Clinical Significance of Rapid and Sensitive Quantification of Pseudomonas aeruginosa by Quantitative Reverse Transcription PCR Targeting of rRNA Molecules

Abstract

Background: For Pseudomonas aeruginosa, nosocomial infection control and appropriate antimicrobial treatment have become important issues. Diagnosis is critical in managing P. aeruginosa infection, but conventional methods are not highly accurate or rapid. A novel P. aeruginosa quantification system based on 23S rRNA-targeted quantitative reverse transcription PCR (qRT-PCR) is a candidate diagnostic tool for managing P. aeruginosa infection. Here, we assessed the performance and potential impact of qRT-PCR on antibiotic therapy administered to ICU patients.Methods: We first evaluated specificity and detection sensitivity of the 23S rRNA-targeted qRT-PCR system in vitro and determined whether P. aeruginosa viable counts detected by this system reflect the inflammatory response of infected cells. Next, we utilized this system on fecal samples collected from 65 septic ICU patients and 44 healthy volunteers to identify the ICU infection status. Additionally, we monitored drug-resistant P. aeruginosa in 4 ICU patients. The trend was compared with trends in fecal microbiota composition, antibiotic use, and mechanical ventilator use.Results: The 23S rRNA-targeted qRT-PCR system quantified P. aeruginosa directly from clinical samples with high sensitivity (blood, 1 cell/mL; stool, 100 cells/g) without cross-reaction (within 6 h). Additionally, P. aeruginosa numbers detected under antibacterial treatment correlated well with the inflammatory response compared to other detection methods such as culturing and qPCR. Using this system, we confirmed that the P. aeruginosa detection ratio in ICU patients was significantly higher than that in healthy volunteers (49.2% vs. 13.6%, P<0.05). While some ICU patients had high P. aeruginosa numbers in feces (108 cells/g of feces), some patients had very low P. aeruginosa numbers (102 cells/g of feces) as observed in healthy volunteers. P. aeruginosa counts in feces of ICU patients monitored by this system correlated negatively with the proportion of total obligate anaerobes and positively with facultative anaerobes and aerobes. Additionally, trends in P. aeruginosa counts accurately reflected various treatment backgrounds of ICU patients.Conclusion: Our results suggest that this new 23S rRNA-targeted qRT-PCR system is beneficial for early diagnosis and evaluation of appropriate antibacterial treatment and may be a useful tool in combating P. aeruginosa infection.