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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
Published on: November 10, 2015
Rapid Identification of Pseudomonas aeruginosa International High-Risk Clones Based on High-Resolution Melting
Kristyna Dufkova1,2, Matej Bezdicek1, Marketa Nykrynova1,3
1Department of Internal Medicine - Haematology and Oncology, University Hospital Brno, Brno, Czech Republic.
Abstract:
The Pseudomonas aeruginosa population has a nonclonal epidemic structure. It is generally composed of a limited number of widespread clones selected from a background of many rare and unrelated genotypes recombining at high frequency. Due to the increasing prevalence of nosocomial infections caused by multidrug-resistant/extensively drug-resistant (MDR/XDR) strains, it is advisable to implement infection control measures. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) are considered the gold standard methods in bacterial typing, despite being limited by cost, staff, and instrumental demands. Here, we present a novel mini-MLST scheme for P. aeruginosa rapid genotyping based on high-resolution melting analysis. Using the proposed mini-MLST scheme, 3,955 existing sequence types (STs) were converted into 701 melting types (MelTs), resulting in a discriminatory power of D = 0.993 (95% confidence interval [CI], 0.992 to 0.994). Whole-genome sequencing of 18 clinical isolates was performed to support the newly designed mini-MLST scheme. The clonal analysis of STs belonging to MelTs associated with international high-risk clones (HRCs) performed by goeBURST software revealed that a high proportion of the included STs are highly related to HRCs and have also been witnessed as responsible for serious infections. Therefore, mini-MLST provides a clear warning for the potential spread of P. aeruginosa clones recognized as MDR/XDR strains with possible serious outcomes. IMPORTANCE In this study, we designed a novel mini-MLST typing scheme for Pseudomonas aeruginosa. Its great discriminatory power, together with ease of performance and short processing time, makes this approach attractive for prospective typing of large isolate sets. Integrating the novel P. aeruginosa molecular typing scheme enables the development and spread of MDR/XDR high-risk clones to be investigated.
Insights
A new mini-MLST method rapidly genotypes Pseudomonas aeruginosa, identifying high-risk multidrug-resistant (MDR/XDR) clones. This approach aids in tracking the spread of dangerous bacterial strains and implementing infection control measures.
Area of Science:
- Microbiology
- Molecular Biology
- Epidemiology
Background:
- Pseudomonas aeruginosa exhibits a nonclonal epidemic structure with widespread clones and rare genotypes.
- Nosocomial infections caused by multidrug-resistant/extensively drug-resistant (MDR/XDR) P. aeruginosa strains are increasing.
- Gold standard typing methods like PFGE and MLST are resource-intensive.
Purpose of the Study:
- To develop a novel, rapid, and cost-effective mini-MLST scheme for P. aeruginosa genotyping.
- To assess the discriminatory power and efficiency of the new mini-MLST scheme.
- To support the investigation of high-risk clone spread and associated infections.
Main Methods:
- A novel mini-MLST scheme was designed for P. aeruginosa.
- High-resolution melting analysis was employed for genotyping.
- Whole-genome sequencing of 18 clinical isolates was performed for validation.
- goeBURST software was used for clonal analysis of sequence types (STs) and melting types (MelTs).
Main Results:
- The mini-MLST scheme converted 3,955 STs into 701 MelTs with a high discriminatory power (D=0.993).
- Whole-genome sequencing supported the validity of the mini-MLST scheme.
- A significant proportion of MelTs were related to international high-risk clones (HRCs) responsible for serious infections.
Conclusions:
- The mini-MLST scheme offers a rapid, highly discriminatory, and practical approach for P. aeruginosa typing.
- This method can effectively identify and monitor the spread of MDR/XDR P. aeruginosa high-risk clones.
- The scheme facilitates timely infection control interventions against dangerous bacterial strains.
