University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, Australia.
J Antimicrob Chemother. 2014 Apr;69(4):959-63. doi: 10.1093/jac/dkt480. Epub 2013 Nov 27.
Bacteria of the genus Acinetobacter are increasingly being isolated in hospitals and are recognized as emerging nosocomial pathogens. Species identification is difficult and there is a need for simple molecular methods to differentiate between the species. Naturally occurring oxacillinase genes (blaOXA) have been identified in several Acinetobacter species and their detection by PCR can aid in species identification. The aim of this study was to develop a multiplex PCR to identify intrinsic blaOXA genes (i.e. bla(OXA-134-like), bla(OXA-211-like), bla(OXA-213-like), bla(OXA-214-like) and bla(OXA-228-like)) from Acinetobacter spp. for use as a tool for rapid species identification.
Primers were designed to selectively amplify internal fragments of intrinsic blaOXA from Acinetobacter lwoffii/Acinetobacter schindleri (bla(OXA-134-like)), Acinetobacter johnsonii (bla(OXA-211-like)), Acinetobacter calcoaceticus (bla(OXA-213-like)), Acinetobacter haemolyticus (bla(OXA-214-like)) and Acinetobacter bereziniae (bla(OXA-228-like)). Multiplex PCR was performed in a total of 100 Acinetobacter isolates. Flanking primers were designed for each blaOXA subgroup and products were sequenced.
All A. lwoffii, A. schindleri, A. johnsonii, A. calcoaceticus, A. haemolyticus and A. bereziniae isolates were positive for their species-specific amplicons while other Acinetobacter species were negative. Thirty blaOXA novel variants were identified; the majority of these (21/30) were from A. calcoaceticus. ISAba11 was found upstream of bla(OXA-214) in four A. haemolyticus isolates, but was not associated with carbapenem resistance.
This multiplex PCR specifically detected each of the five different blaOXA subgroups. Therefore, this method has the potential to aid in the identification of these species and monitor the spread of these genes into other Acinetobacter species.
不动杆菌属的细菌越来越多地在医院中被分离出来，并且被认为是新兴的医院获得性病原体。种属鉴定困难，需要简单的分子方法来区分这些种属。已经在几种不动杆菌种中鉴定出天然存在的青霉素酶基因（blaOXA），PCR 检测可以帮助鉴定种属。本研究旨在开发一种多重 PCR 来鉴定不动杆菌属内源性 blaOXA 基因（即 bla(OXA-134-like)、bla(OXA-211-like)、bla(OXA-213-like)、bla(OXA-214-like)和 bla(OXA-228-like)），作为快速种属鉴定的工具。
设计引物以选择性扩增来自洛菲不动杆菌/辛德毕斯不动杆菌（bla(OXA-134-like)）、约翰逊不动杆菌（bla(OXA-211-like)）、醋酸钙不动杆菌（bla(OXA-213-like)）、溶血不动杆菌（bla(OXA-214-like)）和贝日阿托不动杆菌（bla(OXA-228-like)）的内源性 blaOXA 的内部片段。在总共 100 株不动杆菌分离株中进行多重 PCR。为每个 blaOXA 亚群设计侧翼引物，并对产物进行测序。
所有的洛菲不动杆菌、辛德毕斯不动杆菌、约翰逊不动杆菌、醋酸钙不动杆菌、溶血不动杆菌和贝日阿托不动杆菌分离株均对其种特异性扩增子呈阳性，而其他不动杆菌种则为阴性。鉴定出 30 种 blaOXA 新变体，其中大多数（21/30）来自醋酸钙不动杆菌。在 4 株溶血不动杆菌中发现 bla(OXA-214)上游有 ISAba11，但与碳青霉烯类耐药无关。
该多重 PCR 特异性检测到 5 种不同 blaOXA 亚群中的每一种。因此，该方法有可能有助于鉴定这些种属，并监测这些基因传播到其他不动杆菌种。
