Listeria Detection from Enrichment to Identification: Evaluating 24 Species with Enrichment Media, Screening Media, and Rapid Identification Using MALDI-TOF MS Proteomics Method

Význam tématu

Rapid and reliable detection of Listeria species is critical for food safety and public health. While Listeria monocytogenes is the main human pathogen, the genus now includes over two dozen species that may contaminate food environments. Comprehensive screening and precise identification of all relevant species help prevent outbreaks and ensure regulatory compliance.

Cíle a přehled studie / článku

This study assessed the performance of standard enrichment broths and the CompactDry LS screening medium for the growth of 24 Listeria species. In addition, a MALDI-TOF MS proteomics workflow using the MicrobialTrack software was evaluated for rapid species-level identification. The goal was to validate combined methods for broad-spectrum detection and accurate differentiation.

Použitá metodika a instrumentace

The study workflow comprised two phases:

• Enrichment testing in five media: Half-Fraser broth (HFB), Buffered Listeria Enrichment broth (BLEB), LPT broth (USDA MLG 8.15), modified MSB broth (mMSB), and Trypticase Soy Broth (TSB).
• Screening on three agar plates: CompactDry LS (CD LS), Agar Listeria according to Ottaviani and Agosti (ALOA), and sheep blood agar (SBA).

Growth performance was measured by colony counts and 650 nm absorbance in 96-well plates. Identification used a MALDI-TOF MS platform (MALDI-8020 RUO, Shimadzu Corporation) with three sample preparation approaches: direct smear (DS), ethanol–formic acid wash (EW), and bead-crash extraction (BC). Spectra were analyzed using MicrobialTrack with theoretical protein masses from public databases.

Hlavní výsledky a diskuse

None of the guideline-specified enrichment media supported all 24 species equally. Modified MSB and non-selective SCD broth enabled growth of every species, whereas LPTB failed to recover Listeria grayi. Screening on CompactDry LS recovered all strains with minimal log differences, while ALOA produced atypical colony morphologies for eight species. In MALDI-TOF identification, the direct smear method achieved 100 percent genus-level accuracy, but species-level errors occurred primarily in EW and BC methods. Misidentifications involved closely related pairs (eg L. innocua vs L. farberi). Combining DS for initial screening and repeated EW or BC runs improved overall reliability.

Přínosy a praktické využití metody

The combined protocol enables broad detection of both common and newly proposed Listeria species, supporting food industry monitoring and regulatory testing. CompactDry LS streamlines colony enumeration, and the MicrobialTrack MALDI-TOF approach delivers rapid genus-level screening with high throughput. Laboratories can adapt the workflow for quality control and outbreak investigations.

Budoucí trendy a možnosti využití

Future work may integrate genetic screening kits with proteomic identification to enhance specificity. Expanding the MALDI database and refining extraction protocols could eliminate remaining species-level ambiguities. Automation of enrichment and spectral analysis will accelerate large-scale surveillance and real-time contamination tracking.

Závěr

This evaluation demonstrates that no single enrichment medium suffices for all Listeria species, but combining mMSB or SCD with CompactDry LS ensures comprehensive growth. The MicrobialTrack MALDI-TOF MS method offers rapid genus-level detection, and a tiered sample preparation strategy maximizes species-level accuracy. The proposed workflow provides a robust framework for routine and outbreak-driven Listeria testing.

Reference

• ISO 11290 2017 Detection and enumeration of Listeria monocytogenes
• FDA Bacteriological Analytical Manual Chapter 10
• USDA Microbiological Laboratory Guidebook 8.15
• Costa-Ribeiro et al 2024, modified MSB broth formulation