厦门部分地区医院感染鲍曼不动杆菌耐药性及基因分型研究

发布时间：2018-06-28 21:13

本文选题：医院感染 + 鲍曼不动杆菌　；参考：《安徽医科大学》2012年硕士论文

【摘要】：研究目的和内容 1.了解厦门部分地区医院感染病原体鲍曼不动杆菌(BA)的耐药情况,掌握耐药谱随时间变化趋势,为临床选择适当的抗生素提供依据。 2.探索鲍曼不动杆菌的脉冲场凝胶电泳条件,了解医院感染病原体以及分离株的基因多态性,闸明其菌株间的遗传亲缘关系。 3.结合流行病学资料分析鲍曼不动杆菌基因图谱的聚类分析结果,鉴别优势克隆菌株的时间、地区以及人群的三间分布,判断爆发菌株和散发菌株。 4.分析广泛耐药鲍曼不动杆菌常见耐药基因的携带情况。研究方法 1.菌株的收集方法：2009年1月-2011年12月,收集厦门部分地区三级医院住院的医院感染患者分离的鲍曼不动杆菌,标本来源于医院住院患者的痰、血液、尿液、粪便和脓液等样本,所有菌株经西门子Walkaway96鉴定定药敏仪(美国,德灵)鉴定,并剔除同一患者相同样本重复菌株。 2.药物敏感性测定方法：按美国临床及实验室标准协会推荐标准,采用纸片扩散法对所有收集的菌株进行药物敏感性试验。 3.基因分型方法：建立适合鲍曼不动杆菌的脉冲场凝胶电泳分型的实验室条件,对所有收集的菌株进行PFGE基因分型,采用BionumericSV4.0软件中的非加权配对算数平均法进行聚类分析,分析菌株间的亲缘关系。 4.广泛耐药菌株耐药基因的检测：PCR扩增法检测多重耐药鲍曼不动杆菌分离株中的超广谱β-内酰胺酶、金属β-内酰胺酶等常见耐药基因。研究结果 1.厦门部分地区2009-2011年医院感染BA的药物敏感性结果 1.1BA药敏的总体情况本研究共收集到386株医院感染鲍曼不动杆菌,对20种常用抗生素(青霉素类及加酶抑制剂类、头孢菌素类及加酶抑制剂类、头霉素类、氨基糖苷类、氟喹诺酮类、磺胺类、多粘菌素类、四环素类)均表现出不同程度的耐药。从高到低依次如下：氨曲南(87.05%)、哌拉西林(77.72%)、头孢吡肟(77.72%)、头孢噻肟(72.28%)、厄他培南(71.50%)、替卡西林/克拉维酸(69.17%)、复方新诺明(68.91%)、左氧氟沙星(67.62%)、妥布霉素(67.62%)、环丙沙星(67.36%)、头孢他啶(66.32%)、庆大霉素(65.54%)、阿莫西林/克拉维酸(64.51%)、美罗培南(63.73%)、哑胺培南(63.47%)、阿米卡星(57.77%)、头孢哌酮/舒巴坦(52.85%)、多粘菌素(41.19%),米诺环素(0.00%)；没有发现泛耐药及全敏感菌株；分离菌株对常见p-内酰胺类抗生素及加酶抑制剂、氨基糖苷类、喹诺酮类抗生素、磺胺类耐药较为严重,均超过50%,有的接近90%；对碳青霉烯类抗生素也呈现较高的耐药性,耐药率均超过60%；多粘菌素耐药率低于50%,米诺环素耐药率为0.00%。 1.2BA药敏的时间变化趋势厦门部分地区分离的BA耐药情况较为严重,3年间,厦门部分地区分离的鲍曼不动杆菌对于多数常见的抗生素的耐药性呈现逐年升高的趋势,尤其以亚胺培南、美罗培南、厄他培南和多粘菌素增长较为明显。 1.3多重耐药及泛耐药鲍曼不动杆菌分离株的检出情况 386株鲍曼不动杆菌中检出多重耐药菌株118株,广泛耐药菌株97株,检出率分别为30.57%和25.13%。 2.医院感染鲍曼不动杆菌基因分型结果 2.1PFGE应用于BA的几个技术关键点：0D值为4.5, Spa I20U酶切3h以上,脉冲角度为120度,电场强度为6V/cm,电泳20h。在此条件下90%以上的鲍曼不动杆菌的图潜条带数目、大小、亮度及长度均非常合适。 2.2在使用相同的试剂、仪器和参数的前提下,386株菌经限制性内切酶酶切后进行PFGE电泳,每株约出现21-33个电泳条带,分子量在30kb-800kb之间。经BionumericSV4.0软件进行聚类分析,相似值在45.9%-100%之间,可分为242个型(相似值为100%视为同一PFGE型别)； 2.3最常见的基因型有40株菌,20,09年12株,2010年15株,2011年13株。 2.497株广泛耐药鲍曼不动杆菌的基因分型 97株广泛耐药的鲍曼不动杆菌杆菌中经BionumericSV4.0软件进行聚类分析,相似值在67.9%-100%之间,可分为67个型。 3.广泛耐药鲍曼不动杆菌分离株的耐药基因的研究 97株泛耐药鲍曼不动杆菌检出TEM基因阳性23例,检出率为23.71%；CTX-M-13基因阳性检出17例,检出率为17.53%。97株泛耐药鲍曼不动杆菌检出VIM-2阳性菌株30例,检出率为30.93%；IMP-1阳性菌株39例,检出率为40.20%；OXA-58阳性菌株10例,检出率为10.31%；OXA-51阳性菌株80例,检出率为82.47%；OXA-23阳性菌株85例,检出率为87.63%；OXA-24阳性11株,检出率为11.34%。没有检出NDM-1基因。研究结论 1、厦门部分地区2009年到2011年鲍曼不动杆菌主要分离自呼吸系统标本,科室分布主要是重症监护病房和呼吸内科,分离患者人群集中在45岁以上人群。 2、对厦门部分地区2009年到2011年分离的医院感染鲍曼不动杆菌的耐药性调查表明：386株分离菌的对20种常用抗生素均表现出不同程度的耐药性,对常见的p-内酰胺类抗生素及加酶抑制剂、氨基糖苷类、喹诺酮类抗生素、磺胺类耐药较为严重,均超过50%,有的接近90%；对碳青霉烯类抗生素也呈现较高的耐药性,耐药率均超过60%；多粘菌素耐药率低于50%,米诺环素耐药率为0.00%。3年间,厦门部分地区分离的鲍曼不动杆菌对于多数常见的抗生素的耐药性呈现逐年升高的趋势,尤其以亚胺培南、美罗培南、厄他培南和多粘菌素增长较为明显。 3、PFGE是一种稳定、精确的基因分型方法,适合作为院内感染的同源性分析。 4、349株的PFGE进行基因分型,结果显示厦门部分地区存在242种基因型的流行,在多家医院病房中不存在优势克隆株；厦门部分地区2009年到2011年未出现过大规模的爆发流行,ICU病房出现频率高,交叉感染出现于不同医院之间；另外,不同医院之间出现基因型完全一致菌株,提示及时的流行病学调查的重要性。 5、厦门部分地区泛耐药鲍曼不动杆菌携带的耐药基因包括ESBLs耐药基因组中的TEM和CTX-M-13亚型和碳青霉烯酶中的OXA-23, OXA-24, OXA-51, OXA-58和VIM-2、IMP-1,主要以OXA-23型和OXA-51型碳青霉烯酶为主。
[Abstract]:Purpose and content of the study

1 . To understand the drug resistance of acinetobacter baumanii ( BA ) infected by nosocomial infection in some parts of Xiamen , and to grasp the trend of drug resistance spectrum over time and provide the basis for clinical selection of appropriate antibiotics .

2 . To explore the genetic polymorphism of the pathogen of nosocomial infection and the genetic polymorphism of isolated strain , and to explore the genetic relationship among the isolates .

3 . Based on the results of clustering analysis , the time , region and three - compartment distribution of the dominant clonal strains were identified , and the outbreak strains and sporadic strains were identified .

4 . To analyze the carrying situation of the common drug resistant gene of broad - resistant acinetobacter baumanii .

Research Methods

1 . Collection method of bacterial strain : From January 2009 to December 2011 , the patient was collected from hospital infected patients at three levels of hospital in Xiamen , and the samples were collected from sputum , blood , urine , feces and pus samples from the hospital in hospital . All the strains were identified by Siemens Walkaway96 ( United States , Germany ) and the same sample repeated strains of the same patient were excluded .

2 . Method for measuring drug sensitivity : According to the recommendations of the American Society for Clinical and Laboratory Standards , all collected strains were tested for drug sensitivity using sheet diffusion method .

3 . genotyping method : establishing a laboratory condition suitable for a pulsed field gel electrophoresis typing of baumanii , performing PFGE genotyping on all collected strains , performing cluster analysis by adopting a non - weighted pair arithmetic mean method in the BionumericSV4.0 software , and analyzing the relationship among strains .

4 . Detection of resistance gene of broad - resistant strains : PCR amplification method is used to detect the common drug - resistant genes such as ultra - wide spectrum 尾 - lactamase , 尾 - lactamase and other common drug resistance genes .

Results of the study

1 . Results of drug sensitivity of BA for nosocomial infection in some parts of Xiamen from 2009 to 2011

1 . A total of 386 nosocomial infections were collected from a total of 386 hospitals in this study . There were 20 common antibiotics ( penicillin and enzyme inhibitors , cephem , aminoglycosides , fluoroquinolone , sulfanime , polymyxin and tetracycline ) . The results showed that there were no significant differences in resistance to antibiotics ( 87.05 % ) , piperacillin ( 77.72 % ) , cefpime ( 66.32 % ) , gentamicin ( 65.54 % ) , amoxicillin / clavianic acid ( 64.51 % ) , meropenem ( 63.73 % ) , dumb - amine penem ( 63.47 % ) , amikaine ( 57.77 % ) , cefperazone / Sulptan ( 52.85 % ) . Polymyxin ( 41.19 % ) , Minoryclines ( 0.00 % ) ;
No drug resistance and all - sensitive strains were found .
The isolates were more resistant to common p - lactam antibiotics and enzyme inhibitors , aminoglycosides , quinolinone antibiotics and sulfanilamide , more than 50 % and nearly 90 % .
The drug resistance of enem antibiotics was higher than that of 60 % .
The resistance rate of polymyxin was lower than 50 % , and the drug resistance rate was 0.00 % .

1.2 Time variation trend of drug sensitivity of 2BA

5 . The drug - resistant genes carried by Pan - drug - resistant acinetobacter baumanii in some parts of Xiamen include TEM and CTX - M - 13 subtype and OXA - 23 , OXA - 24 , OXA - 51 , OXA - 58 and vim - 2 , IMP - 1 , which are mainly OXA - 23 and OXA - 51 .

1.3 Detection of multi - drug resistant and pan - resistant acinetobacter baumanii isolates

Among 386 strains of acinetobacter baumanii , 118 strains of resistant strains and 97 strains of resistant strains were detected , and the detection rates were 30.57 % and 25.13 % , respectively .

2 . Results of the genotyping results of M . baumanii in nosocomial infection

2.1 PFGE is applied to several technical key points of BA : the D value is 4.5 , the Spa I20U enzyme is cut for more than 3h , the pulse angle is 120 degrees , the electric field intensity is 6 V / cm , and the electrophoresis is 20 hours . Under this condition , the number , the size , the brightness and the length of the gram latent bands of the acinetobacter baumanii are very suitable .

2.2 On the premise of using the same reagents , instruments and parameters , 386 strains were subjected to restriction enzyme digestion and PFGE electrophoresis was carried out . The molecular weight was between 30 kb and 800 kb . Cluster analysis was performed by BionumericSV4.0 software . The similarity values were between 45.9 % -100 % , and the similarity values were between 45.9 % -100 % .

2.3 The most common genotypes were 40 strains , 12 strains in 20 and 09 , 15 in 2010 and 13 in 2011 .

Gene typing of 2.497 strains of broad - resistant acinetobacter baumanii

Cluster analysis was performed by BionumericSV4.0 software in 97 broad - resistant strains of Escherichia coli , and the similarity values were between 67.9 % and 100 % , which could be divided into 67 types .

3 . Study on the resistance genes of widely resistant isolates of acinetobacter baumanii

There were 23 cases of TEM gene positive in 97 pan - resistant acinetobacter baumandii , and the detection rate was 23.71 % .
The positive rate of CTX - M - 13 gene was 17.53 % . The positive rate was 30.93 % .
The positive rate of IMP - 1 was 40.20 % .
The positive rate of OXA - 58 was 10.31 % .
The positive rate of OXA - 51 was 82.47 % .
The positive rate of OXA - 23 was 87.63 % .
The positive rate of OXA - 24 was 11.34 % . NDM - 1 gene was not detected .

Conclusions of the study

1 . The main isolated self - respiratory system samples from 2009 to 2011 in Xiamen area were mainly from intensive care unit and respiratory department , and the population of isolated patients was more than 45 years old .

2 . The drug resistance of M . baumanii isolated from 2009 to 2011 in some parts of Xiamen showed that the resistance of 386 isolates showed different levels of resistance to 20 common antibiotics , and more than 50 % of the common p - lactam antibiotics and enzyme inhibitors , aminoglycosides , quinolinone antibiotics and sulfanilamide were more severe , with nearly 90 % of them .
The drug resistance of enem antibiotics was higher than that of 60 % .
The drug resistance rate of polymyxin was lower than 50 % , and the resistance rate of Minoryclines was 0.00 % . In the past three years , the drug resistance of acinetobacter baumanii isolated from some parts of Xiamen was increasing year by year for most common antibiotics , especially in Imipenem , meropenem , UNMEE penem and polymyxin .

3 . PFGE is a stable and accurate genotyping method , which is suitable for the homology analysis of nosocomial infection .

The results showed 242 genotypes were prevalent in some parts of Xiamen , and there were no dominant clones in many hospitals .
In some parts of Xiamen , there were no large - scale outbreaks in 2009 - 2011 , the frequency of ICU wards was high , and the cross - infection occurred between different hospitals ;
In addition , there was a completely identical genotype among different hospitals , which suggested the importance of timely epidemiological investigation .

The resistant condition of isolated BA in some parts of Xiamen was more serious . In the past three years , the drug resistance of acinetobacter baumanii isolated from some parts of Xiamen was increasing year by year for most common antibiotics , especially the growth of Imipenem , meropenem , UNMEE penem and polymyxin .
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R446.5;R3416

【参考文献】