Preliminary investigation on the volatilome of Streptococcus pyogenes SF370

Introduction The objective of the study was to identify microbial volatile compounds (mVOCs) produced by Streptococcus pyogenes, one of the most common human pathogens, and to attempt the determination of its minimal volatilome. In recent years, the characterization of this subset of the bacterial metabolome ha attracted specific interest also in view of the possibility of exploiting specific mVOCs as potential biomarkers of an ongoing bacterial infection. Materials and methods General preparation and analysis of samples were done following procedures described for the study of the Staphylococcus aureus volatilome. The Streptococcus pyogenes strain was the SF370. We performed a triplicate of the S. pyogenes culture and a triplicate of the medium (Todd Hewitt + 0.2% yeast extract, THY) incubated under the same conditions as the bacterial culture. Samples were taken at mid-log and in the pre-stationary phase of the growth curve and centrifuged. Supernatants were then filtered. The mVOCs were extracted using a mixed fiber and subjected to Gas Chromatography/Mass Spectrometry (GC-MS) analysis. GC separation was accomplished using a DB-WAX polar column (60 m x 0.25 mm x 0.00025 mm). The compounds were identified by comparison with the NIST database and with published retention indices. Results The analysis of all profiles showed a good intra-sampling reproducibility. The comparative analysis of the samples allowed groups of compounds to be excluded from further analysis based on: (i) presence in all the experimental conditions or (ii) presence in only one of the replicates or (iii) poor identification score. The results showed that metabolic activity of S. pyogenes grown in THY produces mVOCs which are more polar than the volatiles present in the uncultured medium. At mid- log, the minimal volatilome consisted of 2-nitroethanol, 2-tert-butoxyethanol, 1-butanol, 3,3- dimethylhexane, pentadecane, 10-methylnonadecane and 2-tridecanone while ethanol, 1-butanol and gamma-aminobutyric acid were characteristic of the pre-stationary phase. Conclusions Thanks to this preliminary study, it has been possible to determine the minimum volatilome of S. pyogenes strain SF370 grown in THY. Further studies using different culture media, different clinical strains of S. pyogenes and analytical conditions (e.g. column and fiber type) are needed to broaden the definition of the pool of volatile molecules produced by this bacterial species.