Real-time identification of Pseudomonas aeruginosa direct from clinical samples using a rapid extraction method and polymerase chain reaction (PCR)

Abstract

Pseudomonas aeruginosa has emerged as one of the most problematic Gram-negative nosocomial pathogens. Bacteremia caused by P. aeruginosa is clinically indistinguishable from other Gram-negative infections although the mortality rate is higher. This microorganism is also inherently resistant to common antibiotics. Standard bacterial identification and susceptibility testing is normally a 48-hour process and difficulty sometimes exists in rapidly and accurately identifying antimicrobial resistance. The Polymerase Chain Reaction (PCR) is a rapid and simple process for the amplification of target DNA sequences. However, many sample preparation methods are unsuitable for the clinical laboratory because they are not cost effective, take too long to perform, or do not provide a good template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis. In this report, we have utilized our rapid DNA extraction method to generate bacterial DNA direct from clinical samples for PCR. The lower detection level for P. aeruginosa was estimated to be 10 CFU/ml. In addition, we wanted to compare the results of a new rapid-cycle DNA thermocycler that uses continuous fluorescence monitoring with the results of standard thermocycling. We tested 40 clinical isolates of P. aeruginosa and 18 non-P. aeruginosa isolates received in a blinded fashion. Coded data revealed that there was 100% correlation in both the rapid-cycle DNA thermocycling and standard thermocycling when compared to standard clinical laboratory results. In addition, total results turn-around time was less than 1 hour. Specific identification of P. aeruginosa was determined using intragenic primer sets for bacterial 16S rRNA and Pseudomonas outer-membrane lipoprotein gene sequences. The total cost of our extraction method and PCR was $2.22 per sample. The accuracy and rapidness of this DNA-extraction method, with its PCR-based identification system, make it an ideal candidate for use in the clinical laboratory.