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2025
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04
VAPCI-CMS Combined Technology: Ultra-Early Identification and Dynamic Monitoring Based on Bacterial Metabolism of VOCs!
In the over-70-year-long battle between antibiotics and bacteria, the increasingly serious issue of antimicrobial resistance is driving scientific research to new depths. Studies have revealed that volatile organic compounds (VOCs) released by bacteria—unique “molecular fingerprints” generated through metabolic activity—provide crucial clues for identifying bacterial species and unraveling mechanisms of antibiotic resistance! Using a vAPCI-CMS mass spectrometry coupling approach, the research team at the Longshorewell Institute has successfully captured the volatile organic compounds (VOCs) released by bacterial metabolism. In just 24 hours, they were able to achieve 100% differentiation between Escherichia coli and Staphylococcus aureus, and demonstrated partial differentiation capability in as little as 2 hours!
- Rapid testing: Sample analysis takes only 2 minutes.
- High sensitivity: Distinguishing between Escherichia coli (E. coli) and Staphylococcus aureus.
- Real-time monitoring: Non-destructive sampling, supporting long-term dynamic monitoring and tracking VOC changes during bacterial growth.
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Achieve 100% sensitivity and specificity within 24 hours;
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It takes only 2 hours to partially distinguish between bacterial species.
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Through metabolomic analysis, the dynamic relationship between bacterial growth and VOC release is revealed.
Experimental method
Prepare bacterial plates using bacterial suspensions in liquid medium with an OD600 value ranging from 0.2 to 0.5 AU (absorbance units) (see Figure 1). All inoculated plates are prepared in triplicate. After inoculation, the plates are placed in a constant-temperature incubator at 37°C to ensure data reproducibility. Simultaneously, three sets of blank agar plates are prepared and cultured alongside the inoculated plates to eliminate interference from the medium background.
Figure 1: (A) Agar plate culture of Escherichia coli; (B) Agar plate culture of Staphylococcus aureus; and a plate on which both organisms are grown simultaneously.
Instrument configuration:
Adopt AIS expression CMS mass spectrometer , equipped with vAPCI ion source (Figure 2)
Figure 2: vAPCI-CMS, used for the collection of volatile organic compounds.
The customized flatbed sampler (Figure 3) is thermostatically controlled at 37°C and is directly connected to a 100°C heated transfer line.
Figure 3: Custom VOC sampler designed for 90 mm agar plates, directly connected to the vAPCI.
Core parameters:
Ion source gas temperature: 350°C;
Capillary temperature: 250°C;
Transmission line temperature: 100°C;
Dynamic sampling transmission line flow rate: 600 mL/min;
Mass-to-charge ratio range: m/z 30–300;
Single-scan time: 400 milliseconds;
Samples were collected at 2, 6, 24, 48, 72, and 96 hours post-vaccination; only a single analysis is required. 2-minute, 24-hour continuous sampling of E. coli. Dynamically capturing the evolution of VOCs metabolic fingerprints.
Experimental results
Each sample was subjected to 150 consecutive mass spectrometry scans, resulting in high-signal-to-noise ratio characteristic spectra. Figure 4 shows the blank agar plate and the E. coli plate inoculated for 24 hours; the differential peaks clearly reveal strain-specific metabolic fingerprints.
Figure 4: Spectra of a blank agar plate (left) and an E. coli plate after 24 hours of inoculation.
Multivariate analysis was performed using the SIMCA software package (Umetrics, version 14.1). Statistical models were generated for each m/z response ranging from m/z 30 to 300, employing Partial Least Squares Discriminant Analysis (PLS-DA) and Principal Component Analysis (PCA), respectively. Clustering was evaluated at each time point from 2 to 96 hours for blank samples, Escherichia coli, and Staphylococcus aureus. After 24 hours, the blank group, E. coli, and S. aureus achieved complete separation with 100% sensitivity and specificity. At 2 hours, partial separation was observed: S. aureus exhibited 100% sensitivity and 91.3% specificity, while E. coli showed 87.5% sensitivity and 73.9% specificity (Figure 5), suggesting that metabolic fingerprints already exhibit distinct differences during the latent phase of bacterial species!
Figure 5: PCA score plots of blank agar, Escherichia coli, and Staphylococcus aureus at 2 hours (left) and 48 hours (right) post-inoculation.
Escherichia coli is a bacterium that produces indole (m/z 118), which is the major ion in the E. coli mass spectrum. By monitoring the changes in m/z 118 over a 24-hour period, it is possible to determine the growth curve of E. coli (Figure 6).
Figure 6: Growth curve of Escherichia coli monitored continuously for 24 hours, with indole detected at m/z 118.
Feature markers screened based on multivariate analysis (MVA) enable the simultaneous identification of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Figure 7 shows a superimposed mass spectrum of characteristic peaks from the mixed bacterial strains.
Figure 7: Spectra of Staphylococcus aureus (A) and a simultaneous presence of Staphylococcus aureus and Escherichia coli on a single plate (B).
Summary
The detection protocol for volatile organic compounds (VOCs) in in vitro bacterial cultures, based on the combined use of a vAPCI ion source and an AIS expression mass spectrometer, has been experimentally validated. This technology boasts the following core advantages:
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Support for bacterial metabolic characteristics Long-term dynamic monitoring To avoid sample loss and suitable for continuous culture studies.
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Strain available within 24 hours 100% distinction (Achieves both sensitivity and specificity);
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2-hour early warning: The accuracy of identifying certain strains (such as Staphylococcus aureus) exceeds 91%.
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