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Development of a rapid and sensitive analytical system for Pseudomonas aeruginosa based on reverse transcription
Mai Niikura1, Satomi Atobe1, Akira Takahashi1
1Yakult Central Institute, Yakult Honsha Co., Ltd., Kunitachi, Tokyo, Japan.
Abstract:
For Pseudomonas aeruginosa (PA), infection control and appropriate antimicrobial treatment have become important issues. Diagnosis is critical in managing PA infection, but conventional methods are not highly accurate or rapid. We developed a new PA quantification system based on 23S rRNA-targeted reverse transcription quantitative PCR (RT-qPCR). We confirmed that RT-qPCR can quantify PA directly from clinical samples quickly (within 6 h) and with high sensitivity (blood, 1 cell/mL; stool, 100 cells/g) and without cross-reaction. Also, under antibiotic treatment, PA viable counts detected by this system correlated well with the inflammatory response of infected Caco-2 cells compared to other methods such as culturing and qPCR. Next, we utilized this system on fecal samples collected from 65 septic ICU patients and 44 healthy volunteers to identify ICU infection status. We confirmed that the PA detection ratio in ICU patients was significantly higher than that in healthy volunteers (49.2% vs. 13.6%, P < 0.05). Additionally, we monitored drug-resistant PA in 4 ICU patients by this system. The trends in PA counts accurately reflected various treatment backgrounds such as antibiotic use and mechanical ventilator use. Our results suggest that this RT-qPCR system is beneficial for the early diagnosis and evaluation of appropriate antibacterial treatment and may be a useful tool in combating PA infection.
Insights
A new reverse transcription quantitative PCR (RT-qPCR) system accurately quantifies Pseudomonas aeruginosa (PA) in clinical samples within 6 hours. This rapid diagnostic tool aids in early infection detection and evaluating antimicrobial treatment effectiveness for PA.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Pseudomonas aeruginosa (PA) infections pose significant challenges in healthcare settings.
- Current diagnostic methods for PA lack speed and accuracy, hindering effective management.
- Rapid and sensitive detection is crucial for controlling PA infections and guiding antimicrobial therapy.
Purpose of the Study:
- To develop and validate a novel reverse transcription quantitative PCR (RT-qPCR) system for rapid and sensitive quantification of PA.
- To assess the utility of the RT-qPCR system in diagnosing PA infection in ICU patients.
- To evaluate the system's effectiveness in monitoring treatment response and drug-resistant PA.
Main Methods:
- Development of a 23S rRNA-targeted RT-qPCR assay for PA quantification.
- Validation of the assay's sensitivity, specificity, and speed using clinical samples.
- Application of the RT-qPCR system to fecal samples from septic ICU patients and healthy controls.
- Correlation of RT-qPCR results with inflammatory responses and clinical outcomes.
Main Results:
- The RT-qPCR system accurately quantified PA in clinical samples within 6 hours with high sensitivity (blood: 1 cell/mL; stool: 100 cells/g).
- PA detection was significantly higher in septic ICU patients (49.2%) compared to healthy volunteers (13.6%).
- The system effectively monitored drug-resistant PA and reflected treatment backgrounds in ICU patients.
Conclusions:
- The developed RT-qPCR system offers a rapid, sensitive, and accurate method for PA diagnosis.
- This tool can aid in the early detection of PA infections and the evaluation of antimicrobial treatment efficacy.
- The RT-qPCR system shows promise as a valuable tool for combating Pseudomonas aeruginosa infections.
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