BacCapSeq: a Platform for Diagnosis and Characterization of Bacterial Infections

ABSTRACT We report a platform that increases the sensitivity of high-throughput sequencing for detection and characterization of bacteria, virulence determinants, and antimicrobial resistance (AMR) genes. The system uses a probe set comprised of 4.2 million oligonucleotides based on the Pathosystems Resource Integration Center (PATRIC) database, the Comprehensive Antibiotic Resistance Database (CARD), and the Virulence Factor Database (VFDB), representing 307 bacterial species that include all known human-pathogenic species, known antimicrobial resistance genes, and known virulence factors, respectively. The use of bacterial capture sequencing (BacCapSeq) resulted in an up to 1,000-fold increase in bacterial reads from blood samples and lowered the limit of detection by 1 to 2 orders of magnitude compared to conventional unbiased high-throughput sequencing, down to a level comparable to that of agent-specific real-time PCR with as few as 5 million total reads generated per sample. It detected not only the presence of AMR genes but also biomarkers for AMR that included both constitutive and differentially expressed transcripts. IMPORTANCE BacCapSeq is a method for differential diagnosis of bacterial infections and defining antimicrobial sensitivity profiles that has the potential to reduce morbidity and mortality, health care costs, and the inappropriate use of antibiotics that contributes to the development of antimicrobial resistance.

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Towards routine employment of computational tools for antimicrobial resistance determination via high-throughput sequencing

10.1101/2021.11.03.467126 ◽

2021 ◽

Author(s):

Simone Marini ◽

Rodrigo Mora ◽

Christina Boucher ◽

Noelle Noyes ◽

Mattia Prosperi

Keyword(s):

Antimicrobial Resistance ◽

High Throughput ◽

Bacterial Infections ◽

High Throughput Sequencing ◽

Markov Models ◽

Point Of Care ◽

Economic Loss ◽

Clinical Samples ◽

Sequencing Data ◽

Data Repositories

Antimicrobial resistance (AMR) is a growing threat to public health and farming at large. Without appropriate interventions, it can lead to millions of deaths per year and substantial economic loss worldwide. In clinical and veterinary practice, a timely characterization of the antibiotic susceptibility profile of bacterial infections is a crucial step in optimizing treatment. Fast turnaround of AMR testing is also needed in food safety and infection control surveillance (e.g., contamination of healthcare or long-term nursing facilities). High-throughput sequencing is a promising option for clinical point-of-care and ecological surveillance, opening the opportunity to develop genotyping-based AMR determination as a possibly faster alternative to phenotypic testing. In the present work, we compare the performance of state-of-the-art methods for detection of AMR from high-throughput sequencing data in healthcare settings. We consider five complementary computational approaches --alignment (AMRPlusPlus), deep learning (DeepARG), k-mer genomic signatures (KARGA, ResFinder), and hidden Markov models (Meta-MARC). We use an extensive collection of clinical studies never employed for model training. To do so, we assemble data from multiple, independent AMR high-throughput sequencing experiments collected in a variety of hospital settings, comprising of 585 isolates with a available AMR resistance profiles determined by phenotypic tests across nine antibiotic classes. We show how the prediction landscape of AMR classifiers is highly heterogeneous, with balanced accuracy varying from 0.4 to 0.92. Although some algorithms---ResFinder, KARGA, and AMRPlusPlus-- exhibit overall better balanced accuracy than others, the high per-AMR-class variance and related findings suggest that: (1) all algorithms might be subject to sampling bias present both in data repositories used for training and experimental/clinical settings; and (2) a portion of clinical samples might contain uncharacterized AMR genes that the algorithms---mostly trained on known AMR genes---fail to generalize upon. These results lead us to formulate practical advice for software configuration and application, as well as give suggestions for future study design to further develop AMR prediction tools from proof-of-concept to bedside.

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Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis

mBio ◽

10.1128/mbio.01491-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 150

Author(s):

Thomas Briese ◽

Amit Kapoor ◽

Nischay Mishra ◽

Komal Jain ◽

Arvind Kumar ◽

...

Keyword(s):

Nucleic Acids ◽

High Throughput ◽

High Throughput Sequencing ◽

Viral Infections ◽

Limit Of Detection ◽

Virus Detection ◽

Fold Increase ◽

Sequencing Platform ◽

Oligonucleotide Library ◽

Capture Sequencing

ABSTRACT Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories. Here, we describe the development of a virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) that increases the sensitivity of sequence-based virus detection and characterization. The system uses ~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans. A biotinylated oligonucleotide library was synthesized on the NimbleGen cleavable array platform and used for solution-based capture of viral nucleic acids present in complex samples containing variable proportions of viral and host nucleic acids. The use of VirCapSeq-VERT resulted in a 100- to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero rRNA subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics.IMPORTANCEVirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.

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Clin-mNGS: Automated Pipeline for Pathogen Detection from Clinical Metagenomic Data

Current Bioinformatics ◽

10.2174/1574893615999200608130029 ◽

2020 ◽

Vol 15 ◽

Author(s):

Akshatha Prasanna ◽

Vidya Niranjan

Keyword(s):

Antimicrobial Resistance ◽

High Performance ◽

Pathogen Detection ◽

Bacterial Species ◽

Workflow Management ◽

Metagenomic Data ◽

Antimicrobial Resistance Genes ◽

Culture Independent ◽

Automated Pipeline ◽

User Friendly

Background: Since bacteria are the earliest known organisms, there has been significant interest in their variety and biology, most certainly concerning human health. Recent advances in Metagenomics sequencing (mNGS), a culture-independent sequencing technology have facilitated an accelerated development in clinical microbiology and our understanding of pathogens. Objective: For the implementation of mNGS in routine clinical practice to become feasible, a practical and scalable strategy for the study of mNGS data is essential. This study presents a robust automated pipeline to analyze clinical metagenomic data for pathogen identification and classification. Method: The proposed Clin-mNGS pipeline is an integrated, open-source, scalable, reproducible, and user-friendly framework scripted using the Snakemake workflow management software. The implementation avoids the hassle of manual installation and configuration of the multiple command-line tools and dependencies. The approach directly screens pathogens from clinical raw reads and generates consolidated reports for each sample. Results: The pipeline is demonstrated using publicly available data and is tested on a desktop Linux system and a High-performance cluster. The study compares variability in results from different tools and versions. The versions of the tools are made user modifiable. The pipeline results in quality check, filtered reads, host subtraction, assembled contigs, assembly metrics, relative abundances of bacterial species, antimicrobial resistance genes, plasmid finding, and virulence factors identification. The results obtained from the pipeline are evaluated based on sensitivity and positive predictive value. Conclusion: Clin-mNGS is an automated Snakemake pipeline validated for the analysis of microbial clinical metagenomics reads to perform taxonomic classification and antimicrobial resistance prediction.

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Genotypic Characterization of Antimicrobial Resistant Salmonella spp. Strains from Three Poultry Processing Plants in Colombia

Foods ◽

10.3390/foods10030491 ◽

2021 ◽

Vol 10 (3) ◽

pp. 491

Author(s):

Alejandra Ramirez-Hernandez ◽

Ana K. Carrascal-Camacho ◽

Andrea Varón-García ◽

Mindy M. Brashears ◽

Marcos X. Sanchez-Plata

Keyword(s):

Antimicrobial Resistance ◽

Salmonella Spp ◽

Microbial Profile ◽

Antimicrobial Resistance Genes ◽

Processing Plants ◽

Poultry Processing ◽

Paratyphi B ◽

And Control ◽

Sequence Types

The poultry industry in Colombia has implemented several changes and measures in chicken processing to improve sanitary operations and control pathogens’ prevalence. However, there is no official in-plant microbial profile reference data currently available throughout the processing value chains. Hence, this research aimed to study the microbial profiles and the antimicrobial resistance of Salmonella isolates in three plants. In total, 300 samples were collected in seven processing sites. Prevalence of Salmonella spp. and levels of Enterobacteriaceae were assessed. Additionally, whole-genome sequencing was conducted to characterize the isolated strains genotypically. Overall, the prevalence of Salmonella spp. in each establishment was 77%, 58% and 80% for plant A, B, and C. The mean levels of Enterobacteriaceae in the chicken rinsates were 5.03, 5.74, and 6.41 log CFU/mL for plant A, B, and C. Significant reductions were identified in the counts of post-chilling rinsate samples; however, increased levels were found in chicken parts. There were six distinct Salmonella spp. clusters with the predominant sequence types ST32 and ST28. The serotypes Infantis (54%) and Paratyphi B (25%) were the most commonly identified within the processing plants with a high abundance of antimicrobial resistance genes.

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Emerging Options for the Diagnosis of Bacterial Infections and the Characterization of Antimicrobial Resistance

International Journal of Molecular Sciences ◽

10.3390/ijms22010456 ◽

2021 ◽

Vol 22 (1) ◽

pp. 456

Author(s):

Simone Rentschler ◽

Lars Kaiser ◽

Hans-Peter Deigner

Keyword(s):

Antimicrobial Resistance ◽

Bacterial Infections ◽

Point Of Care ◽

Rapid Identification ◽

Adequate Treatment ◽

Resistance Patterns ◽

Detection And Diagnosis ◽

Patient Prognosis ◽

Point Of Care Detection

Precise and rapid identification and characterization of pathogens and antimicrobial resistance patterns are critical for the adequate treatment of infections, which represent an increasing problem in intensive care medicine. The current situation remains far from satisfactory in terms of turnaround times and overall efficacy. Application of an ineffective antimicrobial agent or the unnecessary use of broad-spectrum antibiotics worsens the patient prognosis and further accelerates the generation of resistant mutants. Here, we provide an overview that includes an evaluation and comparison of existing tools used to diagnose bacterial infections, together with a consideration of the underlying molecular principles and technologies. Special emphasis is placed on emerging developments that may lead to significant improvements in point of care detection and diagnosis of multi-resistant pathogens, and new directions that may be used to guide antibiotic therapy.

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INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing

Science Advances ◽

10.1126/sciadv.abe5054 ◽

2021 ◽

Vol 7 (7) ◽

pp. eabe5054

Author(s):

Qianxin Wu ◽

Chenqu Suo ◽

Tom Brown ◽

Tengyao Wang ◽

Sarah A. Teichmann ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Limit Of Detection ◽

Point Of Care ◽

Population Level ◽

Reaction Products ◽

Testing Strategy ◽

Asymptomatic Patients ◽

Testing Stage ◽

Population Scale

We present INSIGHT [isothermal NASBA (nucleic acid sequence–based amplification) sequencing–based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing–based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.

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Development of a capture sequencing assay for enhanced detection and genotyping of tick-borne pathogens

Scientific Reports ◽

10.1038/s41598-021-91956-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Komal Jain ◽

Teresa Tagliafierro ◽

Adriana Marques ◽

Santiago Sanchez-Vicente ◽

Alper Gokden ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Fold Increase ◽

The United States ◽

Genomic Research ◽

Babesia Microti ◽

Hybridization Capture ◽

Mouse Tissues ◽

Genome Analyses ◽

Capture Sequencing

AbstractInadequate sensitivity has been the primary limitation for implementing high-throughput sequencing for studies of tick-borne agents. Here we describe the development of TBDCapSeq, a sequencing assay that uses hybridization capture probes that cover the complete genomes of the eleven most common tick-borne agents found in the United States. The probes are used for solution-based capture and enrichment of pathogen nucleic acid followed by high-throughput sequencing. We evaluated the performance of TBDCapSeq to surveil samples that included human whole blood, mouse tissues, and field-collected ticks. For Borrelia burgdorferi and Babesia microti, the sensitivity of TBDCapSeq was comparable and occasionally exceeded the performance of agent-specific quantitative PCR and resulted in 25 to > 10,000-fold increase in pathogen reads when compared to standard unbiased sequencing. TBDCapSeq also enabled genome analyses directly within vertebrate and tick hosts. The implementation of TBDCapSeq could have major impact in studies of tick-borne pathogens by improving detection and facilitating genomic research that was previously unachievable with standard sequencing approaches.

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atpD gene sequencing, multidrug resistance traits, virulence-determinants, and antimicrobial resistance genes of emerging XDR and MDR-Proteus mirabilis

Scientific Reports ◽

10.1038/s41598-021-88861-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Abdelazeem M. Algammal ◽

Hany R. Hashem ◽

Khyreyah J. Alfifi ◽

Helal F. Hetta ◽

Norhan S. Sheraba ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Proteus Mirabilis ◽

Biofilm Formation ◽

Gene Sequencing ◽

Opportunistic Pathogen ◽

Severe Illness ◽

Virulence Determinants ◽

Antimicrobial Resistance Genes ◽

Port Said

AbstractProteus mirabilis is a common opportunistic pathogen causing severe illness in humans and animals. To determine the prevalence, antibiogram, biofilm-formation, screening of virulence, and antimicrobial resistance genes in P. mirabilis isolates from ducks; 240 samples were obtained from apparently healthy and diseased ducks from private farms in Port-Said Province, Egypt. The collected samples were examined bacteriologically, and then the recovered isolates were tested for atpD gene sequencing, antimicrobial susceptibility, biofilm-formation, PCR detection of virulence, and antimicrobial resistance genes. The prevalence of P. mirabilis in the examined samples was 14.6% (35/240). The identification of the recovered isolates was confirmed by the atpD gene sequencing, where the tested isolates shared a common ancestor. Besides, 94.3% of P. mirabilis isolates were biofilm producers. The recovered isolates were resistant to penicillins, sulfonamides, β-Lactam-β-lactamase-inhibitor-combinations, tetracyclines, cephalosporins, macrolides, and quinolones. Using PCR, the retrieved strains harbored atpD, ureC, rsbA, and zapA virulence genes with a prevalence of 100%, 100%, 94.3%, and 91.4%, respectively. Moreover, 31.4% (11/35) of the recovered strains were XDR to 8 antimicrobial classes that harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Besides, 22.8% (8/35) of the tested strains were MDR to 3 antimicrobial classes and possessed blaTEM, tetA, and sul1genes. Furthermore, 17.1% (6/35) of the tested strains were MDR to 7 antimicrobial classes and harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Alarmingly, three strains were carbapenem-resistant that exhibited PDR to all the tested 10 antimicrobial classes and shared blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Of them, two strains harbored the blaNDM-1 gene, and one strain carried the blaKPC gene. In brief, to the best of our knowledge, this is the first study demonstrating the emergence of XDR and MDR-P.mirabilis in ducks. Norfloxacin exhibited promising antibacterial activity against the recovered XDR and MDR-P. mirabilis. The emergence of PDR, XDR, and MDR-strains constitutes a threat alarm that indicates the complicated treatment of the infections caused by these superbugs.

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