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

Kristen L Reese,A Daniel Jones,Amy Rasley,Julie R Avila,Matthias Frank

doi:10.1038/s41598-020-66136-0

Kristen L Reese, A Daniel Jones + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-66136-0

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Abstract

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.

Highlights

The measurement of volatile organic compounds (VOCs) and non-volatiles in exhaled breath is becoming an important rapid and non-invasive diagnostic tool to assess human physiology and health as well as a diagnostic tool for infections and systemic disease[2,5,6,7]
Our work presented here focused on the in vitro, non-invasive, untargeted profiling of VOCs from cultures of F. tularensis subspecies novicida (Ft novicida) and B. anthracis Sterne (Ba Sterne), both risk group 2 (RG2) surrogates for more virulent species, and from F. tularensis subspecies tularensis SCHU S4 (Ft SCHUS4) and B. anthracis Ames (Ba Ames), two fully virulent, risk group 3 (RG3) organisms. (For descriptions of risk group (RG) classifications of infectious microorganisms and recommended biosafety level (BSL) for their handling see the U.S Department of Health and Human Services guide on Biosafety in Microbiological and Biomedical Laboratories[23] or the World Health Organization Laboratory Biosafety Manual24)
The same criteria were applied to the data from other bacterial species studied here, resulting in 38 putative VOC biomarkers for Ft SCHUS4, 30 biomarkers in Ba Sterne, and 56 biomarkers in Ba Ames (Table 1)

Summary

Introduction

The measurement of VOCs and non-volatiles in exhaled breath is becoming an important rapid and non-invasive diagnostic tool to assess human physiology and health as well as a diagnostic tool for infections and systemic disease[2,5,6,7]. Horsmon and Crouse[15] used thermal desorption tubes coupled to gas chromatography-mass spectrometry (GC-MS) to describe VOC profiles emitted by cultures of Yersinia pestis (Y. pestis), the causative agent of plague, and several strains from the genus Bacillus. They showed that VOC profiles and relative abundances of individual compounds distinguished bacterial genera as well as species within the same genus. Li et al distinguished Francisella tularensis subspecies novicida, Escherichia coli, and Bacillus subtilis by derivatizing fatty acids to form trimethylsilyl esters[22] These studies required whole bacteria and sample preparation that was destructive to the bacteria, precluding analysis of metabolite changes over time in an unperturbed culture

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