Screening and Quantification of the Expression of Antibiotic Resistance Genes in Acinetobacter baumannii with a Microarray
Screening and Quantification of the Expression of Antibiotic Resistance Genes in Acinetobacter baumannii with a Microarray
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DOI：
10.1128/aac.01037-09
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发表时间：
2010-01-01
影响因子：
4.9
通讯作者：
Perichon, Bruno
中科院分区：
文献类型：
--
作者：
Coyne, Sebastien;Guigon, Ghislaine;Perichon, Bruno
开发了基于寡核苷酸的 DNA 微阵列来评估鲍曼不动杆菌中外排泵基因的表达并检测获得性抗生素耐药性决定因素。该微阵列包含 205 个基因的探针，其中包括 47 个外排系统、55 个耐药决定簇和 35 个管家基因的探针。通过相对于亲本菌株过度表达或缺乏泵的突变体的比较分析来验证微阵列。还使用各种抗生素获得的体外单步突变体评估了微阵列的性能。通过定量逆转录酶 PCR 证实，检测到 RND 外排泵 AdeABC 的过表达（由于庆大霉素获得的多重耐药 (MDR) 菌株中 AdeS 中的 G30D 取代）和头孢噻肟或四环素获得的两个突变体中的 AdeIJK 过表达。发现一种新的外排泵 AdeFGH 在氯霉素获得的突变体中过度表达。对 MDR 临床分离株（包括 AYE 菌株）的研究表明，AdeABC 和染色体编码的头孢菌素酶过度表达，并且存在一些获得性耐药基因。微阵列检测到的过度表达和获得性决定簇几乎可以解释分离株的全部 MDR 表型。该微阵列对于检测鲍曼不动杆菌的耐药性可能有用，并且应该允许检测与抗生素耐药性相关的新的流出系统。
An oligonucleotide-based DNA microarray was developed to evaluate expression of genes for efflux pumps in Acinetobacter baumannii and to detect acquired antibiotic resistance determinants. The microarray contained probes for 205 genes, including those for 47 efflux systems, 55 resistance determinants, and 35 housekeeping genes. The microarray was validated by comparative analysis of mutants overexpressing or deficient in the pumps relative to the parental strain. The performance of the microarray was also evaluated using in vitro single-step mutants obtained on various antibiotics. Overexpression, confirmed by quantitative reverse transcriptase PCR, of RND efflux pumps AdeABC, due to a G30D substitution in AdeS in a multidrug-resistant (MDR) strain obtained on gentamicin, and AdeIJK, in two mutants obtained on cefotaxime or tetracycline, was detected. A new efflux pump, AdeFGH, was found to be overexpressed in a mutant obtained on chloramphenicol. Study of MDR clinical isolates, including the AYE strain, whose entire sequence has been determined, indicated overexpression of AdeABC and of the chromosomally encoded cephalosporinase as well as the presence of several acquired resistance genes. The overexpressed and acquired determinants detected by the microarray could account for nearly the entire MDR phenotype of the isolates. The microarray is potentially useful for detection of resistance in A. baumannii and should allow detection of new efflux systems associated with antibiotic resistance.