofloxacin, levofloxacin, moxifloxacin, penicillin, tetracycline and compound new Nuo Mingnai. The.2. blood source of Staphylococcus human biofilm formation is strong. The psm-mec gene that is located between mecRl and xylR between the biological membrane group and the non biological membrane group.3. exists widely in the SCCmec III subtype of the blood source, the variant type and the new type of Staphylococcus in the Staphylococcus, which carries the psm-mec base in the.4. blood of human Staphylococcus. Because of the diversity of SCCmec components, MEC is conserved in its composition, all of which are Class a, and CCR highly variant.5. has a stronger ability to carry the psm-mec gene than that of the strain that does not carry the pSm-mec gene in the blood source Staphylococcus, and the psm-mec gene is related to the formation of the biofilm and carries the bacteria of the psm-mec gene at the same time. The strain is more resistant to p- lactams, tetracycline and compound sulfamethoxazole.

【學(xué)位授予單位】：四川醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5

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