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

Abstract

Widespread release of Bacillus anthracis (anthrax) or Yersinia pestis (plague) would prompt a 24 public health emergency. During an exposure event, high-quality whole genome sequencing (WGS) can 25 identify genetic engineering, including the introduction of antimicrobial resistance (AMR) genes. Here, 26 we developed rapid WGS laboratory and bioinformatics workflows using a long-read nanopore 27 sequencer (MinION) for Y. pestis (6.5h) and B. anthracis (8.5h) and sequenced strains with different 28 AMR profiles. Both salt-precipitation and silica-membrane extracted DNA were suitable for MinION 29 WGS using both rapid and field library preparation methods. In replicate experiments, nanopore quality 30 metrics were defined for genome assembly and mutation analysis. AMR markers were correctly 31 detected and >99% coverage of chromosomes and plasmids was achieved using 100,000 raw sequencing 32 reads. While chromosomes and large and small plasmids were accurately assembled, including novel 33 multimeric forms of the Y. pestis virulence plasmid, pPCP1, MinION reads were error-prone, 34 particularly in homopolymer regions. MinION sequencing holds promise as a practical, front-line 35 strategy for on-site pathogen characterization to speed the public health response during a biothreat 36 emergency. 37 38 40respectively. Deliberate misuse of these pathogens as bioterrorism agents poses a serious public health 41 and safety threat, due to low infectious doses, high lethality, and the ease of production, dissemination 42 and communicability 1 . Strains of both species were weaponized previously, and the 2001 Amerithrax 43 incident underscores the importance of biological threat preparedness and rapid laboratory response 2, 3 . 44Natural anthrax and plague outbreaks are reported annually in animals, and human cases are often 45 zoonotic. While these diseases continue to cycle in isolated, enzootic foci, re-emergence was reported 46 3 during 2016 anthrax outbreaks in Russia and Sweden 4 , and the 2017 pneumonic plague outbreak in 47 Madagascar 5 . Both naturally-occurring and engineered antimicrobial resistance (AMR) have been 48 reported in Y. pestis and B. anthracis 6 . Most B. anthracis strains are susceptible to antibiotics, but 49 surveys of clinical and environmental isolates indicate penicillin resistance occurs in 2 to 16% of strains 50 7 . Attenutated B. anthracis strains with AMR were isolated following laboratory in vitro passaging 51 (selective pressure) and/or targeted genetic manipulation 8, 9 . Laboratory-selected virulent and avirulent 52 Y. pestis strains with quinolone-resistance were previously reported 10, 11 and engineered multi-drug 53 resistant (MDR) strains are a public health concern 12, 13 . Y. pestis is intrinsically susceptible to every 54 antibiotic recommended for human plague therapy, but a few Y. pestis isolates with transferable 55 plasmid-mediated resistance were reported from Madagascar 13, 14, 15, 16 . 56 A public health response to incidents involving human plague or anthrax requires ...