International Journal of 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.

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.

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.

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.

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.

In many tropical countries, fermentation of palm sap into palm wine is an important fermented beverage contributing to local economies, tradition, and culture. Traditionally made in villages and families, palm sap is not inoculated with starter cultures and fermentation commences spontaneously. It is therefore possible that fermentation is influenced by multiple ecological factors, which affect microbial dynamics and thus flavour outcomes. Here, we studied microbial communities during fermentation of palm sap from three different palm tree species (palmyra, coconut, and sugar palm) on the island of Bali, Indonesia in both the wet and dry seasons. Our results suggest that season of collection has a strong influence on microbial dynamics and succession, and these changes positively correlate to metabolite concentration. The change of the season from the dry to wet season led to the loss of microbial diversity with lower richness in the dry season. The dominance of Saccharomyces cerevisiae was not affected by season and fermentation time and was dominant in all samples. Potential spoilage species, such as Candida tropicalis were negatively correlated to ester production and more abundant in the dry season. As microbial species varied in incidence and thus biochemical activity, the chemical groups of esters from their metabolism related to the change of season and fermentation time, while volatile compounds and small molecules were highly discriminated by season in the resultant wines. Ethyl octanoate was consistently different across all variables through comparison by three-way ANOVA and is proposed as a biomarker of seasonal variation in palm sap fermentation. These findings improve our understanding of microbial dynamics in palm sap fermentation, revealing flavour differentiation within season and suggests that strategies for microbial management, product development and quality assurance will elevate this traditional product into the future.

Noble rot, caused by Botrytis cinerea, profoundly alters grape berry physiology and is essential to produce botrytized wines. In this study, we profiled bacterial and fungal communities associated with Vitis vinifera cv. Semillon berries across four stages of noble rot development and four consecutive vintages using 16S rRNA gene and ITS1 amplicon sequencing. Noble rot stage significantly impacted the structure of bacterial communities across vintages, while fungal communities showed more variable vintage-dependent responses. Bacterial alpha diversity increased consistently at advanced stages of infection (S3), coinciding with a marked shift from Pseudomonas-dominated communities toward acetic acid bacteria, particularly Gluconobacter, which was significantly enriched at S3 in all vintages. Fungal communities remained dominated by Sclerotiniaceae throughout infection, consistent with pervasive B. cinerea colonization, while non-Botrytis fungal taxa shifted from filamentous fungi such as Cladosporium and Alternaria toward fermentative yeasts including Hanseniaspora and Lachancea. Co-occurrence network analysis revealed a positive association between Gluconobacter and these fermentative yeasts, suggesting coordinated enrichment of oxidative and fermentative microorganisms at advanced noble rot stages. Together, these results reveal a reproducible stage-associated microbial succession during noble rot progression and identify acetic acid bacteria as consistent markers of advanced infection.

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.

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.

Microbial colonization is a major cause of deterioration in paintings, leading to discoloration, pigment degradation, and loss of structural integrity. While biodeterioration of artworks has been studied in temperate climates, tropical environments remain underexplored despite their high humidity and temperature, which promote microbial growth. This study assessed the microbiological deterioration of two eighteenth-century oil paintings, La Muerte de San Jose and Virgen de Guadalupe, located in Orosis Colonial Church and Religious Art Museum, Costa Rica. Microorganisms were isolated and identified using VITEK(R) 2, microscopy, and MALDI-ToF analysis, and their biofilm-forming capacity was evaluated. Additionally, the antimicrobial activity of six essential oil components was tested using direct and indirect contact assays. Twenty-three bacterial species and fifteen fungal genera were identified, with Bacillus, Staphylococcus, Cladosporium, and Aspergillus among the most common. Notably, La Virgen de Guadalupe displayed the highest microbial diversity, reflected in a high Shannon index, indicative of a more complex microbial community. Several isolates displayed strong biofilm formation, particularly Bacillus subtilis/amyloliquefaciens/vallismortis and Staphylococcus saprophyticus. Linalool exhibited the strongest inhibitory activity, achieving complete bacterial growth inhibition in non-contact assays. Environmental monitoring revealed persistently elevated relative humidity and CO2 levels during the study period. Together, these results reveal the complex microbial ecology of tropical heritage paintings and demonstrate that volatile essential oil components can serve as candidates for low-impact antimicrobial strategies in preventive conservation. ImportanceUnderstanding the microbiological deterioration of cultural heritage in tropical environments is crucial for designing sustainable conservation strategies. While microbial colonization of artworks has been widely studied in temperate regions, data from tropical climates remain limited despite inherently favorable conditions for microbial proliferation. This study integrates microbiological, environmental, and physicochemical analyses to characterize microbial communities colonizing eighteenth-century oil paintings in Orosi, Costa Rica. By combining microbial identification, biofilm quantification, and essential oil biocide testing, it bridges applied microbiology and cultural heritage conservation. The finding that volatile components such as linalool inhibit biofilm-forming bacteria without direct contact highlights their potential as eco-friendly, noninvasive antimicrobial alternatives to conventional biocides. These results expand the understanding of biodeterioration dynamics under tropical conditions and offer a practical framework for developing sustainable, evidence-based conservation protocols that protect both heritage materials and the environment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/723565v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@16cd608org.highwire.dtl.DTLVardef@57aa00org.highwire.dtl.DTLVardef@159fcbeorg.highwire.dtl.DTLVardef@e0363b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 0.C_FLOATNO Artistic visualization of the geographical context of the studied artworks and the multidisciplinary analytical approaches applied, highlighting the diversity of microorganisms identified (illustration by Keylin Urena-Alvarado). C_FIG

The search for antimicrobials with a low propensity to select resistance has intensified in response to the global antimicrobial resistance crisis. Norway spruce resin (Picea abies) has long been used in Northern European wound care traditions and has shown broad antimicrobial activity in earlier microbiological studies. In the present study, we evaluated whether prolonged exposure to medical-grade spruce resin promotes reduced susceptibility in clinically relevant bacterial species. A 20-day serial-passage experiment was performed with Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis using sub-inhibitory resin concentrations and broth microdilution readouts at baseline, day 10, and day 20. Resistance development was predefined as a [&ge;]4-fold increase in inhibitory concentration. Baseline inhibitory concentrations were 1.25% for S. aureus, 5.0% for P. aeruginosa, and 2.5% for E. faecalis. After 20 days, inhibitory concentrations were 2.5%, 10.0%, and 2.5%, respectively, corresponding to at most 2-fold changes and remaining below the predefined threshold for resistance development. Validation and vehicle-control arms indicated that these shifts were not attributable to medium transfer or solvent-related bias. These findings suggest that medical-grade Norway spruce resin has a low short-term tendency to select for reduced susceptibility under serial-passage conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723837v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@160479forg.highwire.dtl.DTLVardef@1fe1e95org.highwire.dtl.DTLVardef@89dec3org.highwire.dtl.DTLVardef@17ff134_HPS_FORMAT_FIGEXP M_FIG C_FIG

Beetles (order Coleoptera) are a diverse insect group whose associated microbial communities remain understudied despite their ecological, commercial, and biotechnological importance. Larvae of the darkling beetle species Tenebrio molitor (mealworms) and Zophobas atratus (superworms) have gained attention for their reported ability to ingest and possibly degrade plastics such as polyethylene and polystyrene. This degradation is believed to be mediated by enzymes from their gut microbiota, yet the underlying microbial mechanisms remain unclear. Using deep shotgun metagenomics, we generated comprehensive gut metagenomes for mealworms and superworms, recovering 53 and 100 high-quality metagenome-assembled genomes (MAGs), respectively. We found that the gut microbiomes of both insect species are dominated by bacteria from the phyla Bacillota and Pseudomonadota, with superworms trending towards greater bacterial diversity than mealworms. Comparing gut bacterial communities of insects fed polyethylene or polystyrene versus controls revealed no clear plastic ingestion effects beyond those attributable to starvation. On the other hand, insect cohort contributed substantially to variation in bacterial community composition, explaining 23% and 33% of the total variation in mealworms and superworms, respectively. Finally, we computationally identified numerous secreted proteins with sequence similarity to enzymes previously implicated in polyethylene and polystyrene degradation, and we provide support for dye decolorizing peroxidase (DyP)-type peroxidases as being the enzyme class mostly likely to initiate plastic oxidation in the digestive tracts of mealworms and superworms. Overall, this study provides high-quality metagenomes and MAGs for mealworms and superworms, revealing substantial under-explored microbial biodiversity, and highlighting DyP-type peroxidases as promising targets for future plastic biodegradation studies.

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.

Herbivore gut microbiomes may contain a diversity of anaerobic gut fungi (AGF, phylum Neocallimastigomycota), important for fibre degradation. To perform functional studies and elucidate niches of different AGF species, representative fungal isolates must be obtained into axenic culture, which is a resource-intensive process. Here we leverage the integration of morphological and functional assessments of AGF isolates with a newly developed PCR-RFLP strategy, to distinguish and identify isolates of interest from faecal samples from zoo-housed animals. In silico prediction of PCR-RFLP profiles of cultured genera, followed by experimental validation, confirmed that LSU-based PCR-RFLP with AluI and Hyp188I digestion was effective in identification of fungi of distinct genera. Together our workflow resulted in isolation of a so far uncultured Piromyces (NY08) species and Neocallimastix cameroonii from nyala samples, as well as Feramyces austinii from giraffe and Khoyollomyces ramosus from zebra. Amplicon sequencing confirmed that these species dominated AGF communities in their hosts, likely benefiting isolation success, and we identified enrichment conditions which also affected cultivability. The workflow developed here aids efficient AGF isolations, which are instrumental in expanding opportunities for functional studies that provide insight into the physiology and ecology of these fungi and help realise applications in white and green biotechnology. One sentence summaryA validated PCR-RFLP strategy, developed based on genetic diversity data from Neocallimastigomycota, enables efficient identification of isolates of these anaerobic gut fungi from environmental samples, as demonstrated via targeted enrichment of anaerobic gut fungi common in faeces of giraffe, zebra and nyala, resulting in isolation of species of genera Feramyces, Neocallimastix, Khoyollomyces, and a novel Piromyces/NY08 species.

Bacterial canker of tomato, caused by Clavibacter michiganensis (Cm), remains difficult to control due to lack of effective management options. In this study, a collection of over 500 bacterial isolates was screened in vitro for antagonistic activity against Cm and plant growth-promoting (PGP) traits. Based on these results, 32 candidates were evaluated in planta, leading to the identification of three highly effective strains: Pantoea agglomerans SO16PY and two Pseudomonas marginalis sensu lato strains, IRDA16 and SO16PC, which consistently enhanced tomato vegetative growth. Notably, P. agglomerans SO16PY delayed disease onset in Cm-inoculated plants by up to 7 days and significantly reduced wilting severity, lowering the disease severity score from 85% to 45%. Strains IRDA16 and SO16PC also restricted disease development, reducing severity scores to 67.5% and 57.5%, respectively. Whole-genome sequencing and comparative genomics revealed that strains IRDA16 and SO16PC form a distinct, specialized rhizosphere lineage within the Pseudomonas marginalis group, exhibiting average nucleotide identity (ANI {approx} 96%) and digital DNA-DNA hybridization (dDDH {approx} 69.5%) values near species delineation thresholds. Genome mining identified diverse biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), the lipopeptide viscosin, and terpenes, which likely drive the biostimulant and antagonistic traits of this novel Pseudomonas lineage. Together, these findings characterize promising bacterial candidates with dual biostimulant and biocontrol capacities while uncovering a genomically distinct Pseudomonas lineage optimized for beneficial plant-microbe interactions in sustainable agriculture. IMPORTANCEClavibacter michiganensis (Cm) is a major bacterial pathogen of tomato and poses a significant economic threat to global production. It is classified as an A2 quarantine pathogen by the European and Mediterranean Plant Protection Organization (EPPO). Current management strategies rely largely on chemical control, including copper-based compounds (e.g., Bordeaux mixture, copper oxychloride), mancozeb, and antibiotics like streptomycin. However, these approaches raise increasing concerns related to environmental contamination, phytotoxicity, and the development of resistant pathogen populations. As a sustainable alternative, plant growth-promoting bacteria (PGPR) have emerged as promising biocontrol agents. In this study, we identified bacterial strains exhibiting antagonistic activity against Cm both in vitro and in planta. Notably, these strains also enhanced tomato growth parameters, demonstrating their dual functionality. Given the environmental drawbacks associated with chemical inputs, the use of such beneficial microorganisms represents a promising strategy for advancing sustainable and ecofriendly tomato production systems.

ObjectivesListeria monocytogenes is an opportunistic pathogen, associated with foodborne infections that disproportionately affect newborns, elderly and immunocompromised patients. L. monocytogenes can be classified on the antigenic and related structural variation of cell-associated wall teichoic acid (WTA) molecules through conventional serotyping techniques. The WTA structure of serovars (SV) 1/2, 1/2*, 3 and 7 consists of a linear poly-ribitolphosphate (RboP) polymer either with or without decoration with rhamnose (Rha) and/or N-acetylglucosamine (GlcNAc). Of these four SVs, SV1/2 (WTA with GlcNAc and Rha) causes [~] 99% of all listeriosis cases. However, conventional serotyping cannot accurately discriminate between these four SVs, particularly SVs1/2* (WTA with Rha). MethodsHere we applied two identified monoclonal antibodies (mAb), with specificity for the RboP backbone or GlcNAc modification to develop a discriminatory serotyping scheme for SV1/2, 1/2*, 3 and 7. Isogenic mutants for the different SVs were created in L. monocytogenes SV1/2 strain EGD-e. The typing scheme was then adapted to an immnoblot assay and applied to a collection of 317 previously classified listeriosis isolates from the Netherlands Reference Laboratory for Bacterial Meningitis. ResultsBinding of the RboP-specific mAb was limited to EGD-e wild type (SV1/2), but increased significantly for isogenic EGD-e mutants representing SV1/2*, 3 and 7. In contrast, the GlcNAc-specific mAb only recognized EGD-e mutants representing SVs 1/2 and 3. The combined staining profiles of the two mAbs allowed accurate discrimination of the four SVs as verified on clinical isolates. Applying this typing scheme to 317 listeriosis isolates previously typed as SV1/2, we confirmed SV designation in >90% of isolates, but also identified SV1/2* (5.4%), SV3 (0.6%) and SV7 (0.3%) isolates. SV1/2* isolates were also identified among meningitis patients. ConclusionThe increased discriminatory capacity of L. monocytogenes serotyping provides a more detailed insight of the epidemiological landscape and the critical factors for L. monocytogenes infections.

Mushroom production generates large amounts of by-products, particularly stipes, which can represent up to half of the fruiting body biomass. Due to their similar composition to mushroom caps, these residues represent a promising substrate for the development of value-added foods. In this study, oyster mushroom stipes were used as a substrate for solid-state fermentation (SSF) with a Neurospora crassa strain isolated in Albacete to produce a novel meat analogue inspired by the oncom. Fermentation generated a cohesive matrix bound by hyphae that adopted the shape of the mold and exhibited a meat-like color, although with a softer texture. Nutritional analysis revealed a product with relatively low protein content but a complete amino acid profile, enriched in dietary fiber and containing unsaturated fatty acids. These results demonstrate that SSF with N. crassa provides a strategy to upcycle oyster mushroom by-products into fiber-rich meat analogues with potential applications in sustainable food systems.

Soft rot Pectobacteriaceae (SRP) are among the most economically important plant pathogenic bacteria and are especially known to be problematic in potato production. The epidemiology of disease transmission has been investigated for almost a century, and several aspects have been highlighted as plausible infection routes. However, it is generally accepted that the major source of disease is the latently infected mother tuber, but several parameters are still influencing disease prevalence including contaminated equipment, soil water status as well as temperature. Management of the disease is limited to hygiene practices, dry storage and seed certification systems but several studies have also proven biocontrol agents such as bacteriophages (phages) as promising tools. Despite the severity of SRP on potato production, little is known about the genetic diversity of SRPs in Denmark, and since only few isolates are available, the possibility to design a broadly effective phage cocktail is limited. Here we describe a three-year field study utilizing an agri-citizen science approach where Danish farmers provided symptomatic potato plants or tubers, together with metadata such as date, location, potato variety and origin. By using whole genome sequencing (Illumina and Nanopore) together with metadata we were able to investigate and monitor the epidemiological disease spread across the country using 103 complete genomes, sampled across all three years. In this study we provide epidemiological evidence of disease origins and a suite of phages that could be used as a biocontrol tool for early disease intervention. Our results revealed several clonal clades across diverse locations (SNPs < 20) which strongly indicate common origin. A total of 17 Pectobacterium phages were tested and did target > 80% of clonal clades. Based on the clonality across the soft rot isolates we propose the possibility to set in early on using phages targeting strains relevant for soft rot development, with the possibility of a surveillance program together with customizing the phage preference.

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.

Black soldier fly larvae (BSFL) are generalist decomposers with promise for converting agricultural and food-processing by-products into value-added bioproducts, but BSFL performance on lipid-rich waste oil streams and the role of the gut microbiome in this process remains unclear. Here, we evaluated BSFL bioconversion of a standard chicken feed diet supplemented with three chemically distinct waste oils: acidulated vegetable oil (AVO), pork grease (PG), and used cooking oil (UCO). Larval performance, bioconversion rate, gut microbiome composition, total protein and fat content, and fatty-acid profiles were measured across bioconversion. Larval age was a major driver of gut microbiome structure, but waste oil supplementation further reshaped community membership and structure, with the strongest diet-associated effects occurring during early-to-intermediate bioconversion. Most differentially abundant taxa were members of the baseline core gut community, suggesting that oil supplementation primarily altered dominance patterns among resident taxa. PG and UCO supported larval growth and bioconversion performance comparable to the chicken feed control, whereas AVO reduced bioconversion rate and showed weaker growth outcomes. Oil supplementation also increased larval fat content, reduced protein content, and shifted fatty-acid profiles toward the corresponding oil feedstocks, although larval biomass composition remained shaped by basal diet and host or microbial metabolism. These findings show that selected lipid-rich waste streams can support efficient BSFL bioconversion while restructuring resident gut microbiome members that may tolerate, metabolize, or indirectly respond to oil-associated conditions, contributing to substrate-dependent changes in larval lipid accumulation and fatty-acid composition. IMPORTANCEAgricultural and food-processing systems generate large amounts of lipid-rich by-products that are difficult to manage using conventional waste-valorization approaches. Black soldier fly larvae (BSFL) offer a biological route for recovering nutrients from these materials, but efficient conversion depends on interactions among substrate chemistry, larval physiology, and the gut microbiome. This study shows that selected waste oil streams can support larval growth while restructuring resident gut microbial communities and altering larval fatty-acid composition. These findings are important for agricultural biotechnology because they frame BSFL production as a host-microbiome bioconversion system rather than simply an insect-based waste-reduction process. Understanding how gut microbes respond to chemically distinct lipid wastes can guide substrate selection, pretreatment, and microbiome-informed optimization strategies for converting underutilized agricultural and food-processing residues into value-added bioproducts for circular agricultural systems.