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We conducted comprehensive (untargeted) metabolic profiling of volatile organic compounds (VOCs) emitted in culture by bacterial taxa  Francisella tularensis (F. tularensis)  subspecies  novicida  and  Bacillus anthracis (B. anthracis)  Sterne, surrogates for potential bacterial bioterrorism agents, as well as selective measurements of VOCs from their fully virulent counterparts,  F. tularensis  subspecies  tularensis  strain SCHU S4 and  B. anthracis  Ames.  F. tularensis  and  B. anthracis  were grown in liquid broth for time periods that covered logarithmic growth, stationary, and decline phases. VOCs emitted over the course of the growth phases were collected from the headspace above the cultures using solid phase microextraction (SPME) and were analyzed using gas chromatography-mass spectrometry (GC-MS). We developed criteria for distinguishing VOCs originating from bacteria versus background VOCs (originating from growth media only controls or sampling devices). Analyses of collected VOCs revealed methyl ketones, alcohols, esters, carboxylic acids, and nitrogen- and sulfur-containing compounds that were present in the bacterial cultures and absent (or present at only low abundance) in control samples indicating that these compounds originated from the bacteria. Distinct VOC profiles where observed for  F. tularensis  when compared with  B. anthracis  while the observed profiles of each of the two  F. tularensis  and  B. anthracis  strains exhibited some similarities. Furthermore, the relative abundance of VOCs was influenced by bacterial growth phase. These data illustrate the potential for VOC profiles to distinguish pathogens at the genus and species-level and to discriminate bacterial growth phases. The determination of VOC profiles lays the groundwork for non-invasive probes of bacterial metabolism and offers prospects for detection of microbe-specific VOC biomarkers from two potential biowarfare agents.

Metabolic Profiling of Volatile Organic Compounds (VOCs) Emitted by the Pathogens Francisella tularensis and Bacillus anthracis in Liquid Culture