Two Multiplex Real-Time PCR Assays to Detect and Differentiate Acinetobacter baumannii and Non- baumannii Acinetobacter spp. Carrying bla(NDM), bla(OXA-23-Like), bla(OXA-40-Like), bla(OXA-51-Like), and bla(OXA-58-Like) Genes

Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including bla(NDM), bla(OXA-23-like), bla(OXA-40-like), bla(OXA-51-like), and bla(OXA-58-like). We demonstrate the potential utility of these assays for the direct detection of bla(NDM)-, bla(OXA-23-like)-, bla(OXA-40-like)-, bla(OXA-51-like)-, and bla(OXA-58-like)-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S–23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene bla(NDM); and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (bla(OXA-23-like), bla(OXA-40-like), bla(OXA-51-like),and bla(OXA-58-like)). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r(2) > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 10(2) CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of bla(NDM)-, bla(OXA-23-like)-, bla(OXA-40-like)-, bla(OXA-51-like)-, and bla(OXA-58-like)-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections.