生物膜对耐亚胺培南鲍曼不动杆菌中苯唑西林酶和AdeABC外排泵基因表达的影响

发布时间：2018-03-15 09:40

本文选题：鲍曼不动杆菌　切入点：亚胺培南　出处：《宁夏医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的1、通过对OXA、Ade ABC、Car O基因及生物膜形成在临床分离的亚胺培南耐药和敏感鲍曼不动杆菌中的分布特性的研究,进一步探讨鲍曼不动杆菌对亚胺培南的耐药机制。2、通过生物膜对苯唑西林酶(OXA)和Ade ABC外排泵基因表达的影响研究,揭示生物膜的形成对OXA和Ade ABC外排泵相关耐药机制的影响,探讨生物膜在鲍曼不动杆菌对亚胺培南耐药方面的作用机制及作用方式。方法运用多重PCR及PCR技术对2014年11月-2015年4月宁夏医科大学总医院临床分离并经鉴定和确认的106株亚胺培南耐药和102株亚胺培南敏感的鲍曼不动杆菌分别进行OXA、Ade ABC、Car O基因的检测;采用微量滴定板法模拟生物膜在体内的生长制备生物膜模型及定量实验;实时荧光定量PCR技术对不同状态下OXA-23、Ade B基因表达量进行检测;E-test方法对产膜与非产膜菌株亚胺培南的最低抑菌浓度(MIC)进行测定。结果1、OXA-23在亚胺培南耐药和敏感菌株中的检出率分别为99.1%和2.9%,经卡方检验P0.05;在所有检测菌株中OXA-51基因均被检出,而OXA-58基因均未被检出;OXA-24基因仅在一株耐药菌株中检出。相反,Ade ABC基因在耐药和敏感菌株分布具有较大差异,Ade A、Ade B、Ade C基因及三个基因共存在耐药菌株中的检出率均为98.1%(104/106),而敏感菌株中Ade A、Ade B、Ade C的检出率分别为64.7%(66/102)、82.4%(84/102)、66.7%(68/102),三个基因共存的检出率仅为64.7%(66/102),P0.05。Car O基因的检出率较高,分别为97.2%(103/106)和99.0%(101/102),P0.05。产生物膜菌株在亚胺培南耐药和敏感菌株中均具有较高的比重,分别为67.9%和75.5%,P0.05。2、106株亚胺培南耐药鲍曼不动杆菌中产生物膜和不产生物膜菌株分别占67.9%和32.1%;生物膜状态下的菌株OXA-23、Ade B基因的相对表达量明显高于普通肉汤培养和浮游状态下基因的相对表达量(P0.05);产膜比非产膜菌株亚胺培南MIC更高,P0.05结论鲍曼不动杆菌的生物膜形成能够促进OXA-23和Ade ABC外排泵基因的表达,从而增强鲍曼不动杆菌对亚胺培南的耐药性。结论1、OXA-51基因是所有亚胺培南耐药和敏感菌株共有的基因,OXA-23基因是除OXA-51外最流行的基因,是该医院耐亚胺培南鲍曼不动杆菌的最主要OXA亚型,提示其在鲍曼不动杆菌对碳青霉烯类抗生素耐药机制中起到了重要的作用;Ade ABC基因在耐药和敏感菌株分布具有较大差异,Ade ABC外排泵在降低鲍曼不动杆菌对亚胺培南的敏感性,增强细菌耐药性方面起着重要作用;Car O基因几乎在全部菌株中被发现,外膜蛋白Car O的改变对于降低鲍曼不动杆菌对亚胺培南的敏感性的可能性不大;临床鲍曼不动杆菌所引发的的感染多数与生物膜的形成有关。2、鲍曼不动杆菌的生物膜形成能够促进OXA-23和Ade ABC外排泵基因的表达,从而增强鲍曼不动杆菌对亚胺培南的耐药性。
[Abstract]:Objective 1. To study the distribution of OXA Ade ABCCAR O gene and biofilm formation in clinical isolates of imipenem resistant and acinetobacter baumannii. In order to explore the drug resistance mechanism of Acinetobacter baumannii to imipenem, the effect of biofilm on the gene expression of oxa and Ade ABC efflux pump was studied to reveal the effect of biofilm formation on the mechanism of drug resistance related to OXA and Ade ABC efflux pump. To explore the mechanism and mechanism of biofilm on the resistance of Acinetobacter baumannii to imipenem. Methods Multiplex PCR and PCR techniques were used to isolate clinical isolates from November 2014 to April 2015 in the General Hospital of Ningxia Medical University. 106 strains of imipenem resistant and 102 strains of Acinetobacter baumannii which were sensitive to imipenem were detected for the gene of Oxa Ade ABCCARO. The biofilm model and quantitative experiment were established by microtitration plate method to simulate the growth of biofilm in vivo. The expression of OXA-23Ade B gene was detected by real-time fluorescence quantitative PCR. The minimum inhibitory concentration of imipenem was determined by E-test method. Results 1 OXA-23 was found to be resistant to imipenem and sensitive to imipenem. The detection rates of OXA-51 gene were 99.1% and 2.9, respectively, and were detected by Chi-square test (P0.05), and the OXA-51 gene was detected in all the tested strains. However, OXA-58 gene was not detected in only one drug-resistant strain. On the contrary, the distribution of Ade ABC gene in resistant and sensitive strains was significantly different. For 98.1 / 104 / 106, the detectable rate of Ade Amo-Ade Ade Ade C in sensitive strains was 64.7 / 102and 82.44 / 102respectively. The positive rate of the coexistence of the three genes was only 64.766 / 102P0.05.Car O. The biofilm producing strains have a high proportion in imipenem resistance and sensitive strains. The percentage of biofilm-producing and non-biofilm-producing strains in acinetobacter baumannii was 67.9% and 32.1respectively, and the relative expression of OXA-23Ade B gene in biofilm was significantly higher than that in broth culture and phytoplankton. The relative expression of P0.05G gene was higher than that of imipenem MIC. Conclusion the biofilm formation of Acinetobacter baumannii can promote the expression of OXA-23 and Ade ABC efflux pump genes. Conclusion the OXA-51 gene is the most popular gene except OXA-51, which is common to all imipenem resistant and sensitive strains, and can enhance the resistance of Acinetobacter baumannii to imipenem. Is the main OXA subtype of Acinetobacter baumannii resistant to imipenem in this hospital. The results suggest that Ade ABC gene plays an important role in the resistance mechanism of Acinetobacter baumannii to carbapenem antibiotics. The Ade ABC efflux pump can reduce the susceptibility of Acinetobacter baumannii to imipenem. The enhancement of bacterial resistance plays an important role in the bacterial resistance. The change of outer membrane protein Car O is not likely to decrease the sensitivity of Acinetobacter baumannii to imipenem. Most of the infections caused by Acinetobacter baumannii are related to biofilm formation. The biofilm formation of Acinetobacter baumannii can promote the expression of OXA-23 and Ade ABC efflux pump genes and enhance the resistance of Acinetobacter baumannii to imipenem.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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