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.

• Klíčová slova
• Pseudomonas aeruginosa, high-resolution melting, mini-MLST, molecular epidemiology, strain typing,
• MeSH
• buněčné klony MeSH
• genotyp MeSH
• lidé MeSH
• molekulární epidemiologie metody   MeSH
• multilokusová sekvenční typizace MeSH
• prospektivní studie MeSH
• pseudomonádové infekce * epidemiologie   mikrobiologie   MeSH
• Pseudomonas aeruginosa * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• Klíčová slova
• Bacterial genotyping, De novo assembly, Genome assembly, Mini-MLST, Multilocus sequence typing, Unmapped reads,
• MeSH
• Bacteria * genetika   MeSH
• Escherichia coli genetika   MeSH
• genom * MeSH
• genotyp MeSH
• multilokusová sekvenční typizace metody   MeSH
• techniky typizace bakterií metody   MeSH
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• MLST, MRSA, MSSA, high-resolution melting, mini-MLST, spa-typing, whole-genome sequencing,
• MeSH
• kontrola infekce * MeSH
• lidé MeSH
• methicilin rezistentní Staphylococcus aureus genetika   izolace a purifikace   MeSH
• molekulární typizace metody   MeSH
• multilokusová sekvenční typizace MeSH
• prospektivní studie MeSH
• reprodukovatelnost výsledků MeSH
• sekvenování celého genomu MeSH
• stafylokokové infekce diagnóza   mikrobiologie   MeSH
• Staphylococcus aureus klasifikace   genetika   izolace a purifikace   MeSH
• techniky typizace bakterií metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Routinely used typing methods including MLST, rep-PCR and whole genome sequencing (WGS) are time-consuming, costly, and often low throughput. Here, we describe a novel mini-MLST scheme for Eschericha coli as an alternative method for rapid genotyping. Using the proposed mini-MLST scheme, 10,946 existing STs were converted into 1,038 Melting Types (MelTs). To validate the new mini-MLST scheme, in silico analysis was performed on 73,704 strains retrieved from EnteroBase resulting in discriminatory power D = 0.9465 (CI 95% 0.9726-0.9736) for mini-MLST and D = 0.9731 (CI 95% 0.9726-0.9736) for MLST. Moreover, validation on clinical isolates was conducted with a significant concordance between MLST, rep-PCR and WGS. To conclude, the great portability, efficient processing, cost-effectiveness, and high throughput of mini-MLST represents immense benefits, even when accompanied with a slightly lower discriminatory power than other typing methods. This study proved mini-MLST is an ideal method to screen and subgroup large sets of isolates and/or quick strain typing during outbreaks. In addition, our results clearly showed its suitability for prospective surveillance monitoring of emergent and high-risk E. coli clones'.

• MeSH
• bakteriální geny * MeSH
• denaturace nukleových kyselin MeSH
• DNA bakterií chemie   genetika   MeSH
• DNA primery MeSH
• epidemický výskyt choroby MeSH
• Escherichia coli klasifikace   genetika   izolace a purifikace   MeSH
• genom bakteriální MeSH
• genotypizační techniky * MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• jednonukleotidový polymorfismus * MeSH
• multilokusová sekvenční typizace metody   MeSH
• počítačová simulace MeSH
• polymerázová řetězová reakce metody   MeSH
• repetitivní sekvence nukleových kyselin MeSH
• sekvenování celého genomu MeSH
• surveillance populace MeSH
• techniky typizace bakterií * MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• DNA bakterií MeSH
• DNA primery MeSH

Studying bacterial population diversity is important to understand healthcare associated infections' epidemiology and has a significant impact on dealing with multidrug resistant bacterial outbreaks. We characterised the extended-spectrum beta-lactamase producing K. pneumoniae (ESBLp KPN) population in our hospital using mini-MLST. Then we used whole genome sequencing (WGS) to compare selected isolates belonging to the most prevalent melting types (MelTs) and the colonization/infection pair isolates collected from one patient to study the ESBLp KPN population's genetic diversity. A total of 922 ESBLp KPN isolates collected between 7/2016 and 5/2018 were divided into 38 MelTs using mini-MLST with only 6 MelTs forming 82.8% of all isolates. For WGS, 14 isolates from the most prominent MelTs collected in the monitored period and 10 isolates belonging to the same MelTs collected in our hospital in 2014 were randomly selected. Resistome, virulome and ST were MelT specific and stable over time. A maximum of 23 SNV per core genome and 58 SNV per core and accessory genome were found. To determine the SNV relatedness cut-off values, 22 isolates representing colonization/infection pair samples obtained from 11 different patients were analysed by WGS with a maximum of 22 SNV in the core genome and 40 SNV in the core and accessory genome within pairs. The mini-MLST showed its potential for real-time epidemiology in clinical practice. However, for outbreak evaluation in a low diversity bacterial population, mini-MLST should be combined with more sensitive methods like WGS. Our findings showed there were only minimal differences within the core and accessory genome in the low diversity hospital population and gene based SNV analysis does not have enough discriminatory power to differentiate isolate relatedness. Thus, intergenic regions and mobile elements should be incorporated into the analysis scheme to increase discriminatory power.

• MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• dítě MeSH
• DNA bakterií genetika   MeSH
• dospělí MeSH
• infekce bakteriemi rodu Klebsiella enzymologie   epidemiologie   genetika   mikrobiologie   MeSH
• infekce spojené se zdravotní péčí enzymologie   epidemiologie   genetika   mikrobiologie   MeSH
• Klebsiella pneumoniae enzymologie   genetika   izolace a purifikace   MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mnohočetná bakteriální léková rezistence * MeSH
• multilokusová sekvenční typizace * MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• sekvenování celého genomu * MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• DNA bakterií MeSH