BacCapSeq: a Platform for Diagnosis and Characterization of Bacterial Infections

Allicock, Orchid M; Guo, Cheng; Uhlemann, Anne‐Catrin; Whittier, Susan; Chauhan, Lokendra V.; Garcia, Joel A.; Price, Adam; Morse, Stephen S.; Mishra, Nischay; Briese, Thomas; Lipkin, W. Ian

doi:10.1128/mbio.02007-18

Cited by 50 publications

(41 citation statements)

References 31 publications

(37 reference statements)

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“…The proportion of the genomes targeted by our probe set ranged from 0.21 to 0.97%, consisting of 13 to 65 ARGs representing 102 unique genes among the isolates tested (Table S1). Genomic DNA from four different species was tested individually via enrichment on two different library preparations (the NEBNext Ultra II library preparation versus the modified Meyer 23S rRNA methyltransferase (37) Trimethoprim resistance (29) Other 122FIG 2 Design of a probe set to target over 2,000 antibiotic resistance genes. Breakdown of resistance gene classes from CARD that are targeted by probes.…”

Section: Resultsmentioning

confidence: 99%

“…Increased interest in targeted capture approaches has resulted in the design of probe sets for the detection of viruses, bacteria, and, more recently, antibiotic resistance elements (26)(27)(28)(29). Although our study is not the first to employ targeted capture for antibiotic resistance genes, we have focused on a rigorous probe design that includes choosing an appropriate reference database, robust probe set validation, and experimental considerations for enrichment, including reduced input library and probe concentrations (25,(30)(31)(32)(33).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Other approaches targeting ARGs have included probes for species identifiers, plasmid markers, and biocide or metal resistance (27)(28)(29). These probe sets range in target capacity from 5,557 genes (3.34 Mb) (28) to over 78,600 genes (88.13 Mb) (27) and comprise up to 4 million probes (29). Other strategies involve designing one probe per gene, tiling probes across a gene without overlap (1ϫ coverage), or interprobe distances of up to 121 nucleotides (28,29).…”

Section: Discussionmentioning

confidence: 99%

“…This strategy offers significant advantages for the sampling of resistomes in a variety of environments where resistance genes are generally rare and genetically diverse. Indeed, recently, the use of this approach for resistance gene capture has been explored by other groups (27)(28)(29). However, these accounts target many other genes that are not rigorously associated with resistance, increasing the sequencing cost and the opportunity for false-positive gene identification.…”

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confidence: 99%

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Capturing the Resistome: a Targeted Capture Method To Reveal Antibiotic Resistance Determinants in Metagenomes

Guitor

Raphenya

Klunk

et al. 2019

Antimicrob Agents Chemother

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Identification of the nucleotide sequences encoding antibiotic resistance elements and determination of their association with antibiotic resistance are critical to improve surveillance and monitor trends in antibiotic resistance. Current methods to study antibiotic resistance in various environments rely on extensive deep sequencing or laborious culturing of fastidious organisms, both of which are heavily time-consuming operations. An accurate and sensitive method to identify both rare and common resistance elements in complex metagenomic samples is needed. Referencing the sequences in the Comprehensive Antibiotic Resistance Database, we designed a set of 37,826 probes to specifically target over 2,000 nucleotide sequences associated with antibiotic resistance in clinically relevant bacteria. Testing of this probe set on DNA libraries generated from multidrug-resistant bacteria to selectively capture resistance genes reproducibly produced higher numbers of reads on target at a greater length of coverage than shotgun sequencing. We also identified additional resistance gene sequences from human gut microbiome samples that sequencing alone was not able to detect. Our method to capture the resistome enables a sensitive means of gene detection in diverse environments where genes encoding antibiotic resistance represent less than 0.1% of the metagenome.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Capturing the Resistome: a Targeted Capture Method To Reveal Antibiotic Resistance Determinants in Metagenomes

Guitor

Raphenya

Klunk

et al. 2019

Antimicrob Agents Chemother

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Human Polyomavirus 9—An Emerging Cutaneous and Pulmonary Pathogen in Solid Organ Transplant Recipients

Mishra

Strom

et al. 2022

JAMA Dermatol

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IMPORTANCEWe describe the first report to our knowledge of cutaneous and systemic pathogenicity of human polyomavirus 9 in solid organ transplant recipients.OBJECTIVE Three solid organ transplant recipients developed a widespread, progressive, violaceous, and hyperkeratotic skin eruption. All died from pulmonary and multiorgan failure around 1 year from onset of the rash. Routine clinical diagnostic testing could not identify any causative agent; therefore, samples and autopsies were investigated for novel pathogens using high-throughput sequencing.DESIGN, SETTING, AND PARTICIPANTS This case series, including 3 solid organ transplant recipients who developed characteristic pink, violaceous, or brown hyperkeratotic papules and plaques throughout the body, was conducted at the Columbia University Medical Center. Lesional skin biopsies were collected from all 3 patients and subjected to high-throughput illumina sequencing for identification of microbial pathogens. Human polyomavirus 9 was identified in lesional skin biopsies. We subsequently collected ocular swabs, oral swabs, urine samples, and blood samples from patients, and organ tissues at autopsy in 1 patient. We investigated these samples for the presence of human polyomavirus 9 using in situ hybridization and quantitative polymerase chain reaction (PCR) assays. MAIN OUTCOMES AND MEASURES A description of the clinical and pathologic findings of 3 patients.RESULTS This case series study found that human polyomavirus 9 was detected in the skin biopsies of all 3 patients by a capture-based high-throughput sequencing method platform (VirCapSeq-VERT). Human polyomavirus 9 was also detected in blood, oral, ocular swabs, and urine by real-time polymerase chain reaction (PCR) assay. In situ hybridization and quantitative PCR assays were performed on the skin biopsies from 3 patients and lung autopsy of 1 patient, which showed the presence of human polyomavirus 9 messenger RNA transcripts, indicating active viral replication and pathogenesis in the skin and lungs.CONCLUSIONS AND RELEVANCE Human polyomavirus 9 was associated with the widespread cutaneous eruption. All 3 patients had progression of cutaneous disease, accompanied by clinical deterioration, pulmonary failure, and death. One patient underwent autopsy and human polyomavirus 9 was identified in the lungs and paratracheal soft tissue. These findings suggest that human polyomavirus 9 may be associated with cutaneous and possibly pulmonary infection and death in solid organ transplant recipients.

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MBPD: A multiple bacterial pathogen detection pipeline for One Health practices

Yang

Jiang

Zhang

et al. 2023

iMeta

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Bacterial pathogens are one of the major threats to biosafety and environmental health, and advanced assessment is a prerequisite to combating bacterial pathogens. Currently, 16S rRNA gene sequencing is efficient in the open-view detection of bacterial pathogens. However, the taxonomic resolution and applicability of this method are limited by the domain-specific pathogen database, taxonomic profiling method, and sequencing target of 16S variable regions. Here, we present a pipeline of multiple bacterial pathogen detection (MBPD) to identify the animal, plant, and zoonotic pathogens. MBPD is based on a large, curated database of the full-length 16S genes of 1986 reported bacterial pathogen species covering 72,685 sequences. In silico comparison allowed MBPD to provide the appropriate similarity threshold for both full-length and variableregion sequencing platforms, while the subregion of V3−V4 (mean: 88.37%, accuracy rate compared to V1−V9) outperformed other variable regions in pathogen identification compared to full-length sequencing. Benchmarking on real data sets suggested the superiority of MBPD in a broader range of pathogen detections compared with other methods, including 16SPIP and MIP. Beyond detecting the known causal agent of animal, human, and plant diseases, MBPD is capable of identifying cocontaminating pathogens from biological and environmental samples. Overall, we provide a MBPD pipeline for agricultural, veterinary, medical, and environmental monitoring to achieve One Health.

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BacCapSeq: a Platform for Diagnosis and Characterization of Bacterial Infections

Cited by 50 publications

References 31 publications

Capturing the Resistome: a Targeted Capture Method To Reveal Antibiotic Resistance Determinants in Metagenomes

Capturing the Resistome: a Targeted Capture Method To Reveal Antibiotic Resistance Determinants in Metagenomes

Human Polyomavirus 9—An Emerging Cutaneous and Pulmonary Pathogen in Solid Organ Transplant Recipients

MBPD: A multiple bacterial pathogen detection pipeline for One Health practices

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