产ESBLs腹泻病原菌的耐药性和分子流行病学研究
发布时间：2018-07-18 10:25
【摘要】： 自从上世纪八十年代初在德国发现第一例产ESBLs(Extend-Spectrumβ-Lactamases,超广谱β-内酰胺酶)的克雷伯菌以来,在全球各地不断有ESBLs报道。革兰阴性杆菌是导致感染的最重要病原菌,过去的三十年由于大量使用超广谱β-内酰胺类抗生素,产生药物选择性压力,导致越来越多的革兰阴性杆菌产生ESBLs。ESBLs是微生物对头孢类抗生素形成耐药的主要原因,同时也介导对其他抗生素耐药,大多数超广谱β-内酰胺酶由广谱TEM-1、2和SHV-1衍生而来,可以水解氧亚氨基头孢菌素类和单环β-内酰胺类抗生素;近年来,CTX-M型逐渐成为主要流行亚型。产ESBLs菌株既可以发生垂直传播,也可以通过质粒或转座子等进行产酶基因的水平传播,可以在细菌种内和种间进行传播扩散,导致大规模的暴发流行。目前对于腹泻病原菌产ESBLs尚缺乏系统研究。为了了解北京地区腹泻致病菌中产超广谱β-内酰胺酶的革兰阴性杆菌的耐药情况、各基因型的分布和分子流行病学情况,我们收集了2006年北京大学第一附属医院、大兴社区、307医院、总政门诊部的腹泻标本,进行了产ESBLs菌株的检测、基因分型和流行机制的研究。 1.为了了解腹泻病原菌中产ESBLs菌株的发生率、耐药性和流行性,我们对收集的腹泻标本进行分离鉴定,采用了CLSI的双纸片法进行了产ESBLs菌株的检测,使用K-B法测定了产ESBLs菌株对多种抗生素的耐药性。结果显示,407株阴性杆菌中检出ESBLs阳性菌株37株,总的检出率是9.09%,产ESBLs菌株在分离的腹泻病原菌中以大肠杆菌为主,共24株,占64%。产ESBLs菌株对于大多数β-内酰胺类和非β-内酰胺类抗生素(如氨基糖苷类、喹诺酮类、四环素、氯霉素等)耐药性均很高,为多重耐药菌株,尤其是对哌拉西啉、头孢噻肟、头孢唑啉、头孢匹罗、奈啶酸的耐药率达到了90%左右。这些菌株对于复合抗生素(氨苄西林/舒巴坦、阿莫西林/克拉维酸)的耐药性也呈现上升趋势,分别达到了70.27%和51.35%,而对于美洛培南高度敏感,达到了100%。阳性菌主要分离自7岁和45岁免疫力较低人群中。本研究认为ESBLs是继喹诺酮耐药机制后成为腹泻病原菌的一个重要的耐药机制。 2.腹泻病原菌中产ESBLs菌株的亚型的检测和基因型分析。为了了解分离的ESBLs菌株的基因流行情况,我们采用了TEM型、SHV型和CTX-M型ESBLs编码基因的通用引物对产ESBLs菌株进行扩增,并采用PFGE技术对分离的产ESBLs腹泻病原菌进行了DNA指纹图谱分析。结果显示,在分离的产ESBLs的腹泻病原菌中,CTX-M型是最为常见的超广谱β-内酰胺酶型别, TEM型次之,SHV型最少,CTX-M亚型中CTX-M-15最多,部分产ESBLs泻病原菌同时产生一种以上的β内酰胺酶,少数菌株的ESBLs基因型不属于上述三种基因型。分离的24株产ESBLs的大肠杆菌有7种基因型,最多见的一种谱型有8株细菌;5株产ESBLs的宋内菌有两种基因型,变形杆菌和克雷伯菌只有一种基因型。ESBLs不仅可以存在于不同种的细菌中,也可以存在同一种细菌的不同基因型中,推测可能是耐药基因在不同细菌或同一细菌不同基因型中平行转移的结果。 3.产ESBLs菌株接合子的ESBLs表型鉴定、质粒分析、超广谱β-内酰胺酶型别的测定为了了解产ESBLs腹泻病原菌中编码ESBLs的基因位于染色体或是质粒、其传播及扩散特征,我们对分离的37株产ESBLs的腹泻病原菌进行接合试验,受体菌为E.coli.J53 FOXRLAC-;采用采用了CLSI的双纸片法对分离菌株进行了产ESBLs菌株的检测,使用K-B法测定了产ESBLs菌株对多种抗生素的耐药性,采用TEM型、SHV型和CTX-M型ESBLs编码基因的通用引物对产ESBLs菌株进行扩增。 结果显示,产ESBLs腹泻病原菌转移发生率为56.76%,转移频率为2.5×10-5～5×10-4,所有的转移接合子的CISL表型确证试验检测均为ESBLs阳性。转移接合子对头孢类抗生素明显耐药,耐药水平与供体菌相近,克拉维酸可以明显降低接合子对这些抗生素的耐药性。同时与供体菌相比,接合子对头孢他啶、安曲南、阿莫西林/克拉维酸、氨苄西林/舒巴坦、头孢吡肟的耐药性明显降低。琼脂糖电泳显示接合子从供体菌获得了一个23.1Kb的质粒,受体菌E.coliJ53 FOXRLAC-未见上述现象。转移接合子中仍然是以CTX-M型为主,TEM次之,SHV编码基因未能发生转移。本部分的研究表明,产ESBLs腹泻病原菌株能以较高频率通过接合转移方式水平传播ESBLs编码基因,受体菌获得了产酶基因而出现ESBLs的表型;这些编码基因由大的质粒携带,这些质粒往往同时携带其他非β-内酰胺类抗生素的耐药基因,使得转移接合子表现多重耐药性。转移过程中可出现基因分离现象,CTX-M型仍是最常见型。 根据以上三个部分的研究,可以得出如下结论: 1.产ESBLs菌株在腹泻病原菌中的检出率较高,主要在老人和小孩多见,在医院和社区存在传播和流行,ESBLs已经成为了腹泻病原菌的一个重要的耐药机制。 2. ESBLs为多重耐药菌株,对大多数常用的β-内酰胺类抗生素和非β-内酰胺类抗生素显著耐药。 3. ESBLs基因型以CTX-M(CTX-M-15)最常见,还有CTX-M-3、TEM-1较为普遍,部分腹泻病原菌产一种以上的超广谱β-内酰胺酶。 4.产ESBLs菌株可以通过垂直方式传播,也可以通过转移接合方式发生产酶基因的水平传播。编码ESBLs质粒同时携带非β-内酰胺类抗生素的耐药基因,导致多重耐药,这些非β-内酰胺类抗生素的耐药性可以与ESBLs表型发生共转移。 5. ESBLs可以存在于不同种细菌,也可以存在于同种细菌的不同基因型,因此,我们在腹泻高发期应该需要加强产ESBLs菌株的检测及其感染的防治。
[Abstract]:In order to understand the drug resistance of Gram - negative bacilli , which is the most important pathogenic bacterium in the past 30 years , most of the super - broad - spectrum 尾 - lactamase produced by broad - spectrum TEM - 1 , 2 and SHV - 1 can hydrolyze oxyimino - cephalosporin and monocyclic 尾 - lactam antibiotics . In recent years , most of the super - wide - spectrum 尾 - lactamase are derived from broad - spectrum TEM - 1 , 2 and SHV - 1 .
1 . In order to understand the incidence , drug resistance and epidemic of the strains producing strains in the pathogenic bacteria of diarrhea , the drug resistance of the isolates was detected by double - sheet method . The results showed that the drug resistance of the isolates was 9 . 09 % . The results showed that there were 37 strains of 尾 - lactam and 51 . 35 % of the isolates . The strains were mainly isolated from 7 and 45 years old .
The results showed that CTX - M was the most common type of 尾 - lactamase , TEM , SHV and CTX - M producing strains . The results showed that CTX - M was the most common type of 尾 - lactamase .
3 . In order to find out the gene located on chromosome or plasmid , its transmission and diffusion characteristics , the isolates were tested by using double - sheet method . The isolates were tested by using the double - sheet method . The strains were amplified by using the universal primers of TEM , SHV and CTX - M - producing strains .
The results showed that the incidence of pathogenic bacteria transfer was 56.76 % and the transfer frequency was 2.5 脳 10 - 5 锝，
本文编号：2131616
[Abstract]:In order to understand the drug resistance of Gram - negative bacilli , which is the most important pathogenic bacterium in the past 30 years , most of the super - broad - spectrum 尾 - lactamase produced by broad - spectrum TEM - 1 , 2 and SHV - 1 can hydrolyze oxyimino - cephalosporin and monocyclic 尾 - lactam antibiotics . In recent years , most of the super - wide - spectrum 尾 - lactamase are derived from broad - spectrum TEM - 1 , 2 and SHV - 1 .
1 . In order to understand the incidence , drug resistance and epidemic of the strains producing strains in the pathogenic bacteria of diarrhea , the drug resistance of the isolates was detected by double - sheet method . The results showed that the drug resistance of the isolates was 9 . 09 % . The results showed that there were 37 strains of 尾 - lactam and 51 . 35 % of the isolates . The strains were mainly isolated from 7 and 45 years old .
The results showed that CTX - M was the most common type of 尾 - lactamase , TEM , SHV and CTX - M producing strains . The results showed that CTX - M was the most common type of 尾 - lactamase .
3 . In order to find out the gene located on chromosome or plasmid , its transmission and diffusion characteristics , the isolates were tested by using double - sheet method . The isolates were tested by using the double - sheet method . The strains were amplified by using the universal primers of TEM , SHV and CTX - M - producing strains .
The results showed that the incidence of pathogenic bacteria transfer was 56.76 % and the transfer frequency was 2.5 脳 10 - 5 锝，
本文编号：2131616
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