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.

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 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.

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.

Atypical Salmonella enterica strains that evade conventional detection pose significant challenges to food safety surveillance. A hydrogen sulfide (H2S)-negative and serologically untypable S. enterica strain (SF1060) was detected by qPCR from cooked farmed mussels in Galicia, Spain, and characterized using phenotypic and genomic approaches. Despite typical biochemical profiles, SF1060 failed to produce black colonies on XLD agar and lacked detectable somatic antigens by conventional serotyping. Hybrid genome assembly using Nanopore and Illumina sequencing yielded a closed chromosome and five plasmids. In silico analyses identified the strain as S. Senftenberg ST14. Comparative genomics revealed a chromosomal inversion at the rfb operon (encoding enzymes needed to synthesize deoxysugars and O antigens) mediated by IS5-family transposase ISEc68, which truncated the rfbD gene and separated the reminding rfb genes at rfbD, disrupting O-antigen biosynthesis, explaining the serotyping failure. The phs operon responsible for H2S production lacked premature stop codons, suggesting the H2S-negative phenotype results from an alternative mechanism. This study demonstrates how whole-genome sequencing resolves identification of atypical strains that fail culture-based detection and emphasizes the critical need for molecular surveillance methods in seafood safety programs, particularly in regions where atypical S. enterica variants may be endemic. Importance StatementPathogen surveillance is in a constant race against microbial evolution. The phenotypic methods used to detect and isolate foodborne bacteria like Salmonella enterica from foods are effective, but only if the pathogens retain their expected characteristics. This study provides a clear genetic snapshot of how Salmonella enterica can adapt to evade detection. This study characterizes a strain from a marine environment that had undergone genetic rearrangement, leading to the concurrent loss of a key biochemical marker and the somatic antigens essential for serological typing. Crucially, only genomic analysis could provide a definitive explanation for this serotyping failure; the underlying mechanism would have remained unknown using conventional methods alone. This work demonstrates that relying on a fixed set of phenotypic traits is an increasingly fragile strategy and highlights why genomic surveillance must become a routine component of food safety programs to keep pace with pathogen evolution.

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.

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.

Foodborne non-typhoidal Salmonella remains a major public health concern, yet routine surveillance recovers large numbers of isolates from food that are not associated with human illness. Studies have shown foodborne isolates can be genetically linked to clinical cases, highlighting a critical challenge for risk assessment and outbreak prioritisation. This study aimed to determine whether genomic markers can distinguish foodborne Salmonella strains with an increased likelihood of causing infection. Whole-genome sequencing data from over 900 Salmonella isolates recovered from food and the environment through UK Health Security Agency surveillance were analysed using hierarchical clustering to define genetically related groups. These clusters were expanded using the global EnteroBase database to provide broader epidemiological context. Genome-wide association analyses identified genetic markers associated with clusters containing clinical isolates, including phage-associated regions. A highly conserved 7 kb marker identified in S. Agona demonstrated strong predictive performance at a global scale, with high sensitivity and specificity for infection-associated lineages and strict serovar restriction. Comparative genomic analysis revealed that all markers localised to a shared chromosomal hotspot corresponding to a prophage integration site. The 7 kb risk-associated marker formed part of a larger prophage closely related to the well-characterised S. Typhimurium Fels-2 phage, which encodes a DNA invertase linked to phase variation, a mechanism known to promote phenotypic heterogeneity and host adaptation. As these S. Agona isolates are monophasic, our findings indicate that our genome-wide association approach has rediscovered this DNA invertase known to contribute to infection risk but in a different serovar via an alternative regulatory mechanism. Overall, this work demonstrates the potential to move beyond treating all foodborne Salmonella isolates as equivalent hazards, towards a genomics-informed framework for risk stratification. This approach provides a foundation for improved risk-based decision-making, enhance outbreak investigations and enable earlier prioritisation of public health responses during Salmonella surveillance and control. Author summaryFoodborne Salmonella infections remain a major public health concern, but not all strains pose the same risk to human health. Here we investigated whether genetic differences could explain why some foodborne strains are more likely to cause human infection. We analysed over 900 genomes from food and environmental sources, grouping closely related strains before placing them in a global context using EnteroBase. By combining pangenome and genome-wide association analyses, we identified distinct lineages within several serovars that differed in their association with human cases. In Salmonella Agona, all clinical isolates belonged to a single lineage carrying a highly conserved 7 kb marker that was absent from low-risk strains. This marker demonstrated strong sensitivity and specificity across global datasets and was located within a prophage closely related to the well-characterised Fels-2 phage. This region encodes a DNA invertase previously linked to phase variation, a mechanism that promotes bacterial adaptability. Our findings indicate that infection risk can be structured at the lineage level and influenced by mobile genomic elements, particularly prophages, that enhance environmental persistence and host adaptation. This work advances genomic surveillance from retrospective linkage towards mechanistic and predictive risk assessment, with direct relevance for supporting risk-based decision-making during outbreak investigations.

BackgroundThe virulence-persistence trade-off is considered a fundamental organizing principle of Listeria monocytogenes population biology: hypervirulent clonal complexes dominate clinical cases but are rarely found in processing environments, while hypovirulent lineages dominate industrial niches but are underrepresented in severe disease. However, whether this dichotomy operates as an absolute paradigm has not been quantitatively evaluated at the population scale. Here we develop a multi-dimensional genomic scoring approach that simultaneously quantifies virulence potential (V), environmental persistence capacity (P), clonal epidemiological context (C), and antimicrobial resistance (R) across 903 genomes from four independent datasets spanning five countries, and apply it to test the universality of the trade-off and to characterize the ecological strategies of L. monocytogenes at the population level. MethodsThe scoring approach integrates four components into a composite 0-100 score through empirically calibrated weights (V: 30%, P: 40%, C: 20%, R: 10%). Validation employed 903 L. monocytogenes genomes from four public BioProjects: longitudinal industrial surveillance in Norway (Fagerlund et al. 2022, n = 513, PRJNA689484), retail environments in the United States (Stasiewicz et al. 2015, n = 191, PRJNA245909), clinical-environmental context in China (Wang et al. 2021, n = 151, PRJNA759341), and meat processing in Poland (Kurpas et al. 2020, n = 48, PRJNA629756). ResultsThe composite score achieved excellent discriminatory performance for identifying persistent clones (AUC = 0.933; 95% CI: 0.910-0.954) with perfect specificity (1.000; zero false positives). The inverse V-P correlation was statistically significant across all four datasets (Spearman {rho} from -0.144 to -0.713; p < 0.01), providing the first cross-dataset quantitative confirmation of the trade-off. However, simultaneous evaluation of V-P profiles at the population scale revealed that the species does not conform to a binary dichotomy but rather exhibits three quantitatively distinguishable ecological strategies, for which we propose a functional trophic taxonomy: nosotrophic lineages (22.7%; V > 65, P < 35), specialized in the pathogenic niche; saprotrophic lineages (5.8%; V < 30, P > 45), with irreversible virulence attenuation and industrial specialization; and, as the central finding, amphitrophic lineages (39.1%; V [&ge;] 35, P [&ge;] 40), which simultaneously retain functional inlA and stress tolerance determinants (SSI-1) without detectable genomic sacrifice. The three strategies differed significantly (Kruskal-Wallis H = 138.7; p = 7.6 x 10-3{superscript 1}). The correspondence between trophic strategy and CC was predominant but not absolute, demonstrating that this phenotypic classification captures intra-CC functional heterogeneity inaccessible through conventional typing. Furthermore, comparison between genome-based and surveillance-informed classifications revealed that 60 hypervirulent isolates (CC1/CC14), genetically classified as nosotrophic, persisted for up to 8 years in industrial facilities despite lacking any recognized persistence markers -- indicating that their prolonged survival reflects environmental opportunity rather than intrinsic genomic adaptation. ConclusionsMulti-dimensional genomic profiling reveals that the virulence-persistence trade-off, while statistically robust, does not operate as an absolute paradigm. The amphitrophic strategy -- documented here for the first time as a quantitatively distinguishable category encompassing 39.1% of the analyzed population -- challenges the prevailing dichotomous model and identifies a previously unrecognized combined ecological niche. The ability to discriminate between genome-encoded persistence capacity and environmentally facilitated persistence provides a biological framework for understanding the ecological determinants of L. monocytogenes population dynamics in anthropogenic environments.

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.

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.

Microbial communities play a central role in compost-bedded pack (CBP) systems by driving organic matter decomposition and nutrient cycling. The objective of this study was to characterize and compare the bacterial community structure of CBP from two dairy farms in Cordoba, Argentina, using 16S rRNA gene sequencing. Two CBP systems were evaluated: Martin Bono (MB; 30 months in operation) and Angela Teresa (AT; 20 months). The MB system was established on natural soil without bedding addition and included concrete feed alleys, whereas AT was initiated with peanut shell bedding and lacked concrete alleys. In both systems, compost was tilled twice daily. Two samples per farm were collected at a depth of 30 cm during winter 2019. Raw Illumina reads were processed using the DADA2 pipeline, including quality filtering, error modeling, denoising, and chimera removal. A total of four samples yielded 2,503 amplicon sequence variants (ASVs), with approximately 76% of reads retained after filtering and chimera removal, indicating high-quality sequencing data. Taxonomic analysis revealed that bacterial communities in both systems were dominated by phyla typically associated with compost environments, including Actinobacteriota, Proteobacteria, and Firmicutes. Differences in relative abundance between systems suggested shifts in community composition associated with management conditions.

Salmonella spp. represents a leading cause of foodborne disease globally. Wild aquatic birds inhabiting ecosystems impacted by human activities may serve as reservoirs and dispersers of Salmonella and antimicrobial resistance genes (ARGs), posing significant public health risks. This study evaluated the prevalence, serovars, resistance genes, and genomic relationships of Salmonella in fecal samples from wild aquatic birds across three high-Andean lakes in Ecuador. Of 134 samples collected from 10 species, five (3.73%) tested positive, all from Yahuarcocha Lake, isolated from Fulica ardesiaca and Phalacrocorax brasilianus. Two serovars were identified: Salmonella Infantis (ST32, n=4) and Salmonella Newport (ST45, n=1). Three S. Infantis isolates exhibited multidrug resistance (MDR), mediated by a pESI-like plasmid carrying resistance genes against beta-lactams, aminoglycosides, tetracyclines, sulfonamides, trimethoprim, fosfomycin, and chloramphenicol. SNP-based phylogenetic analysis revealed low genetic divergence ([&le;]10 SNPs) between wildlife and poultry-associated isolates, indicating a shared transmission network. These findings support a likely spillover from poultry production systems into wild bird populations, and highlight the role of wild aquatic birds as ecological sentinels and potential disseminators of MDR Salmonella across interconnected human, animal, and environmental systems. These results underscore the need to incorporate human, animal, and environmental health factors within a One Health framework.

Foodborne illness is a significant public health concern worldwide. Shiga toxin-producing Escherichia coli and Campylobacter species are recognized as important zoonotic bacterial pathogens contributing to human infections through the food chain, particularly via foods of animal origin. Although goat meat is in high demand in the southern region of Thailand, studies on foodborne pathogens in this livestock species remain limited. The current study aimed to (i) determine the antimicrobial susceptibility of Campylobacter spp. and STEC isolated from goats, and (ii) analyze the genetic relationships among Campylobacter spp. And E. coli O157 isolates obtained from different sources. Campylobacter jejuni and C. coli isolates were characterized based on sequences of seven housekeeping genes using the Achtman multilocus sequence typing scheme. For E. coli O157:H7, core genome multilocus sequence typing analysis was performed using whole-genome sequencing data. Genetic diversity was observed among C. jejuni, whereas a clonal population structure was detected in C. coli and E. coli O157:H7. Overlapping genetic characteristics were observed between C. jejuni isolates from goats and those previously reported in livestock and humans in Thailand. Among Campylobacter species, resistance to fluoroquinolones, including ciprofloxacin and nalidixic acid, was observed, whereas resistance to fosfomycin was most frequently detected in Shiga toxin-producing E. coli. Tetracycline-resistant isolates were identified in both Campylobacter species and Shiga toxin-producing E. coli at moderate levels. A multidrug-resistant pattern was observed only in C. coli, whereas no multidrug-resistant C. jejuni or Shiga toxin-producing E. coli isolates were detected. These findings indicate that healthy goats may serve as potential reservoirs of zoonotic pathogens and antimicrobial resistance in southern Thailand, where goat meat is frequently consumed.

Antimicrobial resistance alone is a serious threat to global public health, but even more so when multidrug-resistant bacteria harbour heavy metal resistance genes, as these can drive co-selection of antibiotics and produce biofilms. The presence of such genes, combined with the bacterias ability to form biofilms, is strongly linked to treatment failure, persistent infections, and reduced therapeutic options. Here, we used the resazurin-crystal violet combination assay to screen a representative cohort of whole-genome sequenced Escherichia coli isolates (n=20) obtained from wastewater surveillance. The specific biofilm formation (SBF) index was used to grade the intensity of biofilm formation as strong, moderate, weak, and non-biofilm producers. Correlation analysis was used to test the association between the intensity of biofilm formation and genotypic features. The SBF index revealed that most of the wastewater E. coli isolates (n=13/20) were weak/non-biofilm producers, four isolates produced moderate biofilms, and three isolates (ST1434: A-O18ab:H55; ST401:A-H25; and ST399:A-O19:H12) produced strong biofilms. The diversity of virulence factors was similar in most of the isolates, except for the three isolates, which had fewer abundant virulence factors. The correlation analysis showed that there was no association between the expression of virulence genes and the formation of strong biofilms by the isolates (p > 0.05). Drug resistance profile was not correlated with higher biofilm production (p > 0.05), as 68.8% (n=11/16) of multi-drug resistant (MDR) and 50% (n=2/4) of non-MDR isolates had weak or no biofilm formation. Similarly, the SBF index was not associated with the number of plasmids in each of the E. coli genomes (p = 0.334). However, there was a positive association between the presence of two or more heavy metal resistance genes (HMRGs) and the strong biofilm formation in our isolates (p = 0.002). Our findings revealed the low occurrence of strong biofilm producers among wastewater E. coli isolates. Further studies are needed to evaluate the impact of the presence of HMRGs and their direct or indirect contribution to enhancing biofilm production and persistence in environmental reservoirs.

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.

IntroductionThe use of Saccharomyces cerevisiae to ferment alcoholic beverages is an ancient tradition, with genetic evidence indicating origins in Neolithic Asia, although the domestication process of the species is not fully understood. Kveik is a group of traditional yeasts used in farmhouse brewing in western Norway preserved through generations of rural brewing practice. While recent studies have highlighted the distinctiveness of kveik, its precise phylogenetic position, genetic diversity, and domestication history remain unclear. ResultsWe performed whole-genome sequencing on 62 samples representing 25 unique Norwegian strains selected using cultural heritage criteria, and generated telomere-to-telomere (T2T) assemblies for representative isolates. Phylogenomic and population genetic analyses reveal that kveik forms a paraphyletic and early diverging group with respect to other domesticated S. cerevisiae strains. Most strains exhibit low within-strain diversity, strong geographic clustering, and little evidence of gene-flow or admixture. Mitochondrial genomes and Ty1 retrotransposon profiles corroborate this distinct lineage history. We further show that previously reported signals of gene flow between kveik and Asian fermentation strains are likely artifacts caused by population structure and selection. Divergence time estimates suggest that the common ancestor of beer, kveik, and other liquid-phase fermenting strains originated from ancestral populations 4,000 to 8,000 years ago. ConclusionKveik yeasts represent a relic of early S. cerevisiae domestication, shaped by ancient human practices, migrations, and the spread of agriculture. Our genomic resource sheds light on yeast evolution and domestication. They likely comprise some of the oldest domesticated lineages in continuous use until today, connecting endangered intangible cultural heritage to an early genetic origin.

Water-miscible metalworking fluids are widely used in industrial processes. Despite the fact that they contain biocides, they are almost always colonized by microorganisms, which degrade different components of the liquid, may clog machines due to biofilm formation and might pose a health risk to workers. In this study, samples from four metalworking machines operated with the same metalworking concentrate from two different locations, were analyzed with respect to microbial growth. Twenty-seven bacterial species and one fungus were identified. From these, twenty species were not observed before as colonizers of metalworking fluids. Growth of microorganisms, resulting health risks, putative contamination pathways and metabolic pathways involved in biodegradation are analyzed and discussed in this study. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/712622v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@16164e6org.highwire.dtl.DTLVardef@1273ee6org.highwire.dtl.DTLVardef@192aa20org.highwire.dtl.DTLVardef@1df4df2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Agricultural fields in the Mezquital Valley, Mexico, were irrigated with untreated wastewater over several decades. Following the construction of a wastewater treatment plant (WWTP) in Atotonilco de Tula, WWTP effluent is used for irrigation. To evaluate the effects of changed irrigation, a soil incubation experiment was performed. Soils of the Mezquital Valley long-term irrigated with untreated wastewater were irrigated with WWTP influent or effluent, both unspiked and spiked with antibiotics and biocidal compounds and incubated four weeks. We investigated the effects of shifted irrigation on the abundance of cultivable total heterotrophic and resistant bacteria (RB). Additionally, RB were cultivated from Coriandrum sativum (cilantro) sown in soil of the incubation experiment. While wastewater treatment significantly reduced the bacterial abundance in effluent, spiking increased RB abundance in both wastewater types including ciprofloxacin (CIP) RB. Before wastewater addition, all soils contained cultivable RB. Irrigation increased the relative abundance of RB cultivated on Mueller Hinton (MH) agar in Leptosols and Phaeozems, compared to soils prior to wastewater addition irrespective of the water type, but not in Vertisols, suggesting the soil type rather than water qualities influenced the RB abundance. Diverse CIP RB were cultivated including strains of 14 genera of three phyla. Among those, Achromobacter spp. strains related to potentially pathogenic A. spanius originating from soil were abundant in both leaves and roots of cilantro. Our results showed that the implementation of wastewater treatment does not reduce the abundance of cultivable RB in Mezquital Valley soils and cilantro plants. Health risk associated monitoring should include long-term persistent RB colonizing plants cultivated in wastewater irrigated soils.

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