中国碳青霉烯耐药鲍曼不动杆菌多位点序列分型及OXA酶分布研究
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摘要
碳青霉烯类(Carbapenem)抗生素是治疗由鲍曼不动杆菌(Acinetobacter baumannii)引发感染最重要的B-内酰胺类(β-lactam)抗生素之一。而碳青霉烯类抗生素耐药鲍曼不动杆菌在我国各地的广泛流行,给临床治疗与抗生素的选择带来极大的困难。为了制定科学、有效的防控措施,通常采用各种分子分型方法,对其进行分子流行病学研究并明确流行趋势,为进一步探究鲍曼不动杆菌的耐药机制提供基础。
本研究于2009年1月至2010年12月期间在全国23个省市的64家医院收集2197株非重复的鲍曼不动杆菌,并选择12种常见抗菌药物开展体外药物敏感性试验。根据药敏结果,对筛选出的808株碳青霉烯耐药鲍曼不动杆菌应用多位点序列分型(Multilocus sequence typing, MLST)方法进行分子流行病学研究；借助. Linux服务器、DNA序列拼接与比对工具编写Perl脚本,实现对各等位基因测序数据的批量处理,并鉴定出等位基因型(Allele type)与序列型(Sequence type,ST)；应用eBURST软件与Bionumerics软件对MLST数据进行聚类分析,预测各菌株间的进化关系。
体外药物敏感性试验结果显示,所有鲍曼不动杆菌对12种临床常用抗生素的平均耐药率为50%～60%,而米诺环素仅有11.56%。MLST结果显示,碳青霉烯耐药鲍曼不动杆菌的遗传背景相对单一,共分为40个ST型(包括17个已被PubMLST数据库收录及23个新发现的ST型),其中ST92及其单位点变异型(Single locus variant, SLV) ST138、ST75与ST381为最主要流行的ST型。应用eBURST软件与Bionumerics软件分析MLST数据,发现当前中国最广泛流行的鲍曼不动杆菌克隆复合体(Clonal complex, CC)为CC92,该克隆复合体包括ST92、 ST138、ST75等ST型。本研究中的体外药物敏感性试验结果也表明,属于CC92的菌株较其他ST型菌株具有更高的耐药率。
上述研究结果表明,CC92是鲍曼不动杆菌流行最广的克隆复合体,它在我国分布广泛且传播迅速,常伴随多重耐药鲍曼不动杆菌的播散与流行。因此,CC92在我国的大范围流行是导致鲍曼不动杆菌对多种抗菌药物耐药率迅速上升的重要原因。
具有碳青霉烯类抗生素水解能力的D类β-内酰胺酶Carbapenem-hydrolyzing class D β-lactamases, CHDLs)是不动杆菌(Acinetobacter spp.)最常见的碳青霉烯酶,常介导其对碳青霉烯类抗生素耐药。本研究主要应用分子生物学手段,探讨5种常见编码CHDLs的OXA耐药基因在不动杆菌中的分布与耐药表型、传播机制之间的关系。
本研究对收集于2009年1月至2010年12月期间全国23个省市64家医院分离的2880株不动杆菌开展体外药物敏感性试验,测定其对12种抗菌药物的最小抑菌浓度(minimum inhibitory concentration, MIC);并应用PCR扩增与测序技术,对筛选出携带blaOXA-51-like、blaOXA-23-like、blaOXA-24-like、blaOXA-25-like与blaOXA-143-like基因的不动杆菌进行菌种鉴定；应用脉冲场凝胶电泳(Pulsed-field gel electrophoresis, PFGE)与Southern杂交确定OXA基因的定位；应用质粒抽提与电转化确定携带OXA基因质粒的可传播性。
体外药物敏感性试验结果显示,不动杆菌对碳青霉烯类抗生素的耐药率约为50%。多重PCR结果显示,携带blaOXA-51-like基因与blaOXA-23-like基因的不动杆菌分别占76.3%与45.7%。而44.9%的不动杆菌同时携带blaOXA-51-like基因与blaOXA-23-like基因,高达95%携带blaOXA-23-like基因的不动杆菌对碳青霉烯类抗生素耐药。此外,筛查发现携带blaOXA-24-like基因、blaOXA-58-like基因与blaOXA-143-like基因的不动杆菌分别为11株、32株与1株。Southern杂交结果显示,携带blaOXA-24-like基因与blaOXA-58-like基因由质粒编码,其质粒可通过电转化的方法导入受体菌,并使受体菌对碳青霉烯类抗生素的MIC值升高。而blaOXA-143-like基因无法通过上述方法检测到,提示该基因可能位于染色体上。
Carbapenems are important class of β-lactam antibiotics, and often participate in antibiotic treatment of Acinetobacter baumannii infections. The carbapenem-resistant Acinetobacter baumannii has widely spread in China, which causing great difficulty in clinical thrapy. In order to formulate a scientific and effective prevention and control measures, different molecular typing method are used to study the epidemiological distribution and antibiotic resistance mechanisms of Acinetobacter baumannii.
In this study,2197non-duplicated Acinetobacter baumannii were collected from64hospitals of23different provinces, and the susceptibility testing of12common antimicrobial agents were determined. According the results of susceptibility testing, we chose808strains of carbapenem-resistant Acinetobacter baumannii to perform Multilocus sequence typing (MLST) research. We applied Linux server, DNA fragment assembly tools and developed Perl scripts to achieve the batch processing of each allele sequencing data and identified the allele type with sequence type (ST). eBURST and Bionumerics software were used to analyze the data of MLST and predict the evolutionary relationships among different strains.
The results of susceptibility testing revealed that the average resistance rate to12antibiotics in Acinetobacter baumannii were50%-60%, except minocycline only11.56%. The study of MLST data suggested that the genetic background of Acinetobacter baumannii was relatively simple, including40STs (17recorded by PubMLST database and23novel STs). ST92and its single locus variants (SLVs), ST138, ST75and ST381were the predominant STs. The results of eBURST and Bionumerics software demonstrated that the most widely disseminated Clonal complex (CC) of Acinetobacter baumannii was CC92in current China, and incorporate ST92, ST138and ST75et al. The study of susceptibility testing revealed that strains belonged to CC92with higher resistance compared to other STs.
Results of this study indicated that CC92was the most widely spread and disseminated clonal complex in China, and often accompanied with the appearance and dissemination of multidrug resistance Acinetobacter baumannii. Hence, the widespread of CC92is the principal reason that responsible for the situation of antimicrobial resistance rate rose rapidly in Acinetobacter baumannii.
Carbapenem-hydrolyzing class D β-lactamases (CHDLs) is the most commonly carbapenem in Acinetobacter spp., and often induce the mechanism of carbapenem resistance. In this study, we applied molecular biology method to discuss the relationship between the distribution of five OXA genes, which coding CHDLs, and dissemination mechanism in Acinetobacter spp.
In this study,2880Acinetobacter spp. were collected from64hospitals of23different provinces, and the minimum inhibitory concentration (MIC) of12common antimicrobial agents were determined. We applied PCR-based amplified and sequencing technology to identify Acinetobacter spp., which harboring blaoxA-51-like,blaoXA-23-like, blaOXA-24-like, blaOXA-58-like and blaOXA-143-like gene, to the genus level. Pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were used to determine the location of OXA genes. Plasmid extraction and electrotransformation were applied to confirm the transferability of OXA carrying plasmids.
Susceptibility testing indicated that the average resistance rate to carbapenem in Acinetobacter spp. were approximately50%. Results of multiplex PCR revealed that the proportion of Acinetobacter spp., which harboring biaOXA-51-like and biaOXA-23-like gene, were76.3%and45.7%respectively. Besides,44.9%of Acinetobacter spp. coharbor biaOXA-51-like and biaOXA-23-like gene, and up to95%of them resistant to carbapenems. The biaOXA-24-like, biaOXA-58-like and biaOXA-143-like gene were detected in32,11and1isolate(s) respectively. Southern blot hybridization indicated that the biaOXA-24-like, and biaOXA-58-like gene were located on plasmds, which can be transferred to recipient and increase its MIC values for carbapenems. But biaOXA-143-like gene can't be detected by the method above, which indicated that it might be located on chromosome.
本研究于2009年1月至2010年12月期间在全国23个省市的64家医院收集2197株非重复的鲍曼不动杆菌,并选择12种常见抗菌药物开展体外药物敏感性试验。根据药敏结果,对筛选出的808株碳青霉烯耐药鲍曼不动杆菌应用多位点序列分型(Multilocus sequence typing, MLST)方法进行分子流行病学研究；借助. Linux服务器、DNA序列拼接与比对工具编写Perl脚本,实现对各等位基因测序数据的批量处理,并鉴定出等位基因型(Allele type)与序列型(Sequence type,ST)；应用eBURST软件与Bionumerics软件对MLST数据进行聚类分析,预测各菌株间的进化关系。
体外药物敏感性试验结果显示,所有鲍曼不动杆菌对12种临床常用抗生素的平均耐药率为50%～60%,而米诺环素仅有11.56%。MLST结果显示,碳青霉烯耐药鲍曼不动杆菌的遗传背景相对单一,共分为40个ST型(包括17个已被PubMLST数据库收录及23个新发现的ST型),其中ST92及其单位点变异型(Single locus variant, SLV) ST138、ST75与ST381为最主要流行的ST型。应用eBURST软件与Bionumerics软件分析MLST数据,发现当前中国最广泛流行的鲍曼不动杆菌克隆复合体(Clonal complex, CC)为CC92,该克隆复合体包括ST92、 ST138、ST75等ST型。本研究中的体外药物敏感性试验结果也表明,属于CC92的菌株较其他ST型菌株具有更高的耐药率。
上述研究结果表明,CC92是鲍曼不动杆菌流行最广的克隆复合体,它在我国分布广泛且传播迅速,常伴随多重耐药鲍曼不动杆菌的播散与流行。因此,CC92在我国的大范围流行是导致鲍曼不动杆菌对多种抗菌药物耐药率迅速上升的重要原因。
具有碳青霉烯类抗生素水解能力的D类β-内酰胺酶Carbapenem-hydrolyzing class D β-lactamases, CHDLs)是不动杆菌(Acinetobacter spp.)最常见的碳青霉烯酶,常介导其对碳青霉烯类抗生素耐药。本研究主要应用分子生物学手段,探讨5种常见编码CHDLs的OXA耐药基因在不动杆菌中的分布与耐药表型、传播机制之间的关系。
本研究对收集于2009年1月至2010年12月期间全国23个省市64家医院分离的2880株不动杆菌开展体外药物敏感性试验,测定其对12种抗菌药物的最小抑菌浓度(minimum inhibitory concentration, MIC);并应用PCR扩增与测序技术,对筛选出携带blaOXA-51-like、blaOXA-23-like、blaOXA-24-like、blaOXA-25-like与blaOXA-143-like基因的不动杆菌进行菌种鉴定；应用脉冲场凝胶电泳(Pulsed-field gel electrophoresis, PFGE)与Southern杂交确定OXA基因的定位；应用质粒抽提与电转化确定携带OXA基因质粒的可传播性。
体外药物敏感性试验结果显示,不动杆菌对碳青霉烯类抗生素的耐药率约为50%。多重PCR结果显示,携带blaOXA-51-like基因与blaOXA-23-like基因的不动杆菌分别占76.3%与45.7%。而44.9%的不动杆菌同时携带blaOXA-51-like基因与blaOXA-23-like基因,高达95%携带blaOXA-23-like基因的不动杆菌对碳青霉烯类抗生素耐药。此外,筛查发现携带blaOXA-24-like基因、blaOXA-58-like基因与blaOXA-143-like基因的不动杆菌分别为11株、32株与1株。Southern杂交结果显示,携带blaOXA-24-like基因与blaOXA-58-like基因由质粒编码,其质粒可通过电转化的方法导入受体菌,并使受体菌对碳青霉烯类抗生素的MIC值升高。而blaOXA-143-like基因无法通过上述方法检测到,提示该基因可能位于染色体上。
Carbapenems are important class of β-lactam antibiotics, and often participate in antibiotic treatment of Acinetobacter baumannii infections. The carbapenem-resistant Acinetobacter baumannii has widely spread in China, which causing great difficulty in clinical thrapy. In order to formulate a scientific and effective prevention and control measures, different molecular typing method are used to study the epidemiological distribution and antibiotic resistance mechanisms of Acinetobacter baumannii.
In this study,2197non-duplicated Acinetobacter baumannii were collected from64hospitals of23different provinces, and the susceptibility testing of12common antimicrobial agents were determined. According the results of susceptibility testing, we chose808strains of carbapenem-resistant Acinetobacter baumannii to perform Multilocus sequence typing (MLST) research. We applied Linux server, DNA fragment assembly tools and developed Perl scripts to achieve the batch processing of each allele sequencing data and identified the allele type with sequence type (ST). eBURST and Bionumerics software were used to analyze the data of MLST and predict the evolutionary relationships among different strains.
The results of susceptibility testing revealed that the average resistance rate to12antibiotics in Acinetobacter baumannii were50%-60%, except minocycline only11.56%. The study of MLST data suggested that the genetic background of Acinetobacter baumannii was relatively simple, including40STs (17recorded by PubMLST database and23novel STs). ST92and its single locus variants (SLVs), ST138, ST75and ST381were the predominant STs. The results of eBURST and Bionumerics software demonstrated that the most widely disseminated Clonal complex (CC) of Acinetobacter baumannii was CC92in current China, and incorporate ST92, ST138and ST75et al. The study of susceptibility testing revealed that strains belonged to CC92with higher resistance compared to other STs.
Results of this study indicated that CC92was the most widely spread and disseminated clonal complex in China, and often accompanied with the appearance and dissemination of multidrug resistance Acinetobacter baumannii. Hence, the widespread of CC92is the principal reason that responsible for the situation of antimicrobial resistance rate rose rapidly in Acinetobacter baumannii.
Carbapenem-hydrolyzing class D β-lactamases (CHDLs) is the most commonly carbapenem in Acinetobacter spp., and often induce the mechanism of carbapenem resistance. In this study, we applied molecular biology method to discuss the relationship between the distribution of five OXA genes, which coding CHDLs, and dissemination mechanism in Acinetobacter spp.
In this study,2880Acinetobacter spp. were collected from64hospitals of23different provinces, and the minimum inhibitory concentration (MIC) of12common antimicrobial agents were determined. We applied PCR-based amplified and sequencing technology to identify Acinetobacter spp., which harboring blaoxA-51-like,blaoXA-23-like, blaOXA-24-like, blaOXA-58-like and blaOXA-143-like gene, to the genus level. Pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were used to determine the location of OXA genes. Plasmid extraction and electrotransformation were applied to confirm the transferability of OXA carrying plasmids.
Susceptibility testing indicated that the average resistance rate to carbapenem in Acinetobacter spp. were approximately50%. Results of multiplex PCR revealed that the proportion of Acinetobacter spp., which harboring biaOXA-51-like and biaOXA-23-like gene, were76.3%and45.7%respectively. Besides,44.9%of Acinetobacter spp. coharbor biaOXA-51-like and biaOXA-23-like gene, and up to95%of them resistant to carbapenems. The biaOXA-24-like, biaOXA-58-like and biaOXA-143-like gene were detected in32,11and1isolate(s) respectively. Southern blot hybridization indicated that the biaOXA-24-like, and biaOXA-58-like gene were located on plasmds, which can be transferred to recipient and increase its MIC values for carbapenems. But biaOXA-143-like gene can't be detected by the method above, which indicated that it might be located on chromosome.
引文
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