International Journal of Food Microbiology

Consumption of sprouted seeds, such as alfalfa sprouts, has increased in recent years due to their perceived health benefits. However, these food products have repeatedly been associated with outbreaks of foodborne pathogens, including Salmonella enterica serovars. An S. enterica serovar Choleraesuis strain previously isolated from melon fruit internal tissues was selected as a model to explore plant-pathogen interactions on alfalfa sprouts. Using this strain, we generated a barcoded transposon mutant library comprising approximately 33,000 unique insertions. This library and a collection of individual insertion mutants derived from it were used to identify genetic mechanisms contributing to the fitness of this S. Choleraesuis strain on alfalfa sprouts. The library was screened on sprouts during cold storage at 8{degrees}C. Negative selection for mutants with insertions in eda, fabF, lpp1_2, pnp, stpA, SCHChr_03621 and two intergenic regions were identified. Competition experiments between individual insertion mutants and the wild type confirmed the phenotype of three genes: eda, coding for a keto-hydroxyglutarate-aldolase/keto-deoxy-phosphogluconate aldolase involved in the Entner-Doudoroff pathway, mnmG, encoding the glucose-inhibited division protein, and fabF, involved in fatty acid biogenesis. This study offers a genome-wide perspective on the genes enabling a plant-associated Salmonella strain to persist on alfalfa sprouts. We highlight factors that are critical not only for persistence throughout the entire cold-storage period under conditions that closely simulate real shelf-life conditions in this high-risk food matrix.

Bacteriophages are promising alternatives for antibiotics. One challenge of developing bacteriophage-based preparation for practical application is to understand expected host range of such products. We evaluated the host range and lytic activity of BAFASAL(R), a four-phage cocktail targeting Salmonella in poultry production. For that purpose, we developed a new composite metric, the Combined Lytic Score (CLS), integrating results from two in vitro assays: serial dilutions spot test on semisolid medium and spectrophotometric growth inhibition in liquid culture. Using this approach phage cocktail was tested against collection of 72 Salmonella strains, including 55 S. Enteritidis isolates representing diverse geographic origins and genomic backgrounds. Spot test patterns were transformed into a continuous scale using the Most Probable Number (MPN) approach to estimate the number of phages required for visible lysis. In parallel, growth inhibition was quantified as the area-under-curve-based inhibition score (ANS). Both metrics were normalized and combined into CLS as a projection onto the regression line describing their correlation (R{superscript 2} {approx} 0.82). More than 65% of S. Enteritidis strains, reached normalized CLS values higher 75%, indicating high susceptibility to BAFASAL(R) in vitro. Phage susceptibility did not correlate with either phenotypic antibiotic resistance or the number of resistance and virulence genes. CLS provides a quantitative method to integrate different experimental methods of determination of bacterial susceptibility to bacteriophages and to rank bacterial strains by phage susceptibility. This approach supports robust host range determination and may facilitate regulatory evaluation and rational design of phage-based interventions in food safety and animal production. IMPORTANCEAssessment of bacteriophage host range is an important step in characterization of bacteriophage strains both in basic and translational research, yet it is still commonly based on qualitative or poorly standardized assays. This lack of harmonization limits reproducibility and complicates comparisons across studies, laboratories, and application contexts. In this work, we propose the Combined Lytic Score (CLS) as a quantitative framework that integrates outcomes from two widely used experimental approaches: serial-dilution spot assays and microtiter-based growth inhibition kinetics. By converting spot-test results into a continuous, concentration-dependent metric and combining them with normalized kinetic inhibition data, CLS enables more consistent interpretation of phage-host interaction outcomes. Application of CLS to a diverse collection of Salmonella enterica strains demonstrates how this approach can support systematic, scalable host range analyses. The CLS framework provides a practical step toward improved standardization of phage susceptibility testing, facilitating clearer data interpretation and comparison in both environmental and applied microbiology research.

There is a growing concern that chronic, low-level exposure to organophosphate insecticides is a threat to human health. These synthetic chemicals are used in crop and livestock production all over the world, and the general population are exposed to them through consuming the residues that remain in food. Evidence is emerging that fermentation with lactic acid bacteria may be an effective way of reducing organophosphate insecticide residues. However, while several studies have investigated this topic, outcome measures have varied, and there has been no research to date which has consolidated this data to better understand the half-lives of organophosphate insecticides in fermented foods and the factors affecting degradation. The aim of this review was to synthesise the evidence on organophosphate insecticide degradation during lactic acid fermentation, and analyse organophosphate insecticide half-lives, in order to determine the effectiveness of lactic acid fermentation in reducing organophosphate insecticide residues in food. Furthermore, the study aimed to explore the factors that impact the rate of degradation. CAB Abstracts, Food Science and Technology Abstracts, Scopus and Web of Science were searched for eligible laboratory-based studies, which were published after 2000. The literature search and screening process resulted in the inclusion of 14 eligible studies. Studies were screened for Risk of Bias (ROB) using the RoBDMAT tool. Collated results showed that organophosphate insecticides degraded over time, and this was irrespective of fermentation. However, out of the 249 experiments that involved a controlled fermentation, 232 demonstrated that fermentation with lactic acid bacteria could speed up the degradation of organophosphate insecticides in food, beyond the rate of inherent degradation in the food matrix, leading to shorter half-lives. The half-lives of organophosphate insecticides in apple juice, milk and wheat ranged from 9.5 hours to 21 days in fermented foods and ranged from 21.4 hours to 36.5 days in non-fermented foods. Single species of lactic acid bacteria that demonstrated strong potential for organophosphate insecticide degradation were Lpb.plantarum subsp.plantarum, Lab.delbrueckii subsp.bulgaricus and Lvb. brevis, where the median percentage change in organophosphate insecticide half-life during fermentation was -42.3%, -25.0% and -22.9%, respectively. Organophosphate insecticide degradation during natural fermentation was less clear because of fewer studies and less consistent results. Whilst the collated data shows that fermentation with lactic acid bacteria is an effective method to reduce organophosphate insecticide residues in food, reflected in shorter half-lives, the small number of studies and variability among studies does limit the conclusions that can be drawn, and further research is needed to strengthen these findings. The results of our analysis may help to inform more reliable organophosphate exposure assessments for the population as well as provide novel insights for both consumers and food manufacturers, expanding the market potential for fermented foods.

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.

Quorum sensing (QS) is a bacterial cell-cell communication system that coordinates group behaviors via self-produced signaling molecules. Serratia liquefaciens, a common mutton spoilage bacterium, precisely regulates its spoilage capacity through the AHL-mediated QS system. The results demonstrated that 20 mol/mL C6-HSL concentration-dependently enhanced AHL activity 1.26-fold, increased biofilm formation by 51.55%, and elevated protease activity and siderophore production by 37% and 26.78%, respectively. In contrast, 80 g/mL chlorogenic acid significantly inhibited AHL activity (by 26.19%), biofilm formation (by 42.54%), protease activity (by 28.92%), and motility (by 38.34%). In stored mutton, chlorogenic acid treatment reduced total plate counts by 6.1% and pH by 5.26%. Transcriptomic analysis revealed that C6-HSL treatment altered metabolic pathways such as flagellar assembly, ABC transporters, two-component systems, and secondary metabolite synthesis, in which spoilage-related genes (slyB, fimA, fliJ, iucD, cheW) were significantly up-regulated. In contrast, chlorogenic acid treatment affected pathways including amino acid metabolism, sulfur metabolism, and carbohydrate metabolism, with spoilage-related genes (fimA, tuf, ibpA, clpS, metQ) significantly down-regulated. These findings demonstrate that AHL activity plays a key role in regulating the spoilage capacity of Serratia liquefaciens, and suggest chlorogenic acid as a potential QS inhibitor with promising application in mutton preservation. ImportanceSerratia liquefaciensis a common spoilage bacterium in refrigerated foods and proliferates extensively in meat, making it one of the primary spoilage organisms. The significance of our research lies in investigating how changes in the activity of the signaling molecule AHL affect both spoilage capacity and mutton quality. Additionally, transcriptomic analysis was employed to elucidate the regulatory mechanisms by which altered AHL activity influences the spoilage potential of S. liquefaciens. The results demonstrated that variations in AHL activity significantly impacted key spoilage-related traits of S. liquefaciens, including biofilm formation, protease activity, and motility, while also contributing to improved meat quality during storage. Furthermore, the study revealed that AHL activity regulates metabolic pathways associated with spoilage as well as the expression of spoilage-related genes. These findings provide a theoretical basis for developing preservation strategies for mutton.

We present a comparative analysis of 13 yeasts available for mead (honey wine) fermentation, a source of Saccharomyces cerevisiae diversity that has not yet been analyzed in detail. Using genomic, phenotyping, and analytic methods, we show that currently available mead yeasts belong to various clades of the species, most commonly to the Commercial Wine clade (5 of 13 samples). Mead yeasts in this group displayed genome structure variations and occasional loss of killer activity, despite being closely related. Historic European and traditional African mead isolates with sequenced genomes were found not to be closely related to any contemporary mead yeast product. The 13 yeasts tested here displayed high variability in oenological characteristics and in aroma production. Maximum ethanol tolerance ranged from 15 to 22% v/v, however, the most tolerant strain produced lower ethanol levels and retained high fructose content in experimental meads. The most abundant aroma components produced in meads were ethyl acetate, ethyl caprylate, isoamyl alcohol, and ethyl caprate, with similar aroma profiles in members of the Commercial Wine clade, and pronounced differences among other yeasts. Our results contribute to the knowledge of Saccharomyces yeasts in various fermentation environments, adding mead to the list of alcoholic beverages with a known diversity of starter cultures. Our results may aid strain selection for honey wine fermentations and inspire strain improvement.

The spread of extended-spectrum {beta}-lactamase (ESBL)-producing and fluoroquinolone-resistant Salmonella pose a global public health challenge in addition to the high burden of infections associated with this foodborne pathogen. In this study we aimed to characterize a multidrug-resistant strain of Salmonella serovar Amager isolated from a Chilean river in October 2023. Antimicrobial susceptibility testing revealed a resistance phenotype against multiple antibiotic families, including fluoroquinolones and {beta}-lactams, showing ESBL production. Hybrid genome sequencing allowed the identification of a 311,303 bp plasmid carrying the aadA1, aph(4)-Ia, aac(3)-IVa, floR, sul1, tet(A), and blaCTX-M-65 genes, sharing 99.98% sequence identity with the Salmonella Infantis pESI-like megaplasmid. In addition, the qnrB19 gene was found in a {approx}2.7 kbp plasmid of widespread distribution. Population structure and temporal phylogenetic analysis at the global scale revealed the emergence of a Salmonella Amager lineage from the HC20_35565 cluster, carrying the Salmonella Infantis blaCTX-M-65-positive pESI-like megaplasmid and causing human infections in the United States and the United Kingdom. Our work describes the emergence of a Salmonella lineage with resistance against first-line antibiotics used for treating severe infections, underscoring the relevance of environmental surveillance as a means for detecting emergent pathogens and anticipating human infections.

The dynamics and impact of microbial contaminants in industrial sugarcane bioethanol production in Brazil were investigated through a two-year metagenomic study across two biorefineries. Shotgun metagenomic sequencing revealed that temporal shifts in the contaminant microbiome dynamics within production seasons were more pronounced than inter-annual or inter-mill variations. While Saccharomyces spp. dominated, bacterial communities, primarily within the Firmicutes phylum and dominated by the genera Lactobacillus, Limosilactobacillus, and Bacillus, exhibited dynamic changes. Correlation analyses with industrial process parameters revealed a complex interplay: lower Lactobacillus levels in one mill were associated with increased ethanol yield, whereas higher levels in another mill correlated with reduced yeast viability and increased flocculation. The presence of Limosilactobacillus was linked to decreased yeast viability, whereas Bacillus showed potential for inhibiting both Lactobacillus and Limosilactobacillus. These findings highlight the nuanced and species-specific impacts of bacterial contaminants on bioethanol production, underscoring the need for strain-level functional studies and targeted interventions to optimize fermentation efficiency and stability in industrial settings.

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.

Postharvest losses and rapid nutrient degradation due to fruit spoilage necessitate alternative preservation methods. Wine production presents a viable approach to minimizing fruit waste while retaining essential nutrients. In this study, mixed fruit wines (watermelon, banana, and pineapple) were produced using Saccharomyces cerevisiae isolated from palm wine as a starter culture. After secondary fermentation, the wines maintained an acidic pH range (2.29{+/-}0.1 to 3.25{+/-}0.2), a stable fermentation temperature (26.50{+/-}1.1{degrees}C to 27.00{+/-}1.1{degrees}C), specific gravity values of 1.021{+/-}0.02 kg/L and 1.027{+/-}0.03 kg/L, and total acidity levels of 1.57{+/-}0.2% and 2.11{+/-}0.1% for Wines A and B, respectively. The final alcohol content was 8.40{+/-}2.9% in Wine A and 9.84{+/-}3.6% in Wine B. Proximate analysis demonstrated the retention of key nutrients post-clarification and maturation, and sensory evaluation indicated a higher consumer preference for Wine B (P>0.05). These findings highlight the potential of indigenous S. cerevisiae strains from palm win for efficient wine fermentation and support the utilization of mixed fruits as a sustainable raw material for value-added wine production. This approach not only mitigates fruit wastage but also provides an economic avenue for enhancing fruit utilization.

Microplastics (MPs), resulting from plastic objects and waste degradation, are increasingly abundant, particularly in marine environments. They exhibit a hydrophobic surface on which biofilms form easily. Metagenomic analyses of these biofilms have revealed that they often contain bacterial species potentially pathogenic to humans or animals. For this reason, MPs are suspected to present a risk for public health by acting as a vector for pathogenic bacteria species. To better understand this hazard, we studied different factors potentially affecting the bacterial attachment rate to MPs. Focusing on the fish pathogen Vibrio anguillarum, a collection of 16 strains was assembled and GFP-labelled. Their attachment rates were measured using fluorescence microscopy on three types of MPs (milled polypropylene and polyethylene terephthalate particles and commercially available polystyrene beads). A strong effect of the particle type was found, likely linked to both the chemical composition of the particles and the surface characteristics, with higher attachment rates on rough particles. Our results also revealed a strong intra-specific variation in attachment rate, highlighting the need of testing several strains of the same species to assess attachment rate and related hazards. Finally, it was observed that when a biofilm already formed on the MPs (by field-incubation of the MPs along the Mediterranean French coast), differences in attachment rates between particle types were erased. It was concluded that the attachment rate of V. anguillarum depends on a combination of biotic and abiotic factors, which makes risk assessment of MPs as vectors of pathogenic bacteria species a very complex task.

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.

The gut microbiota plays an essential role in the conversion of anthocyanins and (epi-)catechins into smaller phenolic acids, which are then absorbed into the blood stream. The phenolic composition of a commercial bilberry extract and grape seed extract was assessed, as well as a formulation extract containing a combination of both extracts. The extracts were subjected to an in vitro salivary, gastric and intestinal digestion environment, based on the INFOGEST Model. The solid fraction end-product of the combined extract from the in vitro digestion was further fermented with faecal samples from six healthy donors, for 72 hours, to assess phenolic acid metabolism, short-chain fatty acid formation and changes in microbial composition. During the in vitro digestion, flavonoid content in all three extract samples (bilberry, grape seed and the formulation extracts) decreased significantly. In the process of anthocyanin and flavonoid digestion, smaller phenolic acid compounds such as benzoic acid, cinnamic acid and mandelic acid increased in bilberry, grape seed and formulation extract samples. All faecal donors harboured unique microbiota compositions, however all faecal microbiota were able to fully convert catechin/epicatechin, the most prominent flavonoids in the formulation extract sample, into smaller phenolic metabolites (phenylacetic, phenylpropionic and benzoic acids) within 24 hours. Using 16S rRNA gene amplicon sequencing, Anaerobutyricum and Enterocloster spp. were correlated with catechin/epicatechin metabolism in the fermentation procedure, however, in single bacterial strain fermentation experiments with the formulation extract or catechin standard, these bacteria were not capable of metabolising flavonoids. HighlightsO_LIFaecal microbiota converted (epi-)catechin to phenolic metabolites within 24 h. C_LIO_LI(Epi-)catechin correlated negatively with Anaerobutyricum and Enterocloster spp. C_LIO_LIFaecal bacterial cultures did not show (epi-)catechin metabolism capacity. C_LI

ImportanceMolecular confirmation of Listeria monocytogenes typically employs a multiplex PCR method that targets both genus-specific prs and species-specific hlyA genes. This study assessed the specificity of this assay within Nigeria, where local microbial diversity may influence performance outcomes. MethodsOut of eight phenotypically presumptive L. monocytogenes food isolates tested, six produced the expected prs and hlyA amplicons, with five classified as serogroup 1/2b. However, all six isolates tested negative for the crucial virulence regulator prfA, necessitating further investigation. ResultsDefinitive 16S rRNA gene sequencing revealed that only two of the six PCR-positive isolates were identified as L. monocytogenes, while the remaining four were identified as Enterococcus faecium. This results in a false-positive rate of 66.7% (4/6) for the assay in this particular context. Phylogenetic analysis corroborated the taxonomic distinction, exhibiting a robust clustering of the four E. faecium isolates with reference strains. In contrast, the two confirmed L. monocytogenes isolates formed a separate sub-clade, indicating regional divergence and further underscoring the assays inability to differentiate between L. monocytogenes and Enterococcus species. ConclusionThese findings highlight a significant lack of specificity, as the prs/hlyA primers exhibited cross-reactivity with non-target E. faecium. The anomalous negative result for prfA served as a critical diagnostic indicator. Consequently, the positive outcomes from this widely utilized confirmatory assay should be regarded as presumptive and necessitate additional verification.

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.

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

Yeasts ability to invade surfaces has important implications for infections and food contamination. Invasive growth in yeast is influenced by genetic and environmental factors. In this exploratory study, we investigated the effects of sodium sulfide, gene deletions, and environmental conditions on the invasive behaviour of the wine yeast strain AWRI 796. Sodium sulfide enhanced invasion in the (parent) AWRI 796 strain under nitrogen-limiting conditions, although its effect was obscured by experimental variability and pre-culture conditions. Genetic factors had a major effect on the overall invasive phenotype, with deletion of key genes suppressing invasion. Most gene-deletion mutants did not significantly affect how the colony responded to sulfide. In addition to sulfide and genotype, environmental conditions also influenced invasive behaviour. The pre-2xSLAD pre-culture condition was best for detecting sulfide-induced growth, and later plate washing time and decreased nutrient levels enhanced invasiveness. Our experimental design and findings provide a framework for understanding the determinants of yeast invasiveness, which may inform future studies on filamentous yeast behaviour.

Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) and other microorganisms have attracted considerable interest due to their structural diversity and physicochemical properties, which makes them valuable across various industrial applications. To achieve high cell densities and maximize EPS yields, microorganisms are typically cultivated in nutrient-rich media containing yeast extract. However, yeast extract may contain high molecular weight polysaccharides that are not metabolized by the bacteria. This can lead to an overestimation of EPS yields and contamination of the bacterial EPS, potentially resulting in misinterpretation of their structure and biological activity. In this study, we demonstrate the presence of high molecular weight -mannan and {beta}-glucan in yeast extract in EPS isolates using both ultrafiltration and the commonly used trichloroacetic acid/ethanol (TCA/EtOH) precipitation method. These polysaccharides were characterized by size-exclusion chromatography, high-performance anion-exchange chromatography, and nuclear magnetic resonance spectroscopy. Their abundances were estimated to range from 10 to 50 mg/L in MRS medium, depending on the supplier of the yeast extract. The main contaminant identified was yeast -mannan. By cultivating L. rhamnosus GG (ATCC 53103) and L. pentosus KW1 and isolating their respective EPS, we illustrate how these yeast extract contaminants affect the structural interpretation of the EPS and that the contaminants can be completely removed by ultrafiltration of the growth medium prior to bacterial cultivation. In conclusion, we emphasize the necessity of stringent controls in the production and purification of microbial EPS, with particular attention to the chemical purity of medium constituents.