Abstract
Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species. Underscored by the impact of the 2020 SARS-CoV-2 outbreak, 2016 Zika pandemic, 2014 Ebola outbreak, 2001 anthrax letter attacks, and 1984 Rajneeshee Salmonella attacks, the threat of future epidemics/pandemics and/or terrorist/criminal use of pathogenic organisms warrants continued exploration and development of both classic and alternative methods of detecting biothreat agents. Volatile organic compounds (VOCs) comprise a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature. Recently, the diagnostic potential of VOCs has been realized, as correlations between the microbial VOC metabolome and specific bacterial pathogens have been identified. Herein, we describe the use of microbial VOC profiles as fingerprints for the identification of biothreat-relevant microbes, and for differentiating between a kanamycin susceptible and resistant strain. Additionally, we demonstrate microbial VOC profiling using a rapid-throughput VOC metabolomics method we refer to as ‘simultaneous multifiber headspace solid-phase microextraction’ (simulti-hSPME). Finally, through VOC analysis, we illustrate a rapid non-invasive approach to the diagnosis of BALB/c mice infected with either F. tularensis SCHU S4 or Y. pestis CO92.
Highlights
Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species
Using gas chromatography coupled with mass spectrometry (GC–MS), we have previously demonstrated the diagnostic potential of volatile organic compounds (VOCs) emanating from biological s amples[4,5,6,7]
F. tularensis spp. novicida, F. tularensis spp. tularensis, Y. pestis A1122, Y. pestis CO92, B. cenocepacia, and B. neotomae can be used to identify and differentiate these bacteria, aliquots from liquid cultures of each were acquired and analyzed by multi-hSPME coupled with GC-FID, as detailed in the “Materials and methods” section
Summary
Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species. VOC profiling has been shown to be a valuable tool in the differentiation of antibiotic resistant strains of Klebsiella pneumoniae and Enterobacter cloacae[13] In light of their eminence as Category A/B biothreat agents, we sought here to assess if microbial VOC (mVOC) fingerprints, generated by GC-based global (untargeted) metabolomic profiling, can uniquely differentiate liquid cultures of Francisella tularensis (the causative agent of tularemia), Burkholderia pseudomallei (melioidosis), Brucella melitensis (brucellosis), and Yersinia pestis (plague). We used simulti-hSPME, in conjunction with both GC-FID and GC–MS, to derive mVOC fingerprints that differentiate between shake flask cultures of bacteria, including antibiotic sensitive and antibiotic resistant strains of F. tularensis and Y. pestis. Thermal desorption (TD) coupled with GC–MS enabled us to derive mouse-associated VOC profiles that differentiate the healthy mice from infected mice, and mice infected with an antibiotic sensitive strain of Y. pestis from those infected with an antibiotic resistant strain
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