Longitudinal RNA Seq analyses reveal the prominent role of Vagococcus in broiler meat spoilage microbiome

Technical Summary: Longitudinal RNA Seq Analyses Reveal the Prominent Role of Vagococcus in Broiler Meat Spoilage Microbiome

Problem Statement
Raw broiler meat is highly perishable, with microbial activity being the primary factor limiting shelf life. Historically, spoilage microbiomes have been characterized using traditional culturing methods and 16S rRNA gene amplicon sequencing. However, these approaches fail to distinguish between living, active species and dormant or dead cells, nor do they reveal the functional metabolic activities of the microbiome. Previous metatranscriptomic studies on broiler spoilage have been limited to specific spoilage organisms rather than analyzing the entire active microbiome. Consequently, the interplay of microbes and their functional succession during the shelf life of modified atmosphere (MA) packaged broiler meat remains poorly understood.

Methodology
The study employed a longitudinal experimental design using MA-packaged skinless broiler leg meat from two commercial production lots. Samples were stored at two temperatures: 4°C and 6°C, extending beyond the use-by date until advanced spoilage occurred.

• Sampling Strategy: RNA was extracted daily from the meat samples. At 4°C, sampling occurred from day 5 to day 14; at 6°C, due to faster spoilage, sampling occurred from day 6 to day 10.
• Multi-Omics Approach: The study integrated four analytical streams:
  1. Metatranscriptomics (RNA-seq): Total RNA was extracted, ribosomal RNA depleted (using chicken and bacteria-specific probes), and sequenced on Illumina NextSeq. A four-level bioinformatics pipeline was implemented:
     • Level 0-1: Removal of host (Gallus gallus) and rRNA reads.
     • Level 2: Taxonomic profiling using Kraken2 and Bracken to estimate species abundance.
     • Level 3-4: Functional profiling using HUMAnN to map gene families to KEGG Orthology (KO) pathways, generating species-resolved pathway activity time series.
  2. Non-Targeted Metabolomics: LC–MS analysis (UHPLC-QTOF) was performed to profile metabolites, identifying trends in nucleotide degradation, biogenic amines, and amino acids.
  3. Traditional Microbiology: Bacterial concentrations were estimated via plating on MRS, PCA, STAA, and VRBG agars.
  4. Sensory Evaluation: Trained evaluators scored smell and appearance daily to correlate microbial activity with sensory spoilage stages (acceptable, early spoilage, late spoilage).

Key Results

• Microbiome Succession:

  • At both temperatures, the active microbiome shifted from a diverse initial community (including Gram-negative bacteria and fungi) to a dominance of Lactic Acid Bacteria (LAB).
  • The three most active species identified were Carnobacterium divergens, Carnobacterium maltaromaticum, and Vagococcus proximus.
  • Temperature Effect: At 4°C, C. divergens and C. maltaromaticum were highly dominant, with V. proximus increasing in abundance after the use-by date. At 6°C, V. proximus became the dominant species in the active microbiome, while C. maltaromaticum abundance dropped significantly.
  • Fungal activity was marginal, declining rapidly after day 5.

• Metabolic Pathway Activity:

  • Central carbohydrate metabolism was the most common pathway module across the microbiome.
  • C. divergens and C. maltaromaticum exhibited stable metabolic profiles throughout the spoilage process.
  • Distinct V. proximus Behavior: V. proximus displayed a unique metabolic shift. In the "acceptable product" phase, it showed high ATP synthesis activity. As spoilage advanced (particularly at 4°C), its activity shifted toward increased fatty acid and carbohydrate metabolism, with a concurrent decrease in ATP synthesis. At 6°C, V. proximus exhibited pronounced fatty acid metabolism and antibiotic biosynthesis, with minimal central carbohydrate metabolism.

• Metabolomic Trends:

  • Metabolomic changes were qualitatively similar at both temperatures but occurred at a faster rate at 6°C.
  • Biogenic Amines: Tyramine and spermidine showed greater accumulation at 6°C. Spermidine levels remained stable at 4°C but increased significantly at 6°C, correlating with the faster decline in sensory smell scores.
  • Nucleotide Degradation: Inosine monophosphate (IMP) decreased progressively at both temperatures. Hypoxanthine increased over time, while inosine decreased at 6°C but remained stable at 4°C.

Significance and Claims
The authors claim that this study demonstrates the efficacy of combining RNA-seq with metabolomics to decode the functional dynamics of meat spoilage microbiomes. Key claims include:

1. Identification of Neglected Spoilers: The study identifies Vagococcus proximus, a recently described species, as a key active spoiler in broiler meat. Its prominence was likely overlooked in previous studies due to the limitations of standard culturing methods and 16S rRNA sequencing, which do not capture activity or distinguish closely related species as effectively as metatranscriptomics.
2. Functional Insight: The research links specific metabolic shifts (e.g., the transition from ATP synthesis to fatty acid metabolism in V. proximus) to the progression of spoilage, suggesting that V. proximus may be a primary driver of sensory deterioration in the later stages of storage.
3. Temperature Impact: While the overall trajectory of spoilage is similar at 4°C and 6°C, the rate of metabolic change and the dominance of specific species (particularly V. proximus) differ significantly. The study reinforces that storage temperatures below 6°C are critical for delaying the accumulation of biogenic amines and the onset of late-stage spoilage.
4. Methodological Value: The paper argues that traditional methods overlook the "active" fraction of the microbiome. By focusing on transcriptional activity, the study reveals that spoilage is driven by specific active species and their metabolic adaptations, rather than just total bacterial load.

The authors conclude that future studies should specifically target the metabolism and detection of Vagococcus to better understand its role in food spoilage, noting that current technological limitations in standard methods have led to the neglect of such active species.