The detection of foodborne pathogens is a critical aspect of ensuring food safety. Traditional methods rely on time-intensive enrichment steps and pathogen-specific assays, extending testing timelines and limiting throughput. This study evaluates an enrichment-free, multiplexed pathogen detection workflow combining the Pathotrak system for bacterial separation and the Neogen Molecular Detection System (MDS) for detection. The workflow enables simultaneous detection of Salmonella, Escherichia coli O157, Listeria monocytogenes, Listeria spp., and Campylobacter in poultry samples, significantly reducing the time taken to screen samples requiring further evaluation. The system’s performance was assessed using inoculated chicken samples over a range of bacterial concentrations (10²–10⁸ CFU/mL). The MDS system demonstrated robust detection for most pathogens, with strong correlations between theoretical inoculation levels and MDS-calculated concentrations (R² > 0.85 for all pathogens). However, detection variability was observed at lower concentrations for Salmonella and Listeria monocytogenes. The system maintained high sensitivity and specificity, achieving a Cohen’s Kappa coefficient for E. coli and Campylobacter. This study highlights the potential of enrichment-free, multiplex detection to streamline food safety testing by reducing the time to results, enhancing efficiency, and providing reliable pathogen quantification across multiple targets.

A biofilm is a complex microbial community that protects bacterial cells from various stressors, including harsh environmental conditions, antimicrobial treatments, and host immune responses. This protective capability enhances Campylobacter survival during food processing and storage and facilitates transmission to humans. Despite their importance, the molecular mechanisms underlying Campylobacter biofilm formation and its impact on pathogen persistence remain poorly understood. In this study, we characterized the biofilm-forming ability of 18 C. jejuni and C. coli strains isolated from retail meat and performed whole-genome sequencing and comparative genomic analysis to identify strain-specific genes contributing to biofilm formation and maintenance. Phenotypic analysis revealed that C. jejuni strains YH001 and YH027 exhibited the strongest biofilm-forming capacity, producing the highest biomass among all isolates. Phylogenetic analysis indicated a close genetic relationship between these two strains, while pangenome analysis identified 19 unique genes/proteins specific to these strains. Functional annotation indicated their critical roles in adhesion, extracellular matrix production, and stress response. These findings demonstrate strain-specific biofilm formation in Campylobacter and highlight genetic determinants that may serve as targets for novel therapeutic approaches and intervention strategies to disrupt biofilms, improve food safety, and reduce persistent infections.

Biofilm is a complex microbial community that provides protection to bacterial cells against various stress conditions, including harsh environments, antimicrobial treatments, and host immune responses. This protective mechanism enhances Campylobacter survival during food processing and storage and promotes transmission to humans. Despite its significance, the molecular mechanisms underlying Campylobacter biofilm formation and its impact on pathogen persistence remain poorly understood. In this study, we characterized the biofilm-forming ability of 18 C. jejuni and C. coli strains isolated from retail meat and performed whole-genome sequencing and comparative genomic analysis to identify strain-specific genes contributing to biofilm formation and maintenance. Phenotypic analysis revealed that C. jejuni strains YH001 and YH027 exhibited the strongest biofilm-forming capacity, producing the highest biomass among all isolates. Phylogenetic analysis indicated a close genetic relationship between these two strains, while pangenome analysis identified 19 unique genes/proteins specific to these strains. Functional annotation indicated their critical roles in adhesion, extracellular matrix production, and stress response. These findings demonstrate strain-specific biofilm formation in Campylobacter and highlight genetic determinants that may serve as targets for novel therapeutic approaches and intervention strategies to disrupt biofilms, improve food safety, and reduce persistent infections.

Salmonella in raw poultry drives foodborne outbreaks and recalls, and process-control decisions increasingly turn on quantitative thresholds rather than presence/absence. Acting on a threshold in real time requires accurate quantification near the cutoff and a result within one work shift. Conventional enrichment-based enumeration meets neither: it adds hours of growth before detection, and the growth step biases the count toward faster-growing, unstressed cells. Near-threshold false positives also matter: products below the limit tests positive, triggering costly and avoidable holds and recalls. We evaluated a same-shift, enrichment-free workflow that couples the Pathotrak concentrator with real-time PCR (Bio-Rad iQ-Check Salmonella II) for quantifying Salmonella in raw poultry, with sample preparation in about 30 min instead of 6–24 h of enrichment. The evaluation spanned 313 independent inoculations across three laboratories, three calendar years (2023–2025), and multiple seasons, technicians, instruments, and chicken sources. A latent-truth framework propagated inoculation uncertainty into sensitivity (Se) and specificity (Sp) estimates and made the half-log gray zone around each cutoff tractable. At 10 CFU/g, the workflow achieved Se 100% (20/20; 95% CI 83.2–100.0%) for samples at least 0.5 log₁₀ above the threshold and Sp 94.4% (185/196; 95% CI 90.2–97.2%) below it; within the half-log gray zone, classification was Se 88.6% (95% CI 68.8–99.1%)/Sp 76.5% (95% CI 62.1–88.1%) (n = 77). In a cross-study comparison, gray-zone performance at 10 CFU/g fell within the range reported for MPN, with inoculation strain, chicken source, and plate-count method differing by site. At 1 CFU/g, the workflow returned a calibrated quantitative result within a single shift, which, to our knowledge, no enrichment-free real-time-PCR workflow has previously achieved without an incubation step. Samples more than 0.5 log₁₀ CFU/g above or below a threshold were classified correctly in 205/216 (94.9%) cases at 10 CFU/g and 109/117 (93.2%; 95% CI 87.0–97.0%) at 1 CFU/g, with classification uncertainty confined to the gray zone (Se 63.2% (95% CI 49.9–76.0%)/Sp 66.5% (95% CI 54.2–77.9%), n = 166) as expected at low copy number (Salmonella genome copies estimated by qPCR).

Biofilms contribute significantly to the persistence and transmission of Campylobacter jejuni, yet enzymatic strategies for disrupting these structures remain underexplored. In this study, we evaluated the activity of a glycosyl hydrolase, CAase, against both planktonic and biofilm-associated C. jejuni. CAase showed no inhibitory effect on planktonic growth at concentrations up to 1 mg/mL. However, at 0.1 mg/mL, CAase reduced mature biofilm biomass by more than 93% in the wild-type strain and produced similar reductions in a luxS mutant, demonstrating substantial degradation of conserved glycan components within the extracellular polymeric substance (EPS). Scanning electron microscopy (SEM) imaging revealed substantial extracellular matrix loss, structural disruption, and altered cell morphology following CAase treatment. Rheological measurements demonstrated marked decreases in kinematic viscosity, further reflecting weakened biofilm cohesion. Together, these results indicate that CAase effectively disrupts multiple components of C. jejuni biofilm architecture despite lacking direct antimicrobial activity. This level of biofilm removal suggests potential utility in poultry-processing or surface-sanitation applications where targeted enzymatic disruption could enhance cleaning efficacy.

Listeria monocytogenes forms resilient biofilms that protect cells from sanitizers and environmental stress, allowing the pathogen to persist in food processing environments. This study evaluated the combined effects of an engineered carbohydrate-active enzyme (CAase) and two food-grade bacteriocins, nisin and thermophilin 110 (T110), on biofilm disruption and cell inactivation. Biofilm biomass was quantified by crystal violet staining and visualized by microscopic imaging, with viable cells enumerated in treatment supernatants. Minimum inhibitory concentrations (MIC) for nisin (12.5–25 µg mL⁻¹) and T110 (64 to 512 arbitrary units (AU) mL⁻¹) were determined against all 11 L. monocytogenes strains, while CAase alone showed limited antimicrobial activity. CAase was shown to significantly reduce biofilm biomass, releasing embedded cells from the matrix. Nisin (25 µg mL⁻¹) and T110 (1024 AU mL⁻¹) were observed to decrease biofilm biomass and viable-cell recovery, but results were strain-dependent. Biofilm clearance improved for some L. monocytogenes strains when CAase was combined with either bacteriocin, but nisin was more effective at reducing the number of viable cells recovered. Results from this study demonstrated that enzymatic degradation of the extracellular matrix may enhance bacteriocin efficacy by improving access to target cells and support the potential of enzyme–bacteriocin combinations as food-safe biofilm control strategies in food production environments.