Rapid Identification of Pseudomonas aeruginosa International High-Risk Clones Based on High-Resolution Melting Analysis.

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.

Identification of highly variable sequence fragments in unmapped reads for rapid bacterial genotyping.

BACKGROUND: Bacterial genotyping is a crucial process in outbreak investigation and epidemiological studies. Several typing methods such as pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and whole genome sequencing are currently used in routine clinical practice. However, these methods are costly, time-consuming and have high computational demands. An alternative to these methods is mini-MLST, a quick, cost-effective and robust method based on high-resolution melting analysis. Nevertheless, no standardized approach to identify markers suitable for mini-MLST exists. Here, we present a pipeline for variable fragment detection in unmapped reads based on a modified hybrid assembly approach using data from one sequencing platform. RESULTS: In routine assembly against the reference sequence, high variable reads are not aligned and remain unmapped. If de novo assembly of them is performed, variable genomic regions can be located in created scaffolds. Based on the variability rates calculation, it is possible to find a highly variable region with the same discriminatory power as seven housekeeping gene fragments used in MLST. In the work presented here, we show the capability of identifying one variable fragment in de novo assembled scaffolds of 21 Escherichia coli genomes and three variable regions in scaffolds of 31 Klebsiella pneumoniae genomes. For each identified fragment, the melting temperatures are calculated based on the nearest neighbor method to verify the mini-MLST's discriminatory power. CONCLUSIONS: A pipeline for a modified hybrid assembly approach consisting of reference-based mapping and de novo assembly of unmapped reads is presented. This approach can be employed for the identification of highly variable genomic fragments in unmapped reads. The identified variable regions can then be used in efficient laboratory methods for bacterial typing such as mini-MLST with high discriminatory power, fully replacing expensive methods such as MLST. The results can and will be delivered in a shorter time, which allows immediate and fast infection monitoring in clinical practice.

Sequencing Independent Molecular Typing of Staphylococcus aureus Isolates: Approach for Infection Control and Clonal Characterization.

Staphylococcus aureus is a major bacterial human pathogen that causes a wide variety of clinical manifestations. The main aim of the presented study was to determine and optimize a novel sequencing independent approach that enables molecular typing of S. aureus isolates and elucidates the transmission of emergent clones between patients. In total, 987 S. aureus isolates including both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates were used to evaluate the novel typing approach combining high-resolution melting (HRM) analysis of multilocus sequence typing (MLST) genes (mini-MLST) and spa gene (spa-HRM). The novel approach's discriminatory ability was evaluated by whole-genome sequencing (WGS). The clonal relatedness of tested isolates was set by the BURP and BURST approach using spa and MLST data, respectively. Mini-MLST classified the S. aureus isolates into 38 clusters, followed by spa-HRM classifying the isolates into 101 clusters. The WGS proved HRM-based methods to effectively differentiate between related S. aureus isolates. Visualizing evolutionary relationships among different spa-types provided by the BURP algorithm showed comparable results to MLST/mini-MLST clonal clusters. We proved that the combination of mini-MLST and spa-HRM is rapid, reproducible, and cost-efficient. In addition to high discriminatory ability, the correlation between spa evolutionary relationships and mini-MLST clustering allows the variability in population structure to be monitored. IMPORTANCE Rapid and cost-effective molecular typing tools for Staphylococcus aureus epidemiological applications such as transmission tracking, source attribution and outbreak investigations are highly desirable. High-resolution melting based methods are effective alternative to those based on sequencing. Their good reproducibility and easy performance allow prospective typing of large set of isolates while reaching great discriminatory power. In this study, we established a new epidemiological approach to S. aureus typing. This scheme has the potential to greatly improve epidemiological investigations of S. aureus.