A Pilot Study for Legionella pneumophila Volatilome Characterization Using a Gas Sensor Array and GC/MS Techniques

Abstract

Legionellosis is a generic term describing the pneumonic (Legionnaires' disease, LD) and non-pneumonic (Pontiac fever, PF) forms of infection with bacteria belonging to the genus Legionella. Currently, the techniques used to detect Legionella spp. in water samples have certain limitations and drawbacks, and thus, there is a need to identify new tools to carry out low-cost and rapid analysis. In this regard, several studies demonstrated that a volatolomics approach rapidly detects and discriminates different species of microorganisms via their volatile signature. In this paper, the volatile organic compounds (VOCs) pattern emitted in vitro by Legionella pneumophila cultures is characterized and compared to those produced by other Legionella species and by Pseudomonas aeruginosa, using a gas sensor array and gas chromatograph mass spectrometer (GC-MS). Bacterial cultures were measured at the 3rd and 7th day after the incubation. Sensor array data analyzed via the K-nearest neighbours (k-NN) algorithm showed a sensitivity to Legionella pneumophila identification at around 89%. On the other hand, GC-MS identified a bouquet of VOCs, mainly alcohols and ketones, that enable the differentiation of Legionella pneumophila in respect to other waterborne microorganisms.

Keywords: Legionella pneumophila; gas sensor array; volatile organic compounds; volatilome.

Figure 1

Schematic representation of experimental workflow.

Figure 2

Experimental outline of gas sensor array analysis.

Figure 3

Experimental outline of GC-MS and fast GC-MS analysis.

Figure 4

The portable GC-MS Torion^® T-9 interfaced with its computer.

Figure 5

Boxplot representing the distribution of the sensor responses to control media (CM) and bacteria cultures: L. pneumophila (LP), Legionella species (LSp) and Pseudomonas aeruginosa (PA) considered after 3 (e.g., LP3) and 7 (e.g., LP7) days of incubation.

Figure 6

Comparison of chromatograms obtained from analysis of culture headspace of Legionella species (blue line), Pseudomonas aeruginosa (red line), Legionella pneumophila (pink line) and not-inoculated BYEα broth (CM-black line). Samples were inoculated for 7 days before the analysis.

Figure 7

Bar plot representing compound pattern of each analyzed sample of BYEα broth (CM), L. pneumophila (LP), L. species (LSpp) and P. aeruginosa (PA) incubated for 7 days. Each colored bar represents the compound abundance and bands are sorted with respect to VOC retention time.

Figure 8

Boxplot representing the distribution of different characteristic compound abundances for the different kinds of cultures: BYEα broth (CM), L. pneumophila (LP), L. species (LSpp) and P. aeruginosa (PA).

Figure 9

Chromatogram from L. pneumophila. (1) 1-butanol- 3methyl, (2) Methyl isovalerate.

Figure 10

Chromatogram from L. species. (1) 1butanol- 3methyl.

Figure 11

Chromatogram for P. aeruginosa. (1) 2-nonanone.