Development of a rapid and sensitive analytical system for Pseudomonas aeruginosa based on reverse transcription quantitative PCR targeting of rRNA molecules

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.

KEYWORDS:

• Pseudomonas aeruginosa
• infection
• RT-qPCR
• blood
• feces
• ICU patients
• antimicrobial resistance
• microbiota

Disclosure statement

No potential conflict of interest was reported by the author(s).

Acknowledgments

We thank Yukihiro Yokoyama, MD for critical reading of the manuscript. We also thank Hirotaka Haibara, Toshihiko Takada, and Ryoko Iizuka for assistance with cell culture, FISH analyses, and cytokine assays, respectively.

Data availability

The 23S rRNA gene sequences deposited in the DDBJ (http://www.ddbj.nig.ac.jp/) nucleotide sequence database are listed in Table S3.