Development and Validation of a Real-time Multiplex PCR Assay for Diagnosis of Carbapenem Resistant Acinetobacter baumannii



Cash, Dana Marie

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Bacterial antibiotic resistance, particularly in nosocomial infections, has become an increasing problem over the last few decades. Overuse of antibiotics in medical and agricultural settings has contributed to the alarming rise of multi-drug resistant bacterial species. Of significant concern are multi-drug resistant (MDR) gram-negative hospital acquired infections that are extremely difficult to treat. In particular, the species Acinetobacter baumannii has been a source of many of these infections, not only in the civilian setting, but in military treatment facilities (MTFs) in combat areas and those treating wounded soldiers abroad and at home. A. baumannii harbors many drug resistance genes and mechanisms but the OXA-type carbapenemase encoding genes have been shown to confer resistance to the standard method of treatment for MDR bacteria, carbapenems of the β-lactam group. This project aimed to develop and implement a real-time PCR assay on the SmartCycler platform to detect the presence of relevant multiple carbapenem resistance genes, as well as to identify A. baumannii specifically. The validated assay tests for the carbapenem resistance genes OXA -23, -24, -51 and -58, of which OXA-23 is a reliable indicator of antibiotic resistance while OXA-24 and -58 have also been shown to confer resistance. In our experiments, we also showed that OXA-51 was present in 100% of A. baumannii samples, indicating its use in species identification. This study was successful in producing an assay with a quick turn-around time to detect the presence of multiple carbapenem resistance genes, as well as identifying A. baumannii at the species level.



Acinetobacter baumannii, Real-time PCR, MALDI-TOF MS, 16s rRNA gene sequencing