Food Microbiology

Listeria monocytogenes is a foodborne pathogen of utmost interest to food industry stakeholders because it persists in food processing environments. The ability to form biofilms - bacterial communities of autoaggregated cells embedded in a self-produced matrix - contributes to its persistence. While it is known that biofilm cells exhibit different gene expression than their planktonic counterparts, it remains to be elucidated whether those differences persist once cells detach from the biofilm and what their implications might be for food safety. Therefore, this study examines the differential sigB expression in biofilm-derived cells from three L. monocytogenes strains isolated from the environment within a food model subjected to varying osmotic stress over a 15-day storage period. Under our experimental conditions, biofilm-derived L. monocytogenes cells showed higher sigB expression compared to planktonic counterparts. The upregulation was strain-dependent and transient, suggesting that physiological memory may influence stress adaptation during early storage but dissipates over time. Then, the safety implications of sigB upregulation in biofilm-derived cells were assessed by evaluating cell survival under a simulated gastric environment (pH 1-3). The biofilm-derived cells showed a significant increase in survival under severe gastric conditions compared to the planktonic counterparts. Overall, our findings highlight the need to consider biofilm-derived cells in shelf-life studies and predictive models to more accurately reflect real contamination scenarios. Relying exclusively on planktonic cultures introduces a bias that may compromise risk analysis and decision-making.

Authentication of acai products is increasingly important due to the risk of species substitution among morphologically similar Euterpe taxa, with implications for food quality, labeling accuracy, and consumer trust. Despite advances in molecular methods, rapid and cost-effective tools for discriminating closely related Euterpe species in processed commercial matrices remain limited. This study evaluated High-Resolution Melting (HRM) analysis targeting two complementary chloroplast markers -- psbK-I and ycf1b -- as a practical approach for species-level authentication of acai (Euterpe oleracea and E. precatoria) and jucara (E. edulis) products. In silico specificity analysis confirmed that the ycf1b primer pair shows amplification restricted to the Arecaceae family, supporting the analytical robustness of the method. The combined markers enabled reliable differentiation of all target species, including closely related taxa, with a detection limit of approximately 10% in admixed samples. When applied to 50 commercial products, HRM successfully authenticated 46 samples, substantially outperforming DNA sequencing, which was limited by amplification failure and mixed chromatograms. Mislabeling was detected in one acai sorbet and three frozen acai pulps marketed as acai but molecularly identified as E. edulis, constituting a violation of Brazilian food labeling regulations. These findings demonstrate that HRM analysis provides a robust, rapid, and scalable strategy for routine species authentication in processed plant-based matrices, with potential for integration into food quality control workflows and large-scale commercial monitoring programs.

Manual colony counting remains the rate-limiting, operator-dependent step in culture-based food microbiology quality control (QC). Automated colony analysis using machine learning (ML) offers the potential to standardise, accelerate, and improve the traceability of this process. However, systematic multi-method validation data for AI-based platforms against recognised international standards remain scarce. We conducted a prospective, multi-study validation of the Reshape Smart Incubator which is an automated imaging and ML-based colony analysis system, across eight ISO microbiological reference methods. In total, 887 plates were analysed, spanning qualitative (presence/absence) detection of Listeria spp. (ISO 11290-1) and Salmonella spp. (ISO 6579), and quantitative enumeration of total viable count (ISO 4833), Bacillus cereus (ISO 7932), Enterobacteriaceae (ISO 21528), coagulase-positive Staphylococci (ISO 6888), yeasts and moulds (ISO 21527), and lactic acid bacteria (ISO 15214). Automated results were benchmarked against the consensus of three or more trained technicians. The platform achieved 100% agreement with manual assessment for all both qualitative detection methods (ISO 11290-1, ISO 6579) with zero false positives and zero false negatives. For quantitative enumeration, agreement ranged from 92.97% (ISO 15214, n=122, using ISO-aligned {+/-}10%/>30 CFU thresholds) to 98.46% (ISO 21528, n=130). Where discrepancies occurred, they largely coincided with plates showing high inter-technician variability. Precision testing demonstrated a coefficient of variation of 5.88% and a mean standard deviation of 0.44 CFU for low-count plates. This study presents a comprehensive multi-ISO validation of an AI-based colony analysis system to date. The AI models demonstrated performance comparable to or exceeding that of trained human technicians across a broad range of microbiological targets, agar types, and colony morphologies, thereby supporting their use as a validated and traceable alternative to manual plate reading in accredited food microbiology quality control laboratories.

Listeria monocytogenes can persist in niches of meat processing environments despite routine cleaning and disinfection. Its persistence may depend not only on stress tolerance but also on interactions with resident microbial communities, which may promote or inhibit survival. However, these ecological relationships remain poorly understood. We combined 16S rRNA V3/V4 amplicon sequencing, culture-based detection, and multilocus sequence typing (MLST) to characterize microbial communities in six German meat processing facilities over one year. We examined associations among community structure, sampling sites (drains and food-contact surfaces), and the occurrence of Listeria spp., including L. monocytogenes. Microbial communities were dominated by core genera typical of food-processing environments, particularly Pseudomonas spp. and Acinetobacter spp., but differed significantly among facilities (PERMANOVA, p = 0.001; pairwise R{superscript 2} = 0.023-0.079), indicating facility-specific communities. Culture-based analyses detected Listeria spp. in 51 of 370 environmental samples (13.8%), mainly from drains (44/51, 86.3%). L. monocytogenes was detected in five of six facilities, with 19 of 21 isolates originating from drains (90.5%). MLST of 74 typeable L. monocytogenes isolates revealed high diversity, comprising 21 sequence types across 15 clonal complexes, with lineage II predominating (86.5%). Overall microbial community composition was significantly associated with Listeria spp. and L. monocytogenes presence (PERMANOVA, p = 0.001; R{superscript 2} = 0.0137 and 0.0083). In drains, ASVs assigned to Acinetobacter, Rhizorhapis, and Vagococcus species showed positive associations with Listeria spp.-positive samples. Together these findings suggest that drains are key ecological niches for Listeria spp. and that associated taxa may indicate drain communities linked to Listeria spp. recovery. IMPORTANCEListeria monocytogenes is a major foodborne pathogen that can persist in meat processing environments despite routine cleaning and disinfection. Resident microbial communities may influence its survival, but longitudinal studies linking those communities with culture-based Listeria spp. detection remain limited. Here, we characterized microbial communities in six German meat processing facilities over 1 year using 16S rRNA gene amplicon sequencing and culture-based Listeria spp. detection, and MLST of L. monocytogenes isolates. We identified facility-specific microbial communities, identified floor drains as key niches for Listeria spp., and observed repeated recovery of different L. monocytogenes sequence types across facilities. In drains, ASVs assigned to the genera Acinetobacter, Rhizorhapis, and Vagococcus species were positively associated with culture-positive samples, identifying candidate taxa that may reflect microbial conditions associated with Listeria spp. recovery. These findings highlight the importance of considering not only whether Listeria spp. are detected, but also the resident microbial communities that may support their fitness.

BackgroundRaw broiler meat products are highly perishable, and microbial activity is the main factor limiting their shelf lives. The spoilage microbiomes of broiler meat products have been studied mainly using traditional culturing methods and 16S rRNA gene amplicon sequencing, neither of which can show the activity of whole microbiome. Previous metatranscriptomic research of broiler spoilage has also remained limited to specific spoilage organisms rather than entire microbiomes. ResultsOur longitudinal study of broiler meat spoilage discovered the successions of active bacterial microbiomes and metabolic pathway activities of spoilers at 4{degrees}C and 6{degrees}C. Samples taken daily were subjected to metatranscriptomic analyses in combination with non-targeted metabolomic, traditional microbiology, and sensory analyses until advanced spoilage took place. Carnobacterium divergens, Carnobacterium maltaromaticum and Vagococcus proximus were the most active species at both temperatures. Carnobacteria are known poultry spoilers whereas Vagococcus proximus, a species recently described, played an unexpected active role in the microbiome. It became dominant in samples stored at 6{degrees}C and its activity increased also at 4{degrees}C after the use-by date. Central carbohydrate metabolism was the most common KEGG orthology pathway module of the microbiome at both temperatures. C. divergens and C. maltaromaticum showed stable metabolic profiles during the spoilage process, whereas V. proximus displayed a shift from high ATP synthesis activity to increased fatty acid and carbohydrate metabolism when spoilage advanced in samples stored at 4{degrees}C. Non-targeted metabolomics showed similar metabolomic trends across both temperatures. At 6{degrees}C, time-dependent changes were generally more pronounced, and the spoilage markers tyramine and spermidine showed greater accumulation. ConclusionsAs expected, the rate of spoilage is higher at 6 than 4{degrees}C, however we did not anticipate a similar overall trajectory of the spoilage processes. Our results link V. proximus as a key active spoiler in broiler meat and demonstrate the efficacy of using RNA-seq together with metabolomics to decode the function of a meat spoilage microbiome. This demonstrates that spoilage microbiomes consist of active species we have been neglecting due to the technological limitations of the standard methods. Future studies targeting to the metabolism and detecting of Vagococcus are warranted.

Fungal contamination of food with yeast and moulds is associated with major economic losses due to spoilage and also poses health risks in the form of mycotoxin production. The strain Pantoea agglomerans APC 4211 isolated from leaves of Ilex aquifolium (holly tree) has broad spectrum antifungal activity against a variety of food spoilage fungi. Genomic analysis of the strain confirmed the presence of biosynthetic gene clusters potentially encoding for the enzymatic machinery required for the production of the antifungal lipopeptide herbicolin A. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the cell-free supernatant (CFS) confirmed the presence of molecular masses corresponding to herbicolin A (1300.8 Da), and herbicolin B (1138 Da). Purified herbicolin A has desirable properties for biotechnological applications, including potent antifungal activity against a range of spoilage fungi, thermal stability and resistance to proteases. Herbicolin A has low cytotoxicity against epithelial cell lines and has minimum inhibitory concentrations (MICs) lower than those of some commercial antifungal drugs (0.2 - 2.5 {micro}g/ml). In a model dairy system (10% skim milk), herbicolin A demonstrated excellent solubility and stability, effectively eliminating Aspergillus niger and Penicillium notatum at a concentration of 5 {micro}g/mL. In conclusion, herbicolin A is a potent, naturally occurring antifungal agent with the potential to be applied as a biopreservative in food systems, providing a safe, clean-label, and efficient compound for synthetic preservatives replacement. HighlightsO_LIHerbicolin A has a strong potential as a natural preservative for food protection C_LIO_LIHerbicolin A shows lower MICs than several antifungal agents C_LIO_LIHerbicolin A is stable under heat and resistant to proteolytic degradation C_LIO_LIHerbicolin A has strong solubility and stability in a model dairy system C_LIO_LIHerbicolin A indicates low cytotoxicity against epithelial cell lines C_LI Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

BackgroundReliable detection of Salmonella remains a major challenge for public health surveillance and food safety due to the growing diversity of circulating serovars and the limitations of existing molecular targets. This study aimed to identify an optimal molecular target and develop a TaqMan real-time PCR assay for the detection of Salmonella spp. MethodsBased on the results screening for Salmonella genes suitability as molecular markers, a TaqMan real-time PCR assay targeting the hilA gene was developed and validated. Analytical sensitivity, analytical specificity, and performance on bacterial isolates and artificially contaminated food samples were assessed. ResultsAmong all candidate targets, hilA demonstrated the broadest coverage and was detected in all tested Salmonella isolates, including representatives of rare serological groups, whereas invA conventionally used for this pathogen detection, was absent in a subset of strains. The assay exhibited a limit of detection of 100 bacterial cells/mL and 100 fg/L of genomic DNA. No cross-reactivity was observed with DNA from Shigella flexneri, Shigella sonnei, Yersinia pestis, Y. pseudotuberculosis, Y. enterocolitica, Y. kristensenii, Bacillus anthracis, Vibrio cholerae, or Francisella tularensis. The assay successfully detected Salmonella DNA in all artificially contaminated food samples tested. Evaluation using a collection of 25 bacterial isolates demonstrated positive amplification in all 24 confirmed Salmonella strains, while a strain initially identified by conventional bacteriology as Salmonella but subsequently confirmed by whole-genome sequencing as Proteus mirabilis yielded a negative result. ConclusionsThe hilA gene represents a highly conserved and reliable molecular target for the detection of Salmonella spp. The developed TaqMan real-time PCR assay demonstrated high analytical sensitivity, excellent specificity, and broad serovar coverage, supporting its application in laboratory detection of Salmonella, food safety monitoring, and epidemiological surveillance.

Listeria monocytogenes remains a major foodborne pathogen with high mortality and costly persistence in food-processing environments. Established diagnostics rely on selective enrichment and single-colony isolation, which could introduce strong biases by favouring fast-growing strains or those more tolerant to enrichment broth inhibitors, while suppressing slow-growing, viable-but-nonculturable, and other co-occurring strains. This can obscure true pathogen diversity and may contribute to discrepancies between strains detected in food production environments and those associated with disease. To quantify the bias introduced by established culture-based diagnostics and to assess the potential advantage of metagenomics-based pathogen detection directly from the original sample matrix, we developed and evaluated a rapid nanopore sequencing-based metagenomic framework. We designed an artificial metagenomic community of several Listeria strains, comprising L. monocytogenes lineages I-III (including hypervirulent, persistent, and low-virulence strains), other Listeria spp., and a realistic background microbiome representative of food-processing environments. We then used this mock community to spike standard surveillance sponges and compared three workflows: (i) direct nanopore metagenomic sequencing of the original sample matrix, (ii) quasi-metagenomic sequencing after 4 h, 12 h, 24 h, or 48 h of selective enrichment, and (iii) ISO-based culture followed by whole-genome sequencing of a single presumptive L. monocytogenes isolate. We found that the culture-based approach recovered only a limited subset of strains, consistently underrepresenting diversity and failing to detect multi-strain contamination. These findings were reflected by the quasi-metagenomic results, where we found relative L. monocytogenes enrichment to be strain-dependent, indicating selective enrichment bias favouring specific strains. Metagenomics captured the full spectrum of spiked Listeria strains, enabling comprehensive strain-level resolution at all inoculation levels. We only observed relative enrichment of the L. monocytogenes strains by quasi-metagenomics compared with metagenomics after 48 h of selective enrichment. While driven primarily by the additional enrichment of L. innocua, these results suggest that quasi-metagenomics improves L. monocytogenes recovery only at the cost of a substantial reduction in speed. We finally showed that the sensitivity and accuracy of metagenomics could be improved by utilising different environmental sampling materials. We did not find any significant performance improvements from nanopore sequencing-based enrichment of L. monocytogenes through adaptive sampling approaches. We conclude that integrating long-read metagenomics into routine surveillance shows great promise to improve detection and source attribution in food safety systems.

Across the United States, backyard poultry (BYP) are becoming increasingly popular as a food source as well as pets. Unfortunately, they have also been a source of annual human salmonellosis outbreaks for over a decade. Previous CDC analyses suggest baby poultry are the main source of live poultry-associated outbreaks as opposed to adult birds. However, there are few data on the frequency of pathogens, such as Salmonella enterica, in baby poultry sold to the BYP market. Further, there is a lack of data on the serovars and antimicrobial resistance (AMR) rates in these baby poultry. We collected 643 soiled bedding and shipping box samples from agricultural supply stores primarily located in Vermont. S. enterica was detected in 23.5% (151/643) of samples, with the highest rates of detection in 2021-2022. Rates of S. enterica varied by species. Turkey poult bedding samples had the highest rates of S. enterica (44.4%; 8/18), while laying chick bedding samples had the lowest (19.4%; 68/350). Meat chick bedding samples had an intermediate rate, at 36% (32/89). The most common serovar detected was Salmonella Enteritidis, which represented 51.2% (64/125) of sequenced isolates. AMR genes or AMR-associated point mutations were detected in 21.6% (27/125) of samples, but only in non-Enteritidis serovars. These data indicate that baby poultry intended for the BYP market pose a substantial risk of salmonellosis to consumers.

Bacterial spot is a consistent threat to global tomato and pepper productions; however, Ohios fresh market production currently lacks the updated surveillance data necessary to provide accurate management solutions. While traditional diagnostics focus on identification of a single causal agent, shotgun metagenomic sequencing (MGS) offers a comprehensive view of the infection court. An assignment-first MGS workflow was developed and validated in this study, utilizing Kraken2 databases to extract Xanthomonas species associated with bacterial spot and to characterize the microbial communities of bacterial spot in Ohio production systems. Through in silico spiking experiments, thresholds were established for bacterial spot identification. Species and pathovar identification via average nucleotide identity (ANI) remained accurate at abundance as low as 0.1%. A minimum of 2% Xanthomonas reads were required for high genome completeness (BUSCO >90%) and 3% for reliable type III secretion system (T3SS) effector profiling. Analysis of 63 samples from fresh-market production fields identified Xanthomonas hortorum pv. gardneri, Xanthomonas euvesicatoria pv. euvesicatoria, and Xanthomonas arboricola residing in symptomatic samples, alongside other taxa including Pseudomonas and Stenotrophomonas. Phylogenetic comparisons of metagenome-assembled genomes (MAGs) were comparable to whole genome sequences (WGS) from the same samples, supporting the reliability of culture-independent diagnostics. These results provide a robust framework for utilizing metagenomics as a diagnostic tool, expanding our knowledge of bacterial spot population structure in Ohio, and uncovering the bacterial communities associated with bacterial spot.

Pathogen cross-contamination during food production is primarily controlled through environmental sanitation. However, sanitizer efficacy is often studied in bench-scale experiments that poorly approximate the fluid dynamics of sanitization and limits our understanding of commercial sanitization efficacy. This study paired computational fluid dynamics (CFD) estimates of shear stress with experimental measurements of Listeria innocua reduction on stainless steel following treatment with 100 ppm hypochlorite sanitizer. At the pilot-scale, sanitizer spray manually applied by researchers achieved a 2.6 {+/-} 0.4 log CFU/surface reduction; however, microbial reduction from manual operation of sanitizer spray equipment differed significantly between researchers (p < 0.05). Microbial reduction varied by location following stationary, bench-scale spray application of sanitizer for 3 s. The greatest reduction was at the point of sanitizer spray impingement (7.5 {+/-} 0.5 log CFU/surface) and directly adjacent to the impingement point (6.4 {+/-} 0.7 log CFU/surface) where shear stress was the highest. Significantly less microbial reduction (0.4 {+/-} 0.1 log CFU/surface) occurred where shear stress was lowest in the fluid-film of sanitizer running down from the impingement point (p < 0.05). Static submersion of inoculated coupons in sanitizer for 3 s resulted in a log reduction of 2.3 {+/-} 0.1 log CFU/surface. Discrepancies between bench-scale spraying, pilot-scale spraying, and submerged coupons demonstrate the need for sanitizer efficacy testing under realistic conditions to better estimate the risk reduction achieved through sanitation programs. IMPORTANCESanitation is critical for controlling pathogen cross-contamination during food production. These findings highlight the limitations of traditional approaches to sanitizer efficacy testing, not because they are invalid, but because they do not reflect the level of microbial reduction typically achieved in application. We demonstrate that these differences in outcomes are attributable to fluid dynamics and exposure, which are not well approximated in submerged coupon experiments. Accurate estimation of microbial reduction from sanitizer application is needed to guide food safety policy decisions. For example, overestimation of the risk reduction conferred by sanitizer treatment may result in food safety policies that neglect other sources of microbial reduction within sanitation programs.

Cocoa fermentation is a spontaneous microbe-driven process in which yeasts, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) generate the flavor precursors that determine the sensory quality of chocolate. Although the microbial ecology of cocoa fermentation has been increasingly studied through culture-independent methods, the effect of targeted nutritional interventions on community structure within geographically defined production territories has received limited attention. Here, we employed dual-marker metabarcoding (16S rRNA V4 and ITS1) with Illumina NovaSeq 6000 sequencing to characterize bacterial and fungal communities during spontaneous fermentation of Trinitario cocoa beans subjected to amino acid and zinc supplementation in the Limon province of Costa Rica. Fifteen samples were collected at 0, 24, and 48 h from control, amino acid-supplemented, and zinc-supplemented fermentations, each in duplicate. The bacterial community comprised 292 amplicon sequence variants (ASVs) representing 88 genera across 15 phyla; the fungal community comprised 1,117 ASVs representing 248 genera across 9 phyla. Firmicutes and Proteobacteria dominated the bacterial fraction, with a pronounced shift from Tatumella-dominated fresh pulp toward Weissella- and Leuconostoc-rich assemblages during fermentation. Amino acid supplementation reduced Firmicutes at 48 h while favoring Acetobacter proliferation; zinc supplementation promoted Mucoromycota and Wickerhamomyces while sustaining Liquorilactobacillus abundance. Beta diversity analyses (Aitchison distance, weighted and unweighted UniFrac) confirmed significant compositional differences between treatments (PERMANOVA, p [&le;] 0.01), although alpha diversity indices did not differ between individual treatment pairs. Sparse Estimation of Correlations among Microbiomes (SECOM) revealed structured co-occurrence networks, including positive associations between Gluconobacter and Acetobacter and negative associations between Tatumella and several AAB genera. Predicted functional profiles (PICRUSt2) showed no significant pathway-level differences. Taken together, these results show that nutritional supplementation can reshape microbial community composition without reducing overall diversity. This provides a viable approach for steering fermentation outcomes in cocoa-producing territories that seek quality differentiation.

Climate change and eutrophication are driving the expansion of the range of Vibrio species, including V. parahaemolyticus. This bacterium is a major foodborne pathogen and understanding the biogeography of virulent strains of this species is crucial for ensuring food safety. Whole-genome sequencing (WGS) provides strain-level identification of bacteria and is widely used for tracking bacterial pathogens; however, WGS is costly and labor-intensive. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides a rapid, accurate, and cost-effective method for bacterial identification; however, the resolving power of MALDI-TOF MS and WGS for V. parahaemolyticus has not been systematically compared. In this study, 70 V. parahaemolyticus strains were isolated from oysters (Crassostrea virginica) collected from the Gulf Coast and Massachusetts. Oysters were collected in Galveston Bay (Texas) and aquaculture plots in Massachusetts, and purchased from seafood markets in Texas and Louisiana in the U.S. For comparison, two isolates of V. anguillarum were cultured from the exoskeleton of blue crabs purchased from a seafood market in Seabrook (Texas). All isolates were identified using the MALDI Biotyper system and analyzed with custom R scripts. Cluster analysis of mass spectra generated by MALDI-TOF MS, and phylogenomic analysis revealed distinct clusters corresponding to the source of oysters. In both the mass spectra and WGS analysis, V. parahaemolyticus strains isolated from Massachusetts formed a coherent cluster. For comparisons between species, cosine similarities of mass spectra generated by MALDI-TOF MS ranged from 0.43 to 0.59, and average nucleotide identity (ANI) values generated by WGS ranged from 76% to 77%. For comparisons within species, cosine similarities of mass spectra ranged from 0.68 to 0.91 and ANI values ranged from 98% to 100%. This suggests that MALDI-TOF MS has a resolution comparable to WGS and can be used to track strains of V. parahaemolyticus associated with oysters.

Microbial spoilage of packaged meat is driven by complex microbial succession and related metabolic activity, yet conventional shelf-life assessment is mainly based on shelf-life studies relying on culturing and sensory analysis. In routine quality assurance, results are obtained retrospectively, and they are only indirectly linked to the metabolic activity related to sensory deterioration. Functional, time informative approaches that capture the active metabolic state of the spoilage microbiome and predict the rate of spoilage are lacking. We developed a censoring-aware Gaussian process (CAGP) framework to model longitudinal pathway expression profiles from broiler meat metatranscriptomes collected over consecutive storage days at 4 or 6{degrees}C. Samples were annotated using odor-based sensory scores defining fresh, early-spoilage, and late-spoilage phases. Because observed zeros in pathway-level data may reflect non-detection rather than true absence, the model treats low values as left-censored observations below a soft detection threshold while estimating smooth temporal trajectories with uncertainty. In leave-one-out prediction within the 4{degrees}C time-series, predicted sampling days differed from the true days by an average of 0.43 days, and predicted spoilage phases agreed with the sensory classification. Trajectories learned at 4{degrees}C also transferred to an independent 6{degrees}C time-series at the spoilage-phase level, suggesting that shared functional spoilage programs are preserved despite temperature-dependent changes in spoilage rate. Cross-entropy ranking further identified pathway modules carrying time- and phase-informative signals across temperatures. Overall, this framework provides a probabilistic approach for linking metatranscriptomic functional dynamics to sensory spoilage progression, supporting shelf-life assessment beyond retrospective microbial enumeration. IMPORTANCEShelf-life evaluation of meat products still relies heavily on microbial counts, targeted detection of spoilage organisms, and sensory panels. However, microbial abundance and species-level composition do not always predict when a product becomes unacceptable, because spoilage depends on the active metabolic state of the microbiome and can vary between strains, production lots, and storage conditions. This study shows that longitudinal metatranscriptomics, combined with censoring-aware Bayesian time-series modeling, can recover functional pathway trajectories aligned with sensory spoilage progression. By identifying pathway-level signatures that transfer across refrigeration temperatures, the approach moves shelf-life assessment from retrospective enumeration toward predictive, function-based monitoring. In this study, a spoilage signature refers to a set of microbial pathway trajectories whose expression patterns are informative of storage time and sensory spoilage phase. These signatures could support future tools for earlier spoilage detection, better shelf-life estimation, and improved control of product quality in meat production.

It was recently reported that Methylobacterium extorquens AM1 produces the citrate-hydroxamate siderophore N-deoxyschizokinen A, identified by LC-HRMS. Multiple properties were inconsistent with the assignment: the feature eluted far later than the other schizokinen derivatives (17 min versus 6-8 min), a reversed-phase shift larger than a single-hydroxyl difference in a molecule can explain, further its accurate mass deviated from the calculated one by 28 ppm, well outside the error on the co-analyzed standards and its diagnostic m/z 105 and 77 fragments suggest a molecule with an aromatic moiety. A replicate comparison of identical samples in plastic versus glass autosampler vials was decisive: the m/z 387 feature was reproducibly present with plastic vials and absent with glass. We therefore conclude that the reported detection of N-deoxyschizokinen A in M. extorquens AM1 is an artifact, and recommend glass-vial and solvent-blank controls, an explicit accurate-mass threshold, and narrow MS/MS isolation when assigning trace siderophore-like features from complex extracts.

Edible seaweed has the potential to become a valuable marine resource for food applications due to its potential health benefits and ecological sustainability. The brown seaweed Alaria esculenta is rich in essential minerals, vitamins, and dietary fibers, making it a nutritious food source. Fermentation, as a traditional preservation method, can enhance seaweed shelf-life and be useful for the development of new foods/ beverages. In this study, the effects of fermentation of A. esculenta, by the lactic acid bacterium (LAB) Lactiplantibacillus plantarum, on the nutritional profile, and the content of potentially toxic elements, was investigated. L. plantarum was successfully cultivated on A. esculenta using two modes of operation, submerged (SmF) and solid-state fermentation (SSF), resulting in production of cells and lactic acid, and reduction of the pH to below 4.3 within 3 days, which was not achieved in parallel spontaneous fermentations using indigenous seaweed microbiota. A. esculenta s macro-nutritional profile was altered, reducing mannitol but increasing fucose and glucose content (after acid hydrolysis) while also concentrating the protein content. LAB fermentation significantly increased the concentration of antioxidant phenolic compounds, such as phloroglucinol, syringic acid, and epicatechin, compared to untreated samples. However, lipophilic compounds like carotenoids decreased after both spontaneous and LAB-fermentation. A reduction in total mineral content was observed after LAB fermentation and water soaking, and SmF with L. plantarum effectively reduced arsenic and iodine levels. Overall, fermentation using L. plantarum showed potential as a bio-preservation method for the edible brown seaweed, A. esculenta, improving its nutritional profile and enhancing food safety.

In this research we inoculated Lacticaseibacillus casei (L. casei) into wheat sprouts and studied the viability of the bacteria in the sprout. L. casei (ATCC39392) strain was inoculated into sterilized wheat sprouts. The height of the sprouts and roots were checked and the active phenolic content, flavonoid compounds, and the antioxidant activity were measured. The bacterial viability was determined under simulated gastrointestinal (SGI) conditions. The physicochemical properties of the final product and its organoleptic properties were also investigated. The final confirmation of the presence of bacteria was also done by transmission electron microscope imaging. The number of bacteria increased from 8.18 Log CFU/g to 11.81 {+/-} 0.33 Log CFU/g. The increase in the phenolic content and antioxidant activity indicates the improvement of the nutritional value of the sprout. The physicochemical properties of the product changed due to the activity of bacteria. The inoculated bacteria also survived after exposure to SGI. The organoleptic properties of the product did not reveal a significant difference between the control and treatment groups. The increase in the number of bacteria and their survival after exposure to SGI indicates the suitable condition of wheat sprout as a proper substrate for L. casei bacteria.

Listeria monocytogenes causes listeriosis in humans and animals and contaminates prepared food by attaching to food processing environments. Therefore, closer monitoring of how the organism adheres to surfaces will help identify ways to prevent it from colonising food-processing environments. To develop new attachment assays, clinical and environmental strains of L. monocytogenes were transformed by inserting a plasmid containing lux, gfp reporter genes and an erythromycin-resistant gene into the parent cells. Transformed cells were grown for 48 hours on brain heart infusion agar plates containing 1-5{micro}g/ml of erythromycin, after which the cells were viewed under a molecular light imager and luminometer. Fluorescent cells containing the gfp, lux, and erythromycin-resistant genes were visible, whereas control cells without the plasmid were not. Transformation efficiency was highest with the environmental strains, and subsequent growth and hydrophobicity tests carried out with the transformed cells in different growth conditions showed that they were able to attach well to solvents when compared to the parent cells. However, the growth rate of the transformed cells was poor, indicating a disruption of cell metabolism. Results show the possibility of real-time monitoring of how cells attach to different surfaces and could lead to a better understanding of the initial colonisation of a surface by the organism.

Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes the interaction with negatively charged components of the yeast cell surface, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated the interaction of chitosan with the cell surface and its permeabilizing capacity in three yeast species displaying distinct susceptibility profiles, Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii. We evaluated the correlation between differential susceptibility and chitosan association at the cell surface, as well as cell permeabilization, by integrating growth analyses with surface-binding assays, including FITC-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell surface and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization as a method to measure the activity of selected enzymes in situ, demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast.

Campylobacter jejuni is a leading cause of foodborne gastroenteritis globally and is classified by the CDC as a serious public health threat due to increasing resistance to fluoroquinolones and macrolides. This study used whole-genome sequencing to characterize the virulence and antimicrobial resistance profiles of 2,783 clinical C. jejuni isolates collected from Minnesota residents from 2018 through 2021. More than 90% of the isolates had genes related to stress defense (rpoN and htrB), cytolethal distending toxin (cdtA, cdtB, and cdtC), and cell adhesion and invasion (ciaB, cadF, and flaC). A diverse array of antimicrobial resistance genes was detected, with beta-lactam resistance genes having a particularly high prevalence. The gyrA point mutation associated with quinolone resistance was present in 29% of isolates. To evaluate the correlation between genotypic and phenotypic antimicrobial resistance profiles, the antimicrobial susceptibility testing results from a subset of isolates were compared with genotypic resistance profiles. Results showed a strong overall correlation, particularly for tetracycline and quinolones, though 24 discrepancies were detected. In the majority of discrepancies (n=21), genomic antimicrobial resistance markers were absent in isolates that were phenotypically resistant, suggesting possible unknown resistance mechanisms or limitations in current sequencing methods. The remaining three discrepancies occurred in isolates that had the tet(O) resistance gene but were susceptible to tetracycline phenotypically. These findings highlight the value of whole genome sequencing in improving antimicrobial resistance surveillance and understanding virulence factors in C. jejuni, supporting its integration into routine monitoring practices to better manage and understand antimicrobial resistance in foodborne pathogens.