Evaluation of WGS performance for bacterial pathogen characterization with the Illumina technology optimized for time-critical situations

Whole genome sequencing (WGS) has become the reference standard for bacterial outbreak investigation and pathogen typing, providing a resolution unattainable with conventional molecular methods. Data generated with Illumina sequencers can however only be analysed after the sequencing run has finished, thereby losing valuable time during emergency situations. We evaluated both the effect of decreasing overall run time, and also a protocol to transfer and convert intermediary files generated by Illumina sequencers enabling real-time data analysis for multiple samples part of the same ongoing sequencing run, as soon as the forward reads have been sequenced. To facilitate implementation for laboratories operating under strict quality systems, extensive validation of several bioinformatics assays (16S rRNA species confirmation, gene detection against virulence factor and antimicrobial resistance databases, SNP-based antimicrobial resistance detection, serotype determination, and core genome multilocus sequence typing) for three bacterial pathogens ( Mycobacterium tuberculosis , Neisseria meningitidis , and Shiga-toxin producing Escherichia coli ) was performed by evaluating performance in function of the two most critical sequencing parameters, i.e. read length and coverage. For the majority of evaluated bioinformatics assays, actionable results could be obtained between 14 and 22 h of sequencing, decreasing the overall sequencing-to-results time by more than half. This study aids in reducing the turn-around time of WGS analysis by facilitating a faster response in time-critical scenarios and provides recommendations for time-optimized WGS with respect to required read length and coverage to achieve a minimum level of performance for the considered bioinformatics assay(s), which can also be used to maximize the cost-effectiveness of routine surveillance sequencing when response time is not essential.

Performance of a loop-mediated isothermal amplification assay (Isoplex CRE-ART) to detect common carbapenemase-encoding genes in Gram-negative bacteria

Carbapenem-resistant Gram-negative bacteria (CR-GNB) are a major source of nosocomial infections worldwide. In this study, the ability of a loop-mediated isothermal amplification (LAMP)-based method (Isoplex CRE-ART) to rapidly detect carbapenemase-encoding genes bla OXA-48-like, bla OXA-23-like, bla OXA-24-like, bla KPC, bla VIM, bla NDM and bla IMP in 231 carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii isolates was investigated. The accuracy of the LAMP test was compared to results of molecular isolate characterization using a Laboratory Developed Test multiplex carbapenemase PCR assay. The LAMP test correctly identified the presence of on-panel carbapenemases with a sensitivity of 99.16 % [95 % confidence interval (CI): 95.39–99.96 %] and a specificity of 98.21 % (95 % CI: 93.72–99.68 %) in 60 min. Our findings suggest that the Isoplex CRE-ART assay is able to rapidly identify carbapenemase genes in CR-GNB and improves options for pathogen characterization in the context of clinical microbiological and infection control diagnostics.

SeqScreen: Accurate and Sensitive Functional Screening of Pathogenic Sequences via Ensemble Learning

Modern benchtop DNA synthesis techniques and increased concern of emerging pathogens have elevated the importance of screening oligonucleotides for pathogens of concern. However, accurate and sensitive characterization of oligonucleotides is an open challenge for many of the current techniques and ontology-based tools. To address this gap, we have developed a novel software tool, SeqScreen, that can accurately and sensitively characterize short DNA sequences using a set of curated Functions of Sequences of Concern (FunSoCs), novel functional labels specific to microbial pathogenesis which describe the pathogenic potential of individual proteins. We show that our ensemble machine learning model after training on these curations can label sequences with FunSoCs via an imbalanced multi-class and multi-label classification task with high accuracy. In summary, SeqScreen represents a first step towards a novel paradigm of functionally informed pathogen characterization from genomic and metagenomic datasets. SeqScreen is open-source and freely available for download at: https://www.gitlab.com/treangenlab/seqscreen .

Anthracnose in U.S. Tea: Pathogen Characterization and Susceptibility Among Six Tea Accessions

Tea (Camellia sinensis [L.] O. Kuntze) is under investigation as a specialty crop in the United States. Anthracnose is a serious disease in global tea production, but there is no literature on the susceptibility of U.S. planting materials to this disease. We isolated a Colletotrichum species from symptomatic plants in a field trial and identified the pathogen as Colletotrichum camelliae based on morphology and sequencing of the ITS, GS, GAPDH, TUB2, and ApMat domains. A phylogenetic analysis showed that local field isolates were genetically similar to one another and grouped with isolates from C. sinensis in China, whereas a local isolate from an ornamental camellia (C. japonica) was more closely related to C. camelliae isolated from other Camellia spp. Six commercially available tea accessions were evaluated in detached leaf assays for susceptibility to this anthracnose pathogen. All accessions were susceptible to infection, with Fairhope and Small Leaf having the largest lesion sizes. In field observations, Fairhope, Big Leaf, and Small Leaf consistently had lower disease severity than Georgian over two growing seasons. This work documents the impact of anthracnose on U.S. tea varieties and may help shape future directions of tea research, breeding, and recommendations for growers in establishing a novel industry.

Rapid Detection of Genetic Engineering, Structural Variation, and Antimicrobial Resistance Markers in Bacterial Biothreat Pathogens by Nanopore Sequencing

Widespread release of Bacillus anthracis (anthrax) or Yersinia pestis (plague) would prompt a public health emergency. During an exposure event, high-quality whole genome sequencing (WGS) can identify genetic engineering, including the introduction of antimicrobial resistance (AMR) genes. Here, we developed rapid WGS laboratory and bioinformatics workflows using a long-read nanopore sequencer (MinION) for Y. pestis (6.5 h) and B. anthracis (8.5 h) and sequenced strains with different AMR profiles. Both salt-precipitation and silica-membrane extracted DNA were suitable for MinION WGS using both rapid and field library preparation methods. In replicate experiments, nanopore quality metrics were defined for genome assembly and mutation analysis. AMR markers were correctly detected and >99% coverage of chromosomes and plasmids was achieved using 100,000 raw sequencing reads. While chromosomes and large and small plasmids were accurately assembled, including novel multimeric forms of the Y. pestis virulence plasmid, pPCP1, MinION reads were error-prone, particularly in homopolymer regions. MinION sequencing holds promise as a practical, front-line strategy for on-site pathogen characterization to speed the public health response during a biothreat emergency.

Rapid Detection of Genetic Engineering, Structural Variation, and Antimicrobial Resistance Markers in Bacterial Biothreat Pathogens by Nanopore Sequencing

Widespread release of Bacillus anthracis (anthrax) or Yersinia pestis (plague) would prompt a public health emergency. During an exposure event, high-quality whole genome sequencing (WGS) can identify genetic engineering, including the introduction of antimicrobial resistance (AMR) genes. Here, we developed rapid WGS laboratory and bioinformatics workflows using a long-read nanopore sequencer (MinION) for Y. pestis (6.5h) and B. anthracis (8.5h) and sequenced strains with different AMR profiles. Both salt-precipitation and silica-membrane extracted DNA were suitable for MinION WGS using both rapid and field library preparation methods. In replicate experiments, nanopore quality metrics were defined for genome assembly and mutation analysis. AMR markers were correctly detected and >99% coverage of chromosomes and plasmids was achieved using 100,000 raw sequencing reads. While chromosomes and large and small plasmids were accurately assembled, including novel multimeric forms of the Y. pestis virulence plasmid, pPCP1, MinION reads were error-prone, particularly in homopolymer regions. MinION sequencing holds promise as a practical, front-line strategy for on-site pathogen characterization to speed the public health response during a biothreat emergency.

Recent advances on the characterization and control of sunflower soilborne pathogens under climate change conditions

The control of soilborne crop pathogens is conditioned by the limited management options due to difficult access to active infection courts and to restrictions in the use of synthetic pesticides in Europe. For most soilborne sunflower pathogens, an effective management relies on genetic resistance which is, however, hindered by new pathogen populations (new races). Special emphasis is thus put on updated monitoring and characterization of pathogens and on the enlargement of the set of tools for disease management. Concerning characterization, advances on the population structure of Verticillium dahliae affecting sunflower by means of genetic, molecular and pathogenic approaches are presented. Also in relation to increases of sunflower wilt diseases recently observed, the fungus Cadophora malorum has been identified in Russia and reported as a new pathogen of this crop. Third, new races of Plasmopara halstedii (sunflower downy mildew), have been identified in Spain and Portugal. Most of them have a high virulence, since they overcome several genes for resistance. With regard to alternatives for disease control, entomopathogenic fungi (EF) constitute a novel tool. Used for years in Integrated Pest Management strategies due to their efficacy in controlling insect pests affecting crops, new ecological roles of these fungi have recently been reported. The EF species Beauveria bassiana and Metarhizium brunneum have been assessed by their in vitro effect against V. dahliae and C. malorum by our research group. Our results suggest that antibiosis and/or competition for ecological niche are operating in some EF-pathogen interactions. In summary, pathogen characterization is essential for genetic resistance for worldwide environments of sunflower production. Moreover, the security of sunflower yield and profitability is dependent not only on effective genetic resistance, but also on additional new control options that can be included in successful strategies of sunflower disease management.

Targeted Enrichment for Pathogen Detection and Characterization in Three Felid Species

ABSTRACT Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.