Applying a PCR-based open-reading frame typing method for easy genotyping and molecular epidemiological analysis of
M Suzuki1,2, K Yamada1, M Aoki1
1Laboratory of Bacteriology, Aichi Prefectural Institute of Public Health, Nagoya, Japan.
Aims:
Molecular epidemiological techniques, such as pulsed-field gel electrophoresis (PFGE), or multilocus sequence typing (MLST) have facilitated our understanding of the transmission routes of nosocomial infections by Pseudomonas aeruginosa. However, they are time consuming and technically demanding. To perform molecular epidemiological analysis in a standard microbiology laboratory, we aimed to develop a simpler and effective molecular epidemiological technique based on the open-reading frame (ORF) distribution patterns detected by PCR, which we call PCR-based ORF typing (POT).
Methods And Results:
Ten ORFs from genomic islets, five ORFs from genomic islands, and the metallo-β-lactamases (MBLs) blaIMP and blaVIM were selected by comparing the whole-genome sequences of different Ps. aeruginosa strains (PAO1, PA7, UCBPP-PA14 and LESB58). These 17 ORFs were detected, along with a Ps. aeruginosa marker, using 9-plex and 10-plex PCR systems. The genotypes in the POT were compared to those obtained by using PFGE and MLST.
Conclusions:
Using the POT method, molecular epidemiological analyses of Ps. aeruginosa can be completed in 4 h.
Significance And Impact Of The Study:
Since this method is very easy to perform, even in standard clinical laboratories, it could be a valuable tool for monitoring daily infection control measures.
Insights
A new PCR-based ORF typing (POT) method simplifies Pseudomonas aeruginosa molecular epidemiology. This rapid technique aids infection control in standard labs, offering a faster alternative to PFGE and MLST.
Area of Science:
- Microbiology
- Molecular Epidemiology
- Infectious Diseases
Background:
- Nosocomial infections caused by Pseudomonas aeruginosa are a significant concern.
- Current molecular epidemiological techniques like PFGE and MLST are effective but time-consuming and technically complex.
- There is a need for simpler, faster methods for routine laboratory use.
Purpose of the Study:
- To develop a simpler and effective molecular epidemiological technique for Pseudomonas aeruginosa.
- To establish a method based on PCR detection of open-reading frame (ORF) distribution patterns.
- To name this new technique PCR-based ORF typing (POT).
Main Methods:
- Selected 17 ORFs from genomic islets, genomic islands, and metallo-β-lactamases (MBLs) blaIMP and blaVIM by comparing whole-genome sequences of different P. aeruginosa strains.
- Developed 9-plex and 10-plex PCR systems to detect these ORFs and a P. aeruginosa marker.
- Compared POT-generated genotypes with those from PFGE and MLST.
Main Results:
- The POT method successfully detected the selected ORFs and a P. aeruginosa marker.
- Genotypes obtained by POT were comparable to those from PFGE and MLST.
- Molecular epidemiological analysis using POT could be completed within 4 hours.
Conclusions:
- PCR-based ORF typing (POT) is a rapid and effective method for molecular epidemiological analysis of P. aeruginosa.
- The simplicity of POT makes it suitable for standard clinical microbiology laboratories.
- POT can serve as a valuable tool for monitoring daily infection control measures.
