Genotyping of Pseudomonas aeruginosa Type III Secretion System Using Magnetic Enrichment Multiplex Polymerase Chain Reaction and Chemiluminescence
Abstract:
The pathologic characteristics and toxicity mechanism of Pseudomonas aeruginosa are different in strains with different Type III secretion system (T3SS) genes. The T3SS gene based genotyping of P. aeruginosa strains is important to understand its virulence and predict the clinical outcomes. In this study, a rapid and automatable method for T3SS genotyping was developed using magnetic enrichment multiplex PCR and chemiluminescence. Three P. aeruginosa standard strains were analyzed using this method. The results showed that the chemiluminescent intensity of exoT, exoY, and exoS of these strains were 10 times greater than that of the control, and that their Q values were greater than 2.1. These results were consistent with the regular PCR and electrophoresis results, indicating that the method was reliable. Out of the 22 clinical isolates tested using this method, 100%, 72.7%, 95.5%, and 4.5% of the isolates contained exoT, exoY, exoS, and exoU genes, respectively. The isolates harbored either exoS or exoU gene, but not both. All genotyping results of the isolates were consistent with the information obtained using regular PCR and electrophoresis.
Insights
This study developed a rapid method for Pseudomonas aeruginosa Type III secretion system (T3SS) genotyping. The reliable technique accurately identified virulence genes in clinical isolates, aiding in understanding bacterial pathogenicity.
Area of Science:
- Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- Pseudomonas aeruginosa strains exhibit varying virulence due to differences in Type III secretion system (T3SS) genes.
- Understanding T3SS gene profiles is crucial for assessing P. aeruginosa pathogenicity and predicting clinical outcomes.
Purpose of the Study:
- To develop a rapid, automatable method for T3SS genotyping of P. aeruginosa.
- To validate the reliability and efficiency of the new method using standard and clinical strains.
Main Methods:
- Magnetic enrichment multiplex PCR combined with chemiluminescence was employed for T3SS genotyping.
- The method was validated against standard PCR and electrophoresis techniques.
- Chemiluminescent intensity and Q values were used to assess gene presence and quantity.
Main Results:
- The developed method demonstrated high sensitivity, with chemiluminescent signals 10 times greater than controls for key T3SS genes (exoT, exoY, exoS).
- Genotyping of 22 clinical isolates revealed high prevalence of exoT (100%), exoY (72.7%), and exoS (95.5%), with low prevalence of exoU (4.5%).
- A mutually exclusive presence of exoS or exoU genes was observed in clinical isolates, consistent with conventional methods.
Conclusions:
- The magnetic enrichment multiplex PCR and chemiluminescence method provides a reliable and efficient approach for P. aeruginosa T3SS genotyping.
- This rapid genotyping technique can aid in the clinical management and understanding of P. aeruginosa infections.
