Fast detection of volatile organic compounds from bacterial cultures by secondary electrospray ionization-mass spectrometry

Abstract

We propose a novel application of secondary electrospray ionization-mass spectrometry (SESI-MS) as a real-time clinical diagnostic tool for bacterial infection. It is known that volatile organic compounds (VOCs), produced in different combinations and quantities by bacteria as metabolites, generate characteristic odors for certain bacteria. These VOCs comprise a specific metabolic profile that can be used for species or serovar identification, but rapid and sensitive analytical methods are required for broad utility. In this study, the VOC profiles of five bacterial groups from four genera, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella enterica serovar Typhimurium, and Salmonella enterica serovar Pullorum, were characterized by SESI-MS. Thirteen compounds were identified from these bacterial cultures, and the combination of these VOCs creates a unique pattern for each genus. In addition, principal component analysis (PCA) was applied for the purpose of species or serovar discrimination. The first three principal components exhibit a clear separation between the metabolic volatile profiles of these five bacterial groups that is independent of the growth medium. As a first step toward addressing the complexity of clinical application, in vitro tests for mixed cultures were conducted. The results show that individual species or serovars in a mixed culture are identifiable among a biological VOC background, and the ratios of the detected volatiles reflect the proportion of each bacterium in the mixture. Our data confirm the utility of SESI-MS in real-time identification of bacterial species or serovars in vitro, which, in the future, may play a promising clinical role in diagnosing infections.

FIG. 1.

Positive-ion-mode full-scan spectra (m/z of 40 to 150) of bacterial culture headspace for P. aeruginosa, S. aureus, E. coli, and S. Typhimurium grown aerobically in TSB at 37°C for 24 h. Every spectrum represents an average of spectrum values for nine samples (three biological replicates, each with three technical replicates), with the media blank subtracted and normalization to the peak of greatest intensity.

FIG. 2.

Principal component analysis of the absolute intensities for all peaks (m/z of 40 to 150) of mass spectra of the headspace volatiles of E. coli (E.c.), S. aureus (S.a.), S. Typhimurium (S.T.), S. Pullorum (S.P.), and P. aeruginosa (P.a.) grown in TSB (closed symbols) as well as P. aeruginosa grown in LB-Lennox, MOPS-glucose-succinate, and synthetic cystic fibrosis medium (SCFM). All cultures were grown aerobically at 37°C for 24 h. Each point represents one sample; a total of 72 samples are included.

FIG. 3.

Predicted (gray) and experimental (black) SESI-MS spectra for mixed cultures of S. aureus (S.a.) and P. aeruginosa in TSB. The predicted spectra for the mixed cultures were calculated by scaling the spectra of the monocultures of S. aureus and P. aeruginosa relative to their proportions in the mixture and then adding the two scaled spectra. For the sake of clarity, only the proportion of S. aureus in the mixture is labeled; the remaining percentage of the culture is represented by P. aeruginosa. All of the experimental spectra are plotted on the same scale (y axis; 0 to 9 × 10⁷ cps); the predicted spectra are scaled to the same signal intensity as those of their respective experimental spectra. The trends for the labeled peaks are shown in Fig. S2 in the supplemental material.

FIG. 4.

PCA (PC 2 versus PC 3) of SESI-MS peaks collected in positive mode (m/z of 40 to 150) for pure culture of S. aureus (S.a.), P. aeruginosa (P.a.), E. coli (E.c.), and S. Typhimurium (S.T.) as well as mixed cultures of S. aureus and P. aeruginosa, all grown for a total of 24 h at 37°C in TSB. Mixed cultures of S. aureus and P. aeruginosa were prepared by culturing each species individually in 50 ml of TSB for 23 h at 37°C, followed by mixing in different proportions of S. aureus to P. aeruginosa (3:1, 1:1, and 1:3 [vol/vol]) to give a final volume of 50 ml, and incubating the culture for an additional hour. The three-dimensional (3D) PCA plot is provided in Fig. S3 in the supplemental material.