Dependence of the Staphylococcal Volatilome Composition on Microbial Nutrition

Abstract

In vitro cultivation of staphylococci is fundamental to both clinical and research microbiology, but few studies, to-date, have investigated how the differences in rich media can influence the volatilome of cultivated bacteria. The objective of this study was to determine the influence of rich media composition on the chemical characteristics of the volatilomes of Staphylococcus aureus and Staphylococcus epidermidis. S. aureus (ATCC 12600) and S. epidermidis (ATCC 12228) were cultured in triplicate in four rich complex media (brain heart infusion (BHI), lysogeny broth (LB), Mueller Hinton broth (MHB), and tryptic soy broth (TSB)), and the volatile metabolites produced by each culture were analyzed using headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (HS-SPME-GC×GC-TOFMS). When comparing the chemical compositions of the staph volatilomes by the presence versus absence of volatiles produced in each medium, we observed few differences. However, when the relative abundances of volatiles were included in the analyses, we observed that culturing staph in media containing free glucose (BHI and TSB) resulted in volatilomes dominated by acids and esters (67%). The low-glucose media (LB and MHB) produced ketones in greatest relative abundances, but the volatilome compositions in these two media were highly dissimilar. We conclude that the staphylococcal volatilome is strongly influenced by the nutritional composition of the growth medium, especially the availability of free glucose, which is much more evident when the relative abundances of the volatiles are analyzed, compared to the presence versus absence.

Keywords: GC×GC-TOFMS; Staphylococcus aureus; Staphylococcus epidermidis; catabolite repression control; volatile organic compounds.

Figure 1

Chemical composition of the total staph volatilome (Total), and the volatilome by species (S. aureus, Sa, or S. epidermidis, Se) or by growth medium (brain heart infusion (BHI), tryptic soy broth (TSB), Mueller Hinton broth (MHB), lysogeny broth (LB)). The data are presented as the total number of volatiles detected in each chemical class, unnormalized (a) and normalized (b), and the total relative abundances of volatiles in each chemical class, unnormalized (c) and normalized (d). Level 4 (unknown) compounds were excluded. Each column represents composite data, with the media volatilomes representing the data for both species, the species volatilomes representing the volatilomes of that species in all four media, and the total representing the volatilomes of both species in all four media. The numerical data are provided in Tables S2–S5. HC, hydrocarbons; ALC, alcohols; ALD, aldehydes; KET, ketones; ETH, ethers; A/E, acids and esters; NIT, nitrogen-containing; SULF, sulfur-containing; HALO, halogen-containing; HET-ARO, heteroaromatics; HC-ARO, aromatic hydrocarbons; ALC-ARO, aromatic alcohols; ALD-ARO, aromatic aldehydes; KET-ARO, aromatic ketones; A/E-ARO, aromatic acids and esters; NIT-ARO, aromatic nitrogen-containing.

Figure 2

Media-dependent staph volatilome variation represented as a dissimilarity matrix using Morisita–Horn differences calculated using relative abundances of volatile chemical classes.

Figure 3

Chemical composition of the staph volatilome as a function of species and growth medium. The data are presented as the total number of volatiles detected in each chemical class, unnormalized (a) and normalized (b), and the total relative abundances of volatiles in each chemical class, unnormalized (c) and normalized (d). Level 4 (unknown) compounds were excluded. The numerical data are provided in Tables S2–S5. HC, hydrocarbons; ALC, alcohols; ALD, aldehydes; KET, ketones; ETH, ethers; A/E, acids and esters; NIT, nitrogen-containing; SULF, sulfur-containing; HALO, halogen-containing; HET-ARO, heteroaromatics; HC-ARO, aromatic hydrocarbons; ALC-ARO, aromatic alcohols; ALD-ARO, aromatic aldehydes; KET-ARO, aromatic ketones; A/E-ARO, aromatic acids and esters; NIT-ARO, aromatic nitrogen-containing.