科研成果详情

题名肠球菌对磷霉素耐药性、耐药机制及联合用药方案研究
其他题名Antimicrobial resistance, resistance mechanisms and combined antibiotic regimens of fosfomycin in Enterococci
作者
学位类型硕士
导师曹建明
答辩日期2018-06-07
学位授予单位温州医科大学
学位专业临床检验诊断学
关键词磷霉素 肠球菌 fos基因 MurA靶酶
摘要目的1. 研究临床分离肠球菌对磷霉素的耐药性、耐药机制。2. 分析磷霉素耐药肠球菌的流行特征。3. 探讨磷霉素耐药肠球菌的联合治疗方案,为临床上磷霉素耐药肠球菌的防控和治疗提供理论依据。方法1. 采用琼脂稀释法检测温州医科大学附属第一医院2013年~2016年临床分离肠球菌对磷霉素的耐药性,筛选出磷霉素耐药菌株。2. 随机选取同期临床分离的20株磷霉素敏感菌株作为对照,通过PCR检测菌株fosA、fosB、fosC、fosX耐药基因的携带情况;以磷霉素非耐药标准菌株ATCC29212作为对照,检测磷霉素靶酶MurA是否存在氨基酸突变。3. 通过反向PCR检测fosB基因的周围环境。4. 以RT-PCR方法比较磷霉素耐药菌株与敏感菌株中fosX基因的表达差异。5. 采用PFGE和MLST分析耐药菌株的同源性及分子流行特性。6. 采用氨苄西林+庆大霉素、氨苄西林+头孢哌酮两种药物组合进行ST78型磷霉素耐药肠球菌(对磷霉素表现为不同的耐药程度)的时间杀菌试验,初步探讨两种药物联合使用对耐药菌株的体外抗菌活性。结果1. 临床分离肠球菌对磷霉素具有较好的敏感性,总耐药率仅为2.6%(20/761),屎肠球菌的耐药率高于粪肠球菌,分别为4.9%和0.3%;磷霉素耐药肠球菌对临床常用抗菌药物具有较高的耐药性,仅对达福普丁、利奈唑胺敏感性较好;磷霉素耐药肠球菌主要来源于尿液标本,科室分布广泛。2. 20株磷霉素耐药肠球菌均携带fosX基因,其中有 5株fosB基因阳性,未检出fosA、fosC基因。20株同期分离的敏感株中也均检出fosX基因,未检测到其他fos耐药基因。与磷霉素非耐药标准菌株ATCC29212的MurA序列比对,发现有10株耐药肠球菌存在磷霉素靶酶MurA突变,其中SC263、SC272、SC779、SC811、SC941、SC980菌株发生了MurA 的Cys263Arg、Ser281Gly双突变,SC731、SC965发生Ser281Gly单突变,SC737存在Cys263Arg单突变,SC755则存在MurA的多个氨基酸替代。3. 反向PCR表明fosB基因与tnpA转座子位于同一个环形DNA上。4. fosB基因阴性、MurA未发生突变的6株耐药菌株其fosX基因表达量均高于标准菌株ATCC29212,但表达量水平与磷霉素MIC值不成明显的正相关。5. 磷霉素耐药肠球菌的ST型分布以ST78型为主,占13株,ST1094、ST267、ST789、ST555、ST585、ST187各1株,还有1株为新的未分型ST型。PFGE表明部分耐药菌株具有较高的同源性,临床上存在耐药菌株的克隆传播和不同亚型耐药菌株的散在流行。6.时间杀菌曲线显示:氨苄西林+庆大霉素、氨苄西林+头孢哌酮两种药物组合对2株ST78型磷霉素耐药肠球菌均具有协同作用。其中氨苄西林+头孢哌酮药物组合的联合抑制作用更加明显。结论1. 磷霉素对肠球菌具有良好的抗菌活性,4年总耐药率仅为2.6%,屎肠球菌的耐药率高于粪肠球菌;磷霉素耐药肠球菌多呈多重耐药表型。2. 携带fosB基因、靶酶MurA发生氨基酸替换以及fosX基因表达量升高是肠球菌对磷霉素耐药的主要机制。3. 临床上存在ST78型磷霉素耐药肠球菌的克隆传播及其他克隆型菌株的散在流行。4. 氨苄西林+庆大霉素、氨苄西林+头孢哌酮这两种药物组合对磷霉素耐药肠球菌具有协同杀菌作用,其中后者的联合抑制作用更加明显,可为临床上耐药菌株的治疗提供参考依据。
其他摘要Objective1. Toinvestigate the antimicrobial resistance and resistant mechanisms of enterococci collected fromthe First Affiliated Hospital of Wenzhou Medical University from 2013 to 2016.2. Toanalyze the molecular epidemic characteristics of fosfomycin resistant enterococci.3. Toinvestigate the combined antibiotic regimens of fosfomycin resistant enterococci, and toprovide a theoretical basis for prevention and treatment of fosfomycinresistant isolates.Methods 1. Todetect the drug resistance of enterococci collected from the First Affiliated Hospital of Wenzhou Medical University from2013 to 2016 and collect fosfomycin resistant strains using the agar dilutionmethod.2.Fosfomycin resistance genes (fosA, fosB, fosC and fosX) weredetected and characterised by PCR, and 20 randomly selectedfosfomycin-susceptible isolates from the same period served as controls. Aminoacid mutation of target enzyme MurA was detected and compared with fosfomycinnon-resistant standard strains ATCC29212.3.Inverse PCR was used to amplify the flanking sequences of fosB.4.RT-PCR was implemented to compare the difference of genomic expression level of fosX in fosfomycin resistant isolatesand fosfomycin susceptible strain.5. PFGEand MLST were performed to analyze the homology and epidemic characteristics offosfomycin resistant isolates.6. Time-killexperimentswere conducted using ampicillin in combination with gentamycin and ampicillinin combination with cefoperazone, and explore preliminarily the antibacterialactivity in vitro of two combined antibiotic regimens to two resistantenterococci which showdifferent level resistant to fosfomycin.Results 1.Clinical isolates of enterococcus maintained good sensitivity to fosfomycin, with low resistant rates of 2.6%(20/761). The resistance rates of enterococcusfaecium against fosfomycin is higher than that of enterococcus faecalis, being 4.9% and 0.3% respectively. Fosfomycinresistant enterococci exhibithigh resistance rates to clinical commonly used antibiotics, and only showedgood sensitivity to dalfopristin and linezolid. The source of specimens offosfomycin resistant enterococci weremainly urine sample, and the division distribution of resistant isolates wereabroad.2. fosX were detected in all of 20resistant isolates, and fosB gene wasdetected in 5 isolates, no fosA and fosC determined. fosX were detected in all of 20 fosfomycin-susceptible isolates from the sameperiod, and no other fos gene weredetermined. Compared with fosfomycin non-resistant standard strains ATCC29212,amino acid mutation of target enzyme MurA was detected in 10 resistantisolates. Cys263Arg、Ser281Gly mutations existed in SC263, SC272, SC779, SC811, SC941, SC980,Ser281Gly mutation existing in SC731 and SC965, Cys263Arg mutation existing inSC737, multiple amino acids substitutions in MurA existing in SC755.3.Inverse PCR indicated that fosB3 and tnpA genes were located in the identicalcircular DNA intermediate.4. Thegenomic expression levels of fosX in6 resistant isolates which were fosB-negativeand possessed no mution in MurA were higher than that in ATCC 29212, however,the expression level were not proportional to the minimal inhibitoryconcentrations.5. ST11was the predominant clone in fosfomycin resistant enterococci, with 13 isolates belonging to ST11. Oneisolates respectively belong to ST1094, ST267, ST789, ST555, ST585 and ST187,and one isolates belong to new ST type.6. Time-killexperiments indicated that both two combined antibiotic regimens,including ampicillin plus gentamycin and ampicillin plus cefoperazone, showedsynergic action, and combined inhibition of the latter was more obvious.Conclusions 1.Fosfomycin had a strong bacteriostatic activity against enterococci, the total resistant rate of four years was only 2.6%. The resistancerate of enterococcus faecium againstfosfomycin is higher than that of enterococcusfaecalis. Fosfomycin resistant enterococci were mainly multiple resistance phenotype.2.Presence of the fosB gene, amino acidsubstitutions in fosfomycin target enzyme MurA and the high genomic expressionlevel of fosX were the mainmechanisms of fosfomycin resistance.3. Theclone spread of ST78 fosfomycin resistant enterococci and other sporadic STs exist in the clinic.4. Bothtwo combined antibiotic regimens, including ampicillin plus gentamycin andampicillin plus cefoperazone, showed synergic action, and combined inhibitionof the latter was more obvious, providing theoretical basis for the treatmentof resistant isolates.
语种中文
学号151004707
发布年限2021-06-06
毕业论文分类号R446
原始专题检验医学院、生命科学学院
学位论文研究方向微生物学
参考文献

正文参考文献

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全文文件名151004707毕文姿2018临床检验诊断学.pdf|151004707毕文姿2018临床检验诊断学.pdf
文献类型学位论文
条目标识符https://kms.wmu.edu.cn/handle/3ETUA0LF/112619
专题温州医科大学
作者单位
溫州医科大学检验医学院、生命科学学院
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毕文姿. 肠球菌对磷霉素耐药性、耐药机制及联合用药方案研究[D]. 温州医科大学,2018.

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