Rapid Nanopore Whole-Genome Sequencing for Anthrax Emergency Preparedness

Heather P. McLaughlin; Julia V. Bugrysheva; Andrew B. Conley; Christopher A. Gulvik; Blake Cherney; Cari B. Kolton; Chung K. Marston; Elke Saile; Erin Swaney; David Lonsway; Amy S. Gargis; Thiphasone Kongphet-Tran; Christine Lascols; Pierre Michel; Julie Villanueva; Alex R. Hoffmaster; Jay E. Gee; David Sue

Disclosures

Emerging Infectious Diseases. 2020;26(2):358-361. 

In This Article

Abstract and Introduction

Abstract

Human anthrax cases necessitate rapid response. We completed Bacillus anthracis nanopore whole-genome sequencing in our high-containment laboratory from a human anthrax isolate hours after receipt. The de novo assembled genome showed no evidence of known antimicrobial resistance genes or introduced plasmid(s). Same-day genomic characterization enhances public health emergency response.

Introduction

Bacillus anthracis causes anthrax, a deadly infectious disease, and is found worldwide, including areas of the United States. Naturally occurring anthrax outbreaks are reported annually in wild and domestic grazing animals, but human transmission is rare.[1] Deliberate misuse of B. anthracis as a bioweapon could pose an immediate risk to human populations. In such instances, a timely response is critical to reduce morbidity and mortality rates.

After the anthrax incidents during 2001, the Centers for Disease Control and Prevention (CDC) published medical countermeasure recommendations for human anthrax treatment and postexposure prophylaxis using antimicrobial drugs, including amoxicillin, ciprofloxacin, doxycycline, levofloxacin, and penicillin.[2] Most B. anthracis strains are susceptible to antimicrobial drugs; however, naturally occurring and engineered antimicrobial-resistant strains have been reported.[3–5] Laboratory antimicrobial susceptibility testing (AST) by broth microdilution (BMD) remains the standard method to determine MIC values but requires ≥16 hours before results are available. During an anthrax emergency, rapid genomic characterization of the implicated B. anthracis strain(s) could identify sequences associated with drug resistance.

Single-nucleotide mutations in chromosomal B. anthracis quinolone resistance–determining regions of gyrA, gyrB, parC, and parE genes can lead to ciprofloxacin resistance, and gene acquisition can lead to tetracycline and doxycycline resistance.[3,4,6] Penicillin resistance can result from a chromosomal mutation in the antisigma factor gene, rsiP.[5] Most B. anthracis strains carrying this signature rsiP mutation are resistant to penicillin and amoxicillin.[5,7,8] Detection of known antimicrobial resistance (AMR) mutations or other novel gene insertions and deletions (indels) in the clonal B. anthracis genome signals genetic anomalies and could influence treatment and postexposure prophylaxis strategies.

Whole-genome sequencing (WGS) can identify gene indels, mutations, or previously undescribed genetic elements, including extrachromosomal plasmid DNA. However, common short-read sequencing (SRS) technologies have difficulty resolving bacterial genome structure because de novo assemblies yield multiple contigs. Long-read nanopore sequencing with the MinION device (Oxford Nanopore Technologies, https://nanoporetech.com) can resolve repetitive sequences and structural genomic rearrangements and enables complete bacterial genome finishing.[9] Although MinION data are error-prone, especially in homopolymeric regions,[10] compared with Illumina (https://www.illumina.com)–based SRS, it is available immediately during the sequencing run, enabling rapid assembly and analysis. The technology enables real-time sequencing, including direct pathogen identification from patient specimens, and holds the promise for future point-of-care applications that speed laboratory results reporting.[11,12] Portable WGS instruments are advantageous for laboratories with limited space and remove the need to transfer DNA out of high-containment laboratories for sequencing, mitigating exposure risks to personnel.[13]

CDC described a rapid nanopore sequencing approach and custom bioinformatics pipeline for B. anthracis that yielded complete chromosome and plasmid assemblies, and detected known AMR genes and mutations in avirulent laboratory strains.[13] On the morning of August 2, 2019, our laboratory received a B. anthracis culture isolate (Ba0914) from a naturally occurring human anthrax case in Texas. Same-day laboratory WGS and bioinformatics analysis were performed. This study describes the laboratory work and demonstrates the usefulness of rapid WGS to inform time-sensitive public health responses.

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