title: Multiplex PCR to detect the genes encoding naturally occurring oxacillinases in Acinetobacter spp.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, Australia.
authors.forename: Witchuda
authors.initials: W
authors.lastname: Kamolvit
authors.forename: Paul G
authors.initials: PG
authors.lastname: Higgins
authors.forename: David L
authors.initials: DL
authors.lastname: Paterson
authors.forename: Harald
authors.initials: H
authors.lastname: Seifert
mesh_terms: D000150:Acinetobacter / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D000151:Acinetobacter Infections / Q000382:microbiology; D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D001618:beta-Lactamases; C108449:oxacillinase
keywords: carbapenemases; intrinsic OXA; species identification