SCCmec伴随基因psm-mec与血液来源人葡萄球菌生物被膜形成能力及耐药性的相关性研究

发布时间：2018-05-04 19:13

本文选题：人葡萄球菌 + 生物被膜　；参考：《四川医科大学》2015年硕士论文

【摘要】：目的：人葡萄球菌是引起血流感染的重要病原菌,分离率逐年增高,耐药性强、易产生物被膜,临床治疗十分棘手,引起医学界广泛关注。本文旨在通过研究血液来源人葡萄球菌生物被膜形成能力、耐药性特点,分析psm-mec基因与生物被膜形成能力和耐药性之间的相关性,为人葡萄球菌感染的防治提供依据。方法：1.收集血液来源经全自动微生物分析系统鉴定的人葡萄球菌。2.采用琼脂稀释法检测血液来源人葡萄球菌对16种抗菌药物的最低抑菌浓度(Minimal inhibitory concentration,MIC),通过PCR扩增mecA基因准确验证和区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),比较二者在耐药谱上的差异。3.半定量粘附实验(Microtite Plate Assay,TCP)检测血液来源人葡萄球菌生物被膜形成能力,分析生物被膜组与非生物被膜组菌株在耐药谱上的差异。4.PCR初筛携带psm-mec基因菌株,扩增fudoh基因和DNA测序验证psm-mec检测结果,分析psm-mec基因与生物被膜形成能力和耐药性之间的相关性。5.PCR扩增mecRl psm-mec与psm-mec/xylR基因间隔序列,探究psm-mec基因在SCCmec上的定位特点。6多重PCR法对携带psm-mec基因菌株SCCmec分型,了解其SCCmec的分型情况。7.采用多重PCR分别检测携带psm-mec基因菌株的mec与ccr型别,分析其SCCmec的构成特点。结果：1.收集血液来源人葡萄球菌共55株。2.药敏试验显示,55株血液来源人葡萄球菌对利奈唑胺、呋喃妥因、普丁/达福、万古霉素和替加环素五种抗菌药物全部敏感,对苯唑西林、青霉素、红霉素、复方新诺明、克林霉素、环丙沙星、左氧氟沙星、莫西沙星、四环素、利福平和庆大霉素的耐药率分别为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株血液来源人葡萄球菌中,46(83.64%)株为MRSHo,其中45株mecA基因阳性,1株阴性,其余9(16.36%)株为MSSHo,均未检出mecA基因。MRSHo对环丙沙星、左旋氧氟沙星、莫西沙星、青霉素、四环素和复方新诺明六种抗菌药物的耐药率高于MSSHo,差异具有统计学意义(P0.05)。4.TCP实验显示,55株血液来源人葡萄球菌中,生物被膜阳性35株,占63.6%,阴性20株,占36.4%。5.生物被膜组细菌对16种抗菌药物的耐药率与非生物被膜组间不存在差异(P0.05)。6. psm-mec基因在MRSHo中的检出率为39.13%(18/46),在MSSHo中未检出。18株psm-mec基因阳性的菌株均扩增出fudoh,测序结果经Blast比对显示,均与psm-mec基因编码序列完全匹配。7.携带psm-mec基因的人葡萄球菌产生物被膜率为83.33%(15/18),高于未携带psm-mec基因人葡萄球菌54.1%(20/37)的产生物被膜率,差异具有统计学意义(P0.05)。分析fudoh基因测序结果发现,3株携带psm-mec未产生物被膜的菌株中,2株在psm-mec上游-12处单碱基突变(GA)。携带psm-mec基因菌株较未携带psm-mec菌株更易对p-内酰胺类、四环素和复方新诺明耐药,差异具有统计学意义(P0.05)。8.所有18株psm-mec阳性菌株,mecRl/psm-mec和psm-mec/xylR两段基因间隔序列扩增均阳性。9.18株携带psm-mec基因的SCCmec型别检测显示,2(11.1%)株为典型SCCmec Ⅲ型(ⅢA, ⅢB各一株),16(88.9%)株无法分型,其中,4株为类ⅢA型ⅢA-Tn554/orfX),3株为类ⅢB型(2株ⅢB+pls,1株ⅢB-IS431-Pub110),9株为SCCmec新型别。10.携带psm-mec基因菌株的mec与ccr型别检测显示,18株菌株均为Class A mec; 11株扩增出ccr,其中6株为ccr type 1,1株为ccr type 1+2,2株为ccr type 1+3,其余7株未扩增出ccr。结论：1.血液来源人葡萄球菌甲氧西林耐药率高,耐甲氧西林菌株易同时对环丙沙星、左旋氧氟沙星、莫西沙星、青霉素、四环素和复方新诺明耐药。2.血液来源人葡萄球菌生物被膜能力形成强,生物被膜组细菌耐药谱与非生物被膜组间无差异。3.定位于mecRl与xylR间的psm-mec基因广泛存在血液来源的SCCmecⅢ亚型、变异型和新型别人葡萄球菌中。4.血液来源人葡萄球菌中,携带psm-mec基因的SCCmec元件具有多样性,其组成中,mec保守,均为Class A类,而ccr高度变异。5.在血液来源人葡萄球菌中,携带psm-mec基因较未携带pSm-mec基因的菌株生物被膜形成能力更强,psm-mec基因与生物被膜的形成存在相关性,同时携带psm-mec基因的菌株更易对p-内酰胺类、四环素和复方新诺明耐药。
[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.

【学位授予单位】：四川医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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