Applied and Environmental Microbiology

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.

Fresh produce encounters pathogens at various stages of production and supply, with the harvesting process serving as one of these stages. To evaluate contamination associated with harvesting, we systematically swabbed zone 1 harvester surfaces and quantified Bacteroidales as a fecal biomarker using quantitative polymerase chain reaction (qPCR). Baseline contamination was dominated by non-detects, with occasional low-level detections (<25 copies/cm2) near the assay limit of detection (LoD). Detection occurred more frequently post-harvest (overall [~]4% pre-harvest and 10% post-harvest), while microbial loads remained low, indicating that harvesting primarily affected the likelihood of low-level contamination rather than increasing contamination abundance. Additionally, we developed and field-deployed a portable loop- mediated isothermal amplification (LAMP) assay for rapid harvester hygiene assessment and benchmarked its field performance against qPCR. Together, these results support a practical molecular tool for monitoring fecal contamination and informing cleaning and sanitization decisions.

Short-read amplicon sequencing is widely used for fungal surveys but can limit taxonomic resolution. Long-read sequencing enables recovery of the full internal transcribed spacer (ITS) region and may improve ecological and taxonomic inference. Here, we conducted a paired comparison of Illumina ITS2 and PacBio HiFi full-length ITS sequencing using identical DNA extracts from built-environmental air and surface samples (n = 68) collected across homes, a dormitory, and laboratories. Both datasets were taxonomically assigned using the same algorithm and reference database. We performed paired statistics, in-silico ITS2 trimming of long-read sequences, and cross-platform mapping at multiple identity thresholds. Full-length ITS provided higher taxonomic resolution, assigning a greater fraction of ASVs at the family (98% vs. 88%) and species (42% vs. 32%) ranks than ITS2 (paired Wilcoxon q = 0.002). Alpha-diversity comparisons showed similar Shannon diversity across pipelines, whereas richness metrics were consistently higher for full-length ITS. Beta-diversity analyses indicated broadly comparable community-level patterns, although full-length ITS revealed stronger sample-type- and location-associated structure (PERMANOVA R{superscript 2} [&ge;] 0.06, p = 0.0001). In-silico ITS2 trimming reduced these differences, indicating that amplicon length is a major contributor to enhanced taxonomic resolution and ecological inference. Cross-platform mapping further showed extensive one-to-many relationships between ITS2 and full-length ITS ASVs, consistent with increased sequence resolution in long-read data. Together, these results show that ITS2 sequencing provides robust community-level profiling, while full-length ITS enables improved richness estimates and finer ecological and taxonomic resolution. This paired, bias-aware framework provides a practical template for selecting fungal amplicon sequencing strategies in built-environment mycobiome studies. ImportanceFungal communities in built environments influence indoor air quality and human exposure, yet their characterization depends strongly on sequencing strategy. This study provides a controlled, paired comparison of short-read ITS2 and long-read full-length ITS sequencing, showing that differences in amplicon length substantially contribute to variation in taxonomic resolution and ecological inference. While both approaches yield comparable community-level patterns, full-length ITS improves richness estimates, species-level assignment, and environmental discrimination by resolving sequence variation collapsed in ITS2 surveys. By integrating paired diversity analyses, in-silico ITS2 trimming, and cross-platform ASV mapping, this work offers a bias-aware framework for evaluating fungal amplicon pipelines. Importantly, improved species-level resolution enables functional interpretation of indoor fungi, for example the identification of taxa associated with pathogenic traits, allergen production, or toxin synthesis, supporting the development of more informative exposure metrics and targeted assays relevant to human health in built environments.

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

Bacillus thuringiensis INTA Mo4-4 was characterized phenotypically, genomically, and for insecticidal activity against Alphitobius diaperinus. Microscopy revealed rare flat rectangular parasporal crystals, and SDS-PAGE identified a ca. 67 kDa protein, similar to B. thuringiensis serovar morrisoni strain tenebrionis DSM-2803, which was proteolytically processed to a ca. 55 kDa fragment. Genomic analysis showed a 5.99 Mb genome with 99.43% completeness, clustering phylogenetically with B. cereus and B. thuringiensis. High genomic similarity was observed with B. thuringiensis svar. morrisoni BGSC 4AA1, confirmed by MLST analysis assigning it to ST-23. The genome encodes an interesting arsenal of pesticidal proteins showing significant similarity to Cry3Aa, Mpp23Aa, Xpp37Aa, Mpp5Ab, Vpb1Ad, Vpb1Ae, Vpa2Ab, Vpa2Ba, Vpa2Bb and Spp1Aa, with demonstrated toxicity against coleopteran pests. Biosynthetic gene clusters for toyoncin, fengycin, and bacillibactin were identified. Dose-response bioassays showed that INTA Mo4-4 was nearly four times more toxic to A. diaperinus larvae (LC50 136.9 {micro}g/ml) than DSM-2803 (LC50 540.5 {micro}g/ml), with the difference being statistically significant. No teratological effects were observed on Musca domestica. These findings suggest that INTA Mo4-4 is a promising candidate for the biological control of A. diaperinus.

Phyllosphere microbiomes are increasingly recognized as key regulators of plant health and stress responses, although they are also known to change considerably over both space and time. In the phyllosphere, members of the genus Methylobacterium are often abundant and ecologically important as plant growth promoting bacteria. However, knowledge about the temporal abundances and community dynamics of Methylobacterium in agricultural systems remains limited. To address this gap, we characterized seasonal shifts in Methylobacterium-specific and total phyllosphere bacterial loads and community structure on two common summer crops and one overwintering cover crop. Leaf samples of Zea mays (corn), Glycine max (soybean), and Thlaspi arvense L. (pennycress) plants were collected over one year in Minnesota, USA and analyzed with host-associated microbial PCR (hamPCR). Microbial loads and community composition varied strongly among hosts and across growing seasons. Corn supported the highest Methylobacterium and total bacterial loads, increasing towards senescence, while pennycress exhibited the lowest loads and the most distinct communities. While there were strong host-specific patterns, a group of most abundant genera were shared across all crops (Methylobacterium, Sphingomonas, Pseudomonas, and Massilia) and the most abundant Methylobacterium amplicon sequence variants were present on all three hosts. Our findings highlight how microbial loads and community composition change during phyllosphere assembly across diverse summer and overwintering crops, with a small core of versatile taxa dominating multiple agricultural hosts. Understanding these host and season-linked patterns provides a foundation of harnessing Methylobacterium strains to enhance crop productivity and resilience.

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

Naphthenic acids are amphipathic compounds whose toxicity has primarily been attributed to narcosis toxicity to cell membranes. However, few methods exist that specifically study the membrane disruption and toxicity of this complex family of cyclic, polycyclic and acyclic alkyl-substituted carboxylic acids. Here we describe a whole cell biosensor approach that relies on the ability of Pseudomonas aeruginosa, a ubiquitous environmental organism and opportunistic pathogen, to sense membrane damage (narcosis) and induce protective genes to repair and protect the outer membrane. Many classes of membrane disrupting antimicrobials induce the expression of two operons that encode protective defense systems against outer membrane (OM) damage, including antimicrobial peptides, chelators, and detergents. We demonstrate that the pmrF and spdE2 transcriptional lux reporters are induced by exposure to individual NA compounds with diverse structures, as well as mixtures and naphthenic acid fraction compounds (NAFCs). To further support the narcosis hypothesis, we demonstrated that NA permeabilizes the outer membrane to assist in lysozyme killing, and disrupts the inner membrane integrity, allowing uptake of the DNA binding dye propidium iodide. The conventional OM permeability assay that measures NPN fluorescence is not applicable to study NAs, because they stimulate NPN fluorescence in the absence of cells. This narcosis biosensor approach constitutes a rapid and simple method to measure narcosis and could be developed as a novel toxicity indicator of oil sands tailings.

Salmonella spp. remains one of the leading foodborne pathogens worldwide, and the circulation of multidrug-resistant strains in the poultry industry poses a significant challenge. In this study, five isolates from poultry litter swabs (commercial broiler chickens) belonging to the Salmonella Heidelberg and Salmonella Minnesota serovars were characterized using an integrated approach involving phenotypic resistance profiling, whole-genome sequencing, structural prioritization of molecular targets, and in silico screening of ligands. All isolates exhibited multidrug resistance phenotypes and genetic repertoires consistent with resistance to {beta}-lactams, sulfonamides, and tetracyclines, as well as determinants linked to efflux systems, virulence, and persistence. Genomic analysis allowed for the prioritization of five proteins for structural investigation: CTX-M-2, CMY-2, Sul2, AcrB, and SpvC. Sequence-structure validation revealed high correspondence between the proteins of the isolates and the experimental structures selected for CMY-2, Sul2, AcrB, and SpvC, while CTX-M-2 was modeled with high structural confidence. Molecular docking analyses with GNINA revealed distinct behaviors among the targets. Sul2 showed biological relevance but a more conservative structural response, with no significant gain after analog generation. In contrast, AcrB stood out as the most promising target, with analogs generated by BRICS yielding better scores and, in some cases, coherent international networks identified by PLIP. The results demonstrate that the integration of phenotype, comparative genomics, and structural prioritization constitutes a rational strategy for selecting targets and molecular candidates in multidrug-resistant avian strains of S. Heidelberg and S. Minnesota.

There is disagreement on whether secondary endosymbionts are found in the major cereal pest aphid, Rhopalosiphum padi. Some papers report a diversity of secondary bacterial endosymbionts while others have failed to find evidence of these bacteria in this species. Here we revisit this issue by summarizing the relevant literature and through additional sampling of the species in Australia, China and Denmark using a combination of molecular approaches. We find a general absence of secondary endosymbionts beyond the obligate endosymbiont Hamiltonella defensa in R. padi. While the inconsistency in survey results may reflect rapid changes in endosymbiont turnover in populations and/or the impact of ecological factors such as host plant type on endosymbiont diversity, we are concerned that technical issues may be at least partly responsible for inconsistencies in the literature. This leads us to emphasize the importance of multiple sources of evidence required to establish and characterize endosymbiont infections, including PCR and qPCR assays, DNA Sanger sequencing and 16SrRNA gene metabarcoding. We note that several major aphid pests show a low incidence of secondary endosymbionts which raises issues about the importance of these endosymbionts in aphids that constitute pests, even though endosymbionts can in some cases increase host fitness and therefore pest impact.

The relationship between Campylobacter levels in broiler caeca and on carcass skin is central to quantitative microbial risk assessment along the poultry production chain, underpinning modelling of intervention impacts, including EFSA assessments of the public health impact of control measures. However, this relationship is typically inferred from monitoring data generated under sampling designs that do not preserve pairing between specimens and may involve pooling. In this study, we used a simulation framework to evaluate whether commonly used sampling strategies allow reliable recovery of the caecal-skin relationship. A simulated broiler population was generated, assigning caecal and skin loads to individual birds based on a specified linear relationship. Sampling was conducted under paired and unpaired designs, with and without pooling, reflecting approaches used in surveillance programmes and in policy-oriented models. Regression models were fitted to sampled data across 1,000 simulations for a range of assumed slopes. Under paired sampling, estimated slopes closely matched the true relationship across most scenarios. In contrast, unpaired sampling consistently failed to recover the association, with estimated slopes centred around zero regardless of the true slope. These findings were robust to variation in within-flock prevalence, residual error, and intercept. The results show that sampling design fundamentally affects identifiability of relationships between stages of the production chain. This has implications for interpretation of parameters derived from monitoring data and used in quantitative Campylobacter risk assessments informing policy. Parameters derived from unpaired and pooled monitoring data should therefore be interpreted with caution when used to support risk assessment and decision-making. Campylobacter; broiler chickens; sampling strategy; unpaired sampling; carcass contamination; quantitative microbial risk assessment; simulation.

Common bean (Phaseolus vulgaris) is an important crop in Canada and globally. Like other legumes, common bean (Phaseolus vulgaris) establishes symbiotic interactions with nitrogen fixing bacteria called rhizobia. However, nitrogen fixation by rhizobia in association with common bean is often suboptimal, constraining its productivity and necessitating the application of nitrogen fertilizer. To support the development of high-performing, locally adapted rhizobial inoculants for Ontario common bean growers, we isolated 216 common bean-nodulating rhizobia from southern Ontario soils using a nodule trapping approach with four common bean cultivars. Whole genome sequencing followed by phylogenomic analyses of the 216 rhizobial isolates revealed substantial diversity, assigning them to 11 Rhizobium species, including two novel species. Nearly all isolates belong to the symbiovar phaseoli, spanning the nodC {gamma}-a, {gamma}-b, and alleles, with four isolates belonging to the symbiovar gallica. Soil origin had a significant impact on the species-level community composition recovered during the nodule trapping experiments, indicative of biogeographical structuring of common bean-nodulating rhizobia across southern Ontario. In contrast, host trapping cultivar had only a minor influence of the recovered Rhizobium population diversity. Greenhouse assays demonstrated that one of the novel Rhizobium species exhibited the highest average symbiotic effectiveness, although high-quality isolates were found across multiple species. Together, these results revealed a diverse and genomically variable Rhizobium community capable of forming effective symbioses with common bean in southern Ontario soils. Importantly, our genome-sequenced Rhizobium collection will serve as a valuable resource for identifying competitive and high-quality strains for the development of inoculants tailored to Ontario common bean production. IMPORTANCECommon bean is a globally important food crop, yet its productivity is often limited by suboptimal nitrogen fixation, forcing growers to rely on synthetic fertilizers. Consequently, identifying high-performing, locally adapted inoculant strains is essential for reducing dependence on synthetic nitrogen fertilizers and improving the sustainability of temperate agroecosystems. Our study provides a genome-sequenced collection of common bean-nodulating Rhizobium from southern Ontario, revealing substantial species and genomic diversity across sampling locations. Greenhouse studies allowed us to identify multiple isolates, including isolates from a novel Rhizobium species, that consistently fix nitrogen with, and enhance the growth of, common bean plants. Our findings highlight strong biogeographical structuring of rhizobial communities and demonstrate that Ontario soils already harbour strains with high symbiotic potential. In addition, our Rhizobium collection represents a foundational resource to support future inoculant development and enables future work on the ecology, evolution, and applied optimization of legume-rhizobium symbioses.

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

Odd-chain carboxylates such as valerate and heptanoate are ecologically relevant metabolites and promising platform chemicals, yet the factors leading to their formation during secondary lactate fermentations remain poorly understood. Here, a continuous anaerobic bioreactor was operated for 297 days under mildly acidic conditions to evaluate how lactate:propionate molar ratios shape product spectrum in lactate fermentations. Valerate was the predominant odd-chain product under all conditions, reaching concentrations up to 110 mM, while heptanoate accumulated only at low levels (<10 mM). At low lactate concentrations (10-20 g/L), product selectivity strongly depended on the lactate:propionate ratio. When lactate:propionate ratios were around 1.2 mol/mol, odd-chain products were favored, whereas higher ratios (up to 4.8 mol/mol) shifted metabolism toward caproate and butyrate formation. However, this trend was not maintained at higher lactate concentrations (30-40 g/L; lactate not fully consumed), where odd-chain selectivities remained high even at lactate:propionate ratios of 4.8 mol/mol. Pathway analysis indicated that under high-lactate conditions up to 30% of lactate was redirected toward propionate and acetate formation, likely via the acrylate pathway. Microbial community analysis revealed a stable dominance of Caproiciproducens spp., that could be correlated to valerate production. Overall, this work provides mechanistic insights into the ecology of lactate fermentations and offers a framework for steering product selectivity in engineered anaerobic systems. HighlightsValerate was the dominant product, reaching up to 110 mM. Lactate:propionate ratios drive product selectivities. High lactate concentrations activated in situ propionate formation pathways. Caproiciproducens dominance was associated with sustained valerate production.

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.

Quantifying the lipid biosynthesis rate of archaea in hot spring sediments is necessary to interpret the abundance, isotopic patterns, and environmental significance of archaeal lipid biosignatures, with implications for modern biogeochemical cycling and astrobiology. Here, we performed lipid hydrogen stable isotope probing (LH-SIP) experiments on whole sediments collected from two high-temperature, suboxic, circumneutral hot springs in Yellowstone National Park (USA) and El Tatio Geyserfield (Chile). We determined the incorporation of 2H2O into intact polar lipids (IPLs) which provides a taxon- and metabolism-agnostic quantification of biosynthesis under near-natural conditions. We targeted isoprenoid glycerol dialkyl glycerol tetraether lipids (IPL iGDGTs) and recovered structures with 0 to 7 cyclopentyl rings from both springs. We observed minor 2H-uptake into archaeal IPLs in spring sediments in Yellowstone, corresponding to decadal-scale apparent generation times (16 {+/-} 7 years), and no uptake in El Tatio sediments (consistent with minimum generation times of 35 {+/-} 5 years). We infer that net production of sedimentary IPL-iGDGTs is very slow, consistent with a combination of slow archaeal growth, persistence of older IPLs, lipid recycling, and/or contributions from recently sedimented planktonic biomass. These are the first direct, ex situ estimates of archaeal lipid production rates in terrestrial hydrothermal systems using LH-SIP incubations and provide critical constraints for interpreting archaeal lipids in ancient hot spring deposits. This research establishes a framework for assessing activity by slow-growing extremophilic archaea in hydrothermal environments and provides support for targeting hydrothermal deposits on Mars for biosignature detection efforts. Plain Language SummaryHot springs on Earth are important natural laboratories for understanding how signs of life might form and be preserved in hydrothermal environments on early Earth or Mars. In this study, we examine the rate of archaeal lipid biosignature production in sediments from two hot springs in Yellowstone National Park and the El Tatio Geyserfield in Chile. We used a method that measures new microbial production by tracing heavy hydrogen from labeled water as microbes incorporate that hydrogen into newly made lipids in their cell membranes. We found that archaeal lipids in hot spring sediments are produced very slowly, on timescales of decades. This result, along with the chemical stability of lipids and the rapid mineralization rate in hot springs, may allow these molecular biosignatures to be entombed and preserved in hot spring mineral deposits. These results help us better interpret ancient hydrothermal deposits on Earth and support the idea that slowly growing microbial communities could still leave detectable molecular traces in similar environments on Mars and other rocky planets. Key PointsO_LILipid hydrogen stable isotope probing is applied to high temperature hot spring sediments for the first time C_LIO_LIIn hot spring sediments, archaeal lipid production occurs on decadal timescales comparable to some marine sediments, but are much faster than the century- to millennia-scale rates observed in the deep subsurface C_LIO_LIConfirmation of archaeal lipid synthesis in hot spring sediments adds additional support for targeting Martian hydrothermal deposits for biosignature detection efforts C_LI

Species of Bacillus bacteria including Bacillus safensis and Bacillus subtilis are finding increasing uses in biotechnology and bioremediation, thanks in part to their metabolic robustness. Malate dehydrogenase (MDH) is at the heart of central metabolism and thus a better understanding of Bacillus MDH proteins could aid in the optimization of these applications. MDH of Bacillus spp. belong to the lactate dehydrogenase (LDH)-like class of MDHs, otherwise known as the MDH3 class. Despite wide prevalence in nature among prokaryotes and archaea, this typically homotetrameric class is understudied compared to the MDH1 and MDH2 classes found in eukaryotes. We therefore recombinantly expressed and purified MDH proteins from two societally relevant Bacillus spp.-B. safensis and B. subtilis-and characterized them biophysically (via Size Exclusion Chromatography-Small Angle X-ray Scattering (SEC-SAXS) and Differential Scanning Fluorimetry (DSF)) and enzymatically (via spectroscopic activity assays). As expected based on their high sequence identity, the two MDH orthologs had similar properties in most regards, including a tetrameric structure and high susceptibility to substrate inhibition. However, we uncovered differences in conditional thermal stability, in addition to subtle differences in enzymatic activity that offer insight into the workings of LDH-like MDH. Summary statementMalate dehydrogenase (MDH) is a fundamental metabolic enzyme, from microbes to mammals, yet comparably little is known about microbial MDH, especially MDH of the tetrameric MDH3 class. We compare the biophysical and enzymatic properties of two such enzymes from the societally relevant bacterial species Bacillus subtilis and Bacillus safensis, offering useful insight with potential biotechnological implications.

Many pathogens, both those with human spillover potential as well as avian-specific viruses, are maintained in wild bird populations. While routine surveillance for influenza A viruses (IAVs) is performed annually, surveillance for other pathogens is limited. Sampling of wild birds is time-consuming, labour-intensive, often limited in sample size, and involves handling of wild and potentially infected birds, posing an increased risk of direct exposure for personnel. Additional methods for surveillance are needed given these significant challenges. Longitudinal fecal and sediment sampling was performed at various sites in southern Manitoba, Canada, particularly focused in Winnipeg from May to October 2025. Sites were chosen based on the suitability of the area for waterfowl habitat, the presence of waterfowl in the area, as well as proximity to reported outbreaks of H5N1 influenza virus. Fecal and sediment samples were collected and screened for the presence of influenza A virus (IAV), Newcastle disease virus (NDV), avian reovirus (ARV), and avian poxvirus (APXV). In total, 782 combined fecal and sediment samples were collected. Of the 714 fecal samples, 34 tested positive for IAV RNA (4.8% prevalence). None of the IAV-positive fecal samples tested positive for H5 RNA. Of the 68 sediments, 15 were positive for IAV RNA (22.1% prevalence), four of which were positive for H5 RNA. NDV RNA positivity was low, with only four positive fecal samples (0.6% prevalence) that were all collected on the same day. ARV RNA positivity was also low, with five positive sediment samples (7.4% prevalence in sediment samples). None of the samples tested positive for APXV DNA. This study builds on previous work showing the utility of environmental sampling for a variety of avian and zoonotic pathogens using a One Health approach that is low-risk, efficient, and high-throughput.

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

Biofilms formed by soil microbes hold immense potential for bioremediation, carbon dioxide sequestration, and the development of sustainable cementitious materials. However, quantifying the complex temporal coupling among bacterial growth, extracellular matrix (ECM) production, and mineralization dynamics remains a significant challenge due to the inherent nonlinearity of these processes and signal noise in high-throughput assays. To address this, we utilized an automated real-time kinetic analysis framework integrating connectivity-based segmentation, automated baseline alignment, and robust sliding-window algorithms to quantify the biomineralization competence of Bacillus subtilis under varying calcium regimes. Crucially, our results demonstrate that calcium carbonate promotes microbial growth as effectively as the highly soluble calcium acetate, providing strong evidence that B. subtilis actively solubilizes this crystalline powder to facilitate its metabolic requirements. Despite this growth efficacy, we found that calcium carbonate is an inadequate source for macro-calcite production compared to organic salts. By quantifying the expression efficiency of the sinI reporter gene, we determined that calcium-acetate-driven ECM expression significantly enhances the structural compatibility required for robust biomineralization. Furthermore, kinetic modeling suggests that ECM overproduction can partially compensate for defects in crystal growth-when provided crystalline calcium carbonate powder. These findings, enabled by high-resolution automated signal processing, underscore the critical role of self-mediated carbonate supply and present new engineering pathways for upcycling mineral-rich construction waste.