Applied and Environmental Microbiology

Vibrio natriegens is an emerging model organism in laboratory and biotechnology research that is known for its fast growth rate and diverse metabolic capabilities. However, little is known about its response to oxidative stress. Reactive oxygen species (ROS) are ubiquitous stressors for most aerobic life and without mitigation can lead to cellular damage and sometimes death. V. natriegens is unusual amongst the Vibrio genus and Proteobacterial phylum in having three copies of katG encoding the bifunctional ROS defense enzyme catalase-peroxidase. The biological significance of having three copies of this protective gene was investigated with phylogenetics and gene inactivation studies. We determined that two of the katG copies arose recently via duplication and subsequent divergence. Analysis of single, double, and triple {Delta}katG constructs revealed each katG gene product contributes to survival during exposure to ecologically relevant concentrations of exogenous hydrogen peroxide (HOOH) under stationary phase conditions but found the genes were dispensable for HOOH resistance during exponential growth. We also demonstrated the involvement of the RpoS regulon in V. natriegens oxidative stress response in stationary phase. As an outcome of this investigation, we identified and repaired several spontaneous loss-of-function mutations of rpoS in laboratory cultures of the V. natriegens type strain ATCC 14048. Together, these results provide physiological and evolutionary insights into V. natriegens ROS response.

Enteric viruses are important contaminants of surface waters and a significant burden on public health. In aquatic ecosystems, the stability of these pathogens is differentially impacted by abiotic and biotic stressors, which can exert inactivating effects. Here, we investigated the fate of two enteroviruses, echovirus 11 (E11) and coxsackievirus B5 (CVB5), and one adenovirus, human adenovirus 2 (HAdV2), in lakewater and explored the mechanisms underlying their microbial inactivation. By combining infectivity assays, genome quantification, and capsid integrity analysis, we characterized virus-specific inactivation kinetics and mechanisms and examined the relationship between infectivity loss and capsid structural integrity. We observed rapid, intermediate, and negligible decay for HAdV2, E11, and CVB5, respectively, and confirmed that microbial proteases contribute to their inactivation. In addition, we revealed that loss of capsid structural integrity drives E11 inactivation, but not HAdV2 inactivation. Genome decay did not consistently correlate with loss of infectivity, highlighting the limitations of genome-based detection for assessing the presence of infectious viruses. These findings provide new insights into the mechanisms governing virus inactivation in aquatic environments and emphasize the importance of understanding viral fate when interpreting molecular detection data to assess virus-associated microbial risks.

BackgroundLarval habitat management is increasingly recognized as a complementary strategy for malaria vector control. While bacterial communities are abundant in mosquito breeding sites, their role in shaping oviposition behavior of Anopheles gambiae (Diptera: Culicidae) remains poorly understood. This study evaluated the relative attractiveness or repellency of water infused with bacterial isolates obtained from mosquito larval habitats to gravid Anopheles gambiae. MethodsWe isolated and taxonomically characterized bacteria from 30 larval habitats in coastal Kenya using 16S rRNA sequencing. Thirty-two representative isolates were tested in two-choice oviposition bioassays with gravid An. gambiae s.s., and volatile organic compounds (VOCs) emitted were analyzed by GC-MS. Statistical analyses included Students t-tests, Mann-Whitney U tests, and calculation of the Oviposition Activity Index (OAI). ResultsProteobacteria dominated larval habitats (78%), with frequent genera including Aeromonas, Acinetobacter, Pseudomonas, Enterobacter, and Cronobacter. Most isolates exerted strong deterrent effects, with Aeromonas hydrophila, Enterobacter hormaechei, and Pseudomonas mendocina completely inhibiting oviposition (OAI = -1; 100% repellency). Other isolates such as Neobacillus drentensis and Aeromonas veronii reduced egg deposition by >85%. In contrast, a small subset (Aquitalea pelogenes, Pseudomonas oleovorans, Cronobacter sakazakii) showed weak, non-significant attraction (OAI = 0.15-0.23). GC-MS revealed that highly repellent isolates emitted abundant benzenoids, ketones, phenols, ethers, oxygenated heterocycles, and aldehydes. ConclusionsBacterial volatiles strongly modulate oviposition site selection in An. gambiae, with most isolates acting as potent repellents. These findings highlight microbial VOCs as promising candidates for novel vector control strategies targeting gravid mosquitoes. Limitations include the laboratory-based bioassay design and lack of field validation of VOC activity. Future work should identify specific bioactive compounds and evaluate their efficacy under natural conditions. Research HighlightsO_LIBacteria from mosquito larval habitats influence oviposition of Anopheles gambiae. C_LIO_LISeveral bacterial isolates significantly repel gravid mosquitoes. C_LIO_LIGC-MS analysis revealed VOCs including benzenoids, ketones, phenols, and aldehydes. C_LIO_LIBacterial volatiles show potential for novel mosquito control strategies. C_LI

Abstract Antimicrobial resistance (AMR) is a pressing global public health challenge. AMR is driven in part by the spread of antibiotic resistance genes (ARGs) through bacterial communities via mobile genetic elements. Influent wastewater is a promising sample type for monitoring AMR because it pools biological inputs shed by individuals across a population. However, untargeted sequencing approaches, such as metagenomic sequencing, often miss low-abundance targets such as clinically important ARGs. In this work, we develop a ligation-mediated PCR (LM-PCR) enrichment strategy that can directionally capture the genomic context surrounding an ARG using long-read sequencing. We applied this method to study the natural genomic context diversity of four clinically relevant ARGs (blaCTX-M, blaKPC, and blaOXA-48-like, and qnrS) across 13 wastewater treatment plants in Washington state, each sampled at two timepoints. Across all timepoints, LM-PCR identified distinct genomic context cluster families associated with each ARG, including seven for blaKPC, 11 for blaCTX-M, 24 for qnrS, and one for blaOXA-48-like. Notably, 11 of the 24 qnrS containing clusters were putatively novel, with no matches to existing sequences in public databases. Genomic contexts associated with blaCTX-M and blaKPC were comparatively conserved across clusters, whereas qnrS was associated with a more diverse set of genetic sequences. Together, these results demonstrate that LM-PCR can resolve low-abundance, ARG-associated genomic variation in complex wastewater samples and provide a scalable framework for tracking the dissemination of clinically relevant AMR determinants.

In anaerobic digestion, formate is a central electron carrier linking primary and secondary fermentation to methanogenesis. Efforts to alter methane production have largely focused on directly targeting methanogens, while disruption of other trophic levels is underexplored. We recently demonstrated that hypophosphite is a naturally occurring inhibitor of formate exchange in syntrophic methanogenic systems. Here, we investigated whether intercepting formate exchange using hypophosphite can modulate methane production in a complex fermentative methanogenic system and what the longer-term consequences are for the structure and function of the microbiome. Using continuous up-flow anaerobic sludge blanket (UASB) columns fed with whey, we found that 100 M hypophosphite resulted in a transient (17 days) reduction in methane production by 20% to 50%, while carbon and electron flow was redirected towards hydrogen and volatile fatty acids. Accumulation of propionate, butyrate, branched-chain fatty acids, and not acetate indicated disruption of syntrophic formate metabolism. Following recovery of methane production in hypophosphite treated columns, we increased hypophosphite concentrations to millimolar levels and saw no repeated suppression of methane production. 16S rDNA amplicon sequencing revealed that hypophosphite treatment did not cause significant changes to microbiome composition. Methanogenic activity assays revealed a loss of formatotrophic methanogenic capacity in hypophosphite treated columns. Together, these results show that specific inhibition of formate exchange between syntrophs and methanogens decouples fermentative and methanogenic processes without altering microbiome composition with carbon and electron flow to methane ultimately redirected through hydrogen or acetate. This work demonstrates that hypophosphite can intercept syntrophic formate exchange in an engineered system and that long- term treatment with hypophosphite redirects electron flow towards electron carriers other than formate.

Microbially-mediated mineral precipitation is important in the natural world and to a number of emerging climate, energy, and water technologies. Central to many microbial mineralization processes is the preferential accumulation of precipitates on microbial surfaces. In some systems, including ureolytic microbially-induced carbonate precipitation (MICP), precipitates may entomb cells in a process termed "encapsulation." Encapsulation is known to greatly affect microbial systems, but understanding these relationships quantitatively has proven difficult, largely because nucleic acid extraction from encapsulated cells yields inconsistent results. Here, a demineralization protocol adapted from studies of natural microbialites is shown to recover genomic DNA from carbonate-encapsulated Sporosarcina pasteurii at yields indistinguishable from unmineralized cells and of sufficient quality for quantitative PCR. This method allows the degree of encapsulation to be expressed quantitatively as the amount of precipitate formed normalized to the estimated number of cells present, and allows growing populations to be tracked with increased precision. Simultaneous growth and mineralization experiments in mixed batch reactors suggest encapsulation-induced growth suppression at calcium concentrations of 250 mM, which are commonly employed in MICP under different physicochemical conditions. Variable rates of precipitate accumulation on cells, possibly stemming from local mass transport constraints, are proposed as a mechanism of calcium concentration-dependent growth suppression. Accordingly, mass transport principles are used to derive a reaction rate scaling function, allowing the observed growth and precipitation behavior to be replicated by a numerical model. These findings lay the groundwork for a more fundamental understanding of how encapsulation shapes microbial mineralization processes.

Acinetobacter sp. strain Tol 5 is an attractive microorganism because of its ability to utilize various aromatic compounds, including toluene. However, because Tol 5 is a non-model microorganism, the utilization of carbon sources in central carbon metabolism remains unclear. In this study, the activity of the central carbon metabolism was characterized using 13C-metabolic flux analysis (13C-MFA). Tol 5 was cultured in synthetic medium using ethanol or acetate as the sole carbon source. In addition, because toluene is degraded into pyruvate and acetyl-CoA via the toluene dioxygenase pathway, this situation was mimicked by culturing with ethanol and lactate as co-substrates. The 13C-MFA confirmed flux through the glyoxylate shunt, which bypasses part of the TCA cycle when either ethanol or acetate was used as the sole carbon source. In ethanol, the flux ratio of isocitrate dehydrogenase at the isocitrate branch point decreased markedly. Unlike acetate, the conversion of ethanol to acetyl-CoA is accompanied by NADH production. Therefore, the observed change in this flux ratio was influenced by the excess NADH generated. Under ethanol-lactate conditions, Tol 5 simultaneously utilized ethanol and lactate. The ethanol consumption rate decreased to less than half of that observed when ethanol was the sole carbon source. Most of the lactate uptake was converted to pyruvate and metabolized via the TCA cycle. The glyoxylate shunt flux was lower than that in ethanol. These metabolic characteristics provide valuable insights into the use of Tol 5 as a next-generation microbial cell factory.

Rinsing-dependent dental hygiene presents a significant public health challenge in water-scarce environments. This study investigated combinations of xylitol (Xyl), chitosan (Chi), glycyrrhizin (Gly), epigallocatechin gallate (EGCG), dicalcium phosphate (DCP), and nano-hydroxyapatite (nHA) on the primary bacteria behind dental caries, S. mutans. These combinations were assessed for markers of dental caries by biofilm reduction, bacterial killing, and acid buffering against S. mutans when applied to an in vitro simulated enamel model using glass bead surfaces for biofilm formation, and gene expression was subsequently examined via RT-qPCR. Separately, mineral retention was also quantified. The EGCG-DCP-Xyl film demonstrated the highest overall efficacy, achieving a significant reduction in biofilm concentration compared to the untreated control and performing similarly in magnitude to the positive toothpaste control. Dead fluorescence staining confirmed that the EGCG-DCP-Xyl film induced the highest rate of non-viable cells, followed by the Chi-Gly film and the Gly-Xyl film. During 10-day pH cycling, the EGCG-DCP-Xyl and DCP-Xyl formulations buffered pH the most, consistently maintaining mean pH levels safely above the demineralization threshold of pH 5.5. The EGCG-DCP-Xyl also optimized mineral stability with the highest retained calcium concentration, significantly outperforming the Chi-Xyl film. At the transcript level, the EGCG-DCP-Xyl film induced substantial downregulation of key virulence genes, yielding decreases in expression for glucosyltransferase B (gtfB), associated with biofilm synthesis, collagen-binding protein (cnm), associated with tissue invasion, and lactate dehydrogenase (ldh), associated with lactic acid production, compared to the untreated control, with effects comparable in magnitude to the positive toothpaste control. This research suggests that targeting bacterial pathways and mineral loss through a portable film may have potential for preventing dental caries, especially in environments where water is limited. However, additional studies are necessary to evaluate real-world effectiveness.

The scourge of cholera remains a major global public health threat. It affects up to 4 million people worldwide and causes tens of thousands of deaths each year. The disease is experiencing a concerning resurgence in many parts of Africa, the Middle East, and Asia. To effectively tackle cholera and circumvent rising antimicrobial resistance, targeted biological and preventive approaches, complementing traditional rehydration, are urgently needed. In this regard, our group has demonstrated the efficacy of L-ascorbic acid in controlling the growth and pathogenesis of Vibrio cholerae in vitro. The present work further provides a mechanistic elucidation of the L-ascorbic acid-mediated physiological changes in V. cholerae and also bolsters such a non-antibiotic approach to control cholera.

Shigella flexneri is an enteric pathogen responsible for causing shigellosis, a type of bacillary dysentery of significant global health concern. The increasing prevalence of antibiotic resistance has driven the search for alternative treatments, including bacteriophage (phage) therapy. However, the effectiveness of orally administered phages for enteric pathogens depends on their ability to survive passage through the gastrointestinal tract. In this study, the ability of bacteriophage Sf14 to maintain infectivity was evaluated under simulated gastrointestinal conditions using a modified INFOGEST 2.0 in vitro digestion model. This model includes salivary, gastric, and intestinal fluids with physiologically relevant compositions of electrolytes, bile salts, and enzymes. Phages were incubated in each simulated phase at standard physiological temperature, then tested for infectivity using quantitative plaque assays. Results show that Sf14 remained stable during the short duration of the oral phase and for the duration of the intestinal phase. By contrast, survival in simulated gastric fluid required a pH of 2.75 or higher, with rapid and substantial loss of infectivity at pH 2.5 and below. These findings demonstrate that while Sf14 is stable in oral and intestinal environments, the gastric phase poses a critical barrier to phage survival. With in vivo fasting gastric pH ranging from 1.5 to 3, encapsulation would enable the most flexible oral phage therapy against enteric pathogens, but ingestion after eating or alongside antacids may be sufficient.

Microbial weathering of minerals represents a key biogeochemical process through which microorganisms access essential nutrients locked within rock and ore substrates. In this study, we investigated the metabolic response of a model fungus during growth on lithium (Li) ore to elucidate the mechanisms underpinning biologically mediated mineral weathering and subsequent metal mobilization. Our results revealed a distinct metabolic shift in the fungus when cultivated on Li ore, characterized by altered patterns of organic acid production and energy metabolism. This shift coincided with measurable weathering of the Li ore matrix and the consequent release of soluble Li into the surrounding environment, demonstrating a direct link between fungal metabolic reprogramming and mineral breakdown. Importantly, these observations are consistent with metabolic shifts documented in previous studies of mineral weathering by this fungus, suggesting that such responses constitute a conserved and reproducible strategy employed during the colonization of mineral substrates. Although the present work was conducted using a single model microorganism under controlled conditions, the findings carry broader ecological implications. Natural microbial communities inhabiting mineral-rich environments may undergo analogous metabolic shifts when weathering minerals to acquire limiting nutrients, thereby contributing to large-scale elemental cycling and metal release. Understanding these processes not only advances fundamental knowledge of microbe-mineral interactions but also informs emerging applications in biomining and bio-based recovery of critical metals such as lithium. Collectively, this study highlights the central role of microbial metabolism in driving mineral weathering and offers a framework for predicting and harnessing similar processes within complex microbial communities. IMPORTANCEThe model fungus exhibits a distinct, reproducible shift in metabolism--particularly in organic acid production and energy pathways--when grown on Li ore, directly linking cellular metabolism to mineral breakdown. This metabolic shift promotes weathering of the ore matrix and the release of soluble lithium, demonstrating a biological route for liberating a critical metal from its mineral host. Because similar shifts occur across different minerals, natural microbial communities likely employ comparable strategies to weather minerals and acquire limiting nutrients, contributing to global elemental cycling. These findings provide a foundation for sustainable biomining and bio-based recovery of lithium and other critical metals, offering a lower-impact alternative to conventional extraction methods.

Vibrio cholerae, the facultative pathogen underlying cholera, naturally inhabits warm aquatic estuaries. Environmental persistence is enhanced by the ability of V. cholerae to colonize host reservoirs and form multicellular biofilms, causing seasonally endemic outbreaks in many tropical regions. Most toxigenic strains utilize the type IVa mannose-sensitive hemagglutinin (MSHA) pilus for host reservoir colonization and biofilm formation. Temperature and salinity can alter V. cholerae biofilm formation, yet their impact on MSHA production specifically remains largely unknown. Here, we utilized transcriptional reporters of predicted msh promoters (msh-P1/msh-P2/msh-P3) and functional assays, to determine temperature and salinity impacts on msh expression and pilus biogenesis. Under standard laboratory conditions (30{degrees}C, 1% NaCl) only msh-P1/P2 are active and inversely-coordinated with one another. Both msh-P1/P2 activity were elevated by high temperature (37{degrees}C) and low salinity (0.25%/0.5% NaCl), and reduced by low temperature (20{degrees}C/25{degrees}C) and high salinity (2%/3% NaCl). Temperature-mediated alterations in promoter activity were not immediately reflected in changes to cell-surface MSHA levels, whereas high salinity led to decreased MSHA production. Combining high temperature (37{degrees}C) and high salinity (2%/3% NaCl), attenuated the salinity-mediated reduction of msh-P1/P2 activity. Biofilm biomass levels were only substantially heightened at 25{degrees}C and 20{degrees}C, likely a result of no temperature-dependent changes in cell-surface MSHA, and additional temperature-controlled biofilm regulation previously described. We also found msh-P1/P2 promoter activity and MSHA production varies widely across toxigenic O1 and O139 serogroups despite complete sequence homology. These results shed new light on how key signals regulate MSHA pilus production to support V. cholerae persistence in aquatic environments.

NicA2 is a flavin-bound amine dehydrogenase from Pseudomonas putida S16 that converts nicotine to the pharmacologically inactive N-methylmyosmine. In animal models of nicotine addiction, injection of NicA2 can decrease nicotine-seeking behavior 10-fold. Accordingly, NicA2-related enzymes have been investigated as smoking-cessation therapeutics. However, efficient catalysis by NicA2 in Pseudomonas putida relies on electron transfer to CycN, a cytochrome c, and not directly to O2. Impractically high amounts of NicA2 are thus necessary to achieve a pharmacological effect in the absence of CycN. Directed evolution has improved the ambient-O2 value of kcat from 0.007 s-1 to 1 s-1 for NicA2, but further improvements have been challenging. Here, we identify a strain of Peribacillus frigoritolerans NIC8 which encodes two flavin amine oxidoreductases, Ncox and Pnox. In the presence of oxygen, Ncox and Pnox act on nicotine and pseudooxynicotine respectively with apparent kcat values of 7.7 s-1 and 3.9 s-1. Transient kinetics establishes bimolecular rate constants of 51100 M-1s-1 and 81000 M-1s-1 for the half-reactions between Ncox and O2 and between Pnox and O2 respectively, consistent with Ncox and Pnox being bona-fide oxidases. Transcriptomics shows enhanced expression of Ncox and Pnox under nicotine-dependent growth as well as supporting the identification of downstream enzymes. Phylogenetic analysis suggests that Ncox and Pnox arose out of repurposing of homologous enzymes found in Bacillus species. The enzymes we describe may be useful for the development of nicotine addiction therapeutics and for bioconversion of nicotine in waste streams.

Shiga toxin-producing Escherichia coli (STEC) persist in cattle and can colonize the bovine recto-anal junction, making it an important reservoir for transmission. Curli are extracellular amyloid fibers associated with biofilm formation and environmental persistence, but their contribution to STEC adherence to bovine epithelial cells is unclear. This study examined the relationship between curli production, biofilm formation, and adherence of O157, O26, and O111 STEC isolates to bovine recto-anal junction (RAJ) squamous epithelial (RSE) cells. Thirty isolates were evaluated for RSE adherence, curli-associated phenotypes, and biofilm formation under environmental and host-associated growth conditions. O157 and O111 isolates predominantly exhibited strong aggregative adherence to RSE cells, whereas O26 isolates showed more heterogeneous, primarily diffuse adherence. O111 isolates produced the strongest biofilms under environmental conditions and displayed relatively stable curli phenotypes, while O157 and O26 isolates showed greater variation. However, environmental biofilm formation did not consistently correlate with RSE adherence. Biofilm formation was also markedly reduced under host-simulated conditions in DMEM-LG at 26, 37, and 39{degrees}C, including among isolates that produced strong environmental biofilms. Differences in Shiga toxin genotype and expression likewise did not account for the major adherence patterns, as O111 isolates lacking stx2 retained strong aggregative adherence. These findings indicate that curli-associated biofilm formation is primarily influenced by environmental conditions and does not directly predict STEC adherence to bovine RSE cells. The results suggest that environmental persistence and host-cell attachment represent distinct phenotypes and that STEC adherence to bovine epithelial cells is likely mediated by additional strain- and serotype-specific factors.

Karst groundwater supplies in Virginia are relied on to varying degrees for domestic, agricultural, and municipal water supplies. Further, Virginian caves harbor an estimated 200 endemic invertebrate species. The microbial occupants of Virginias karst aquifers are largely undescribed; characterizing them promises to inform both the scientific description of these systems and the management of a critical water resource. Karst aquifers are heterogeneous, and much of the water moving through them cannot be reached directly; we profiled cave waters both because cave passages offer direct access to active groundwater and because cave water specifically is relied upon by endemic invertebrate species living in caves. Using 16S rRNA sequencing, we characterized aquatic microbial communities in eight Virginia caves, across Virginias four major karst regions. We identified 3,899 unique amplicon sequence variants (ASVs) and found that caves hosted diverse microbial assemblages that differed markedly among sampled sites. These baseline data provide a starting point for future work to understand how seasonal cycles, weather events, and surface disturbances affect the microbial communities present in cave waters and the cave-endemic invertebrates that depend on these waters.

Urinary tract infections (UTIs) are frequently caused by Escherichia coli and are increasingly associated with antimicrobial resistance, highlighting the need for alternative therapies. Although phage therapy is a promising approach, the rational selection of phages and phage cocktails remains challenging, particularly when detailed genomic information on the infecting strain is unavailable. In this study, we isolated 18 phages from environmental samples collected in Sherbrooke, Quebec, Canada. Genome sequencing identified 15 pure lytic phages representing diverse taxonomic groups and revealed prophage-derived contaminants in three isolates, underscoring the importance of genomic validation during phage discovery. Host range was determined using efficiency-of-plating (EOP) assays against UTI-associated E. coli isolates. Based on host-range overlap and cumulative EOP values, we developed a phenotype-based framework for phage cocktail design and classified combinations as redundant or complementary. Complementary cocktails composed of phages with distinct host ranges and high infection effectiveness consistently outperformed redundant combinations in time-kill assays, demonstrating that host-range diversity and lytic effectiveness are independent properties that should be considered separately during cocktail design. To evaluate cocktail performance under physiologically relevant conditions, the most effective combinations were tested in artificial urine. Both cocktails exhibited reduced activity compared with standard laboratory media, indicating that environmental conditions strongly influence phage-host interactions. Together, our results demonstrate that host-range overlap and cumulative infection effectiveness provide a practical framework for identifying effective phage combinations in the absence of detailed bacterial genomic information and may facilitate the rational development of phage therapies for UTIs.

Salmonella is a pathogen linked to foodborne outbreaks, including low-moisture foods. Its ability to resist desiccation can contribute to the formation of dry surface biofilms (DSB). This study evaluated the impact of 3 DSB formation protocols (P1=48-h hydrated phase/48-h dry phase, P2=24-h/120-h and P3=8-h/48-h) on the resistance of Salmonella DSB to 70% alcohol, a commercial product (based on 0.015% quaternary ammonium and 25% isopropyl alcohol), gaseous ozone (45 ppm), hot air (90 {degrees}C) and UV-C light (254 nm). The type of DSB protocol impacted the efficacy of the sanitizers (p < 0.05). The biofilm with the shortest hydration phase showed the greatest susceptibility; three out of the five sanitizers evaluated (70% alcohol, commercial product, and UV-C) promoted significant reductions in P3, with counts below the detection limit (0.8 log CFU/cm{superscript 2}) after 5 to 15 min exposure. Regarding protocols P1 and P2, in general, the best performance was from UV-C, especially against DSB on polypropylene, where it achieved reductions of 1.3 log CFU/cm{superscript 2} for P1 and 2.9 log CFU/cm{superscript 2} for P2 after 15 to 30 min of exposure. In contrast, hot air and ozone showed less effectiveness, with reductions [&le;]1.2 log CFU/cm{superscript 2}. In most scenarios, confocal microscopy images corroborated the plate count results (log CFU/cm{superscript 2}). In summary, our data indicates limited action of dry sanitizers on Salmonella DSB, requiring validation and optimization of sanitization processes to ensure the microbiological safety of low-moisture products.

Photosynthetic hydrogen (photoH2) production by the cyanobacterium Synechocystis sp. PCC 6803 is an attractive means for storing solar energy. However, photoH2 yields remain limited by competing electron flux pathways. Recent in vitro characterization suggests that photoH2 production requires electrons from both carbohydrate oxidation and photosynthesis. Engineered fusions between photosystem I (PSI) and hydrogenase (PSI-H2ase) aim to divert electrons toward H2 production and rely exclusively on photosynthesis. Thus, photoH2 production differs fundamentally between wildtype (WT) and PSI-H2ase fusion mutants. Here, we show that photoH2 production in WT is enhanced by supplemented glucose, consistent with the recently reported confurcating nature of HoxEFUYH H2ases. PhotoH2 production was further studied in the new psaE-hoxUYH mutant by simultaneously monitoring electron flux through PSI alongside with turnover rates of O2, CO2 and H2. PsaE-hoxUYH achieved the highest photoH2 yield and longest production period among the currently available PSI-H2ase mutants in Synechocystis, prolonged by removing O2. Upon illumination, psaE-hoxUYH exhibited high initial photoH2 production rates, which decreased in parallel with CO2 fixation and ceased immediately in the presence of O2. In absence of O2, photoH2 production still declined slowly. Therefore, in addition to CO2 fixation and O2, other yet unknown factors might limit photoH2 production under these conditions. Moreover, we traced a previously observed high H2 production phase of unclear origin in psaD-hoxYH cultures to contaminating [FeFe]-H2ases from Clostridium intestinale rather than genuine photoH2 production by the mutant. Together, these findings indicate a complex metabolic interplay tuning photoH2 production in Synechocystis WT and PSI-H2ase fusion mutants.

Coastal waters often experience fecal contamination from human (e.g., septic systems, wastewater treatment catchments and outfalls) and non-human (e.g., wildlife, domesticated pets, and livestock) origins, posing human health risks through contaminated fisheries and recreational waters. Quantitative PCR (qPCR) methods can identify and quantify fecal origins for their management by targeting Bacteroides, a dominant group of gastrointestinal bacteria with high host specificity. This study identified major contributors and drivers of fecal contamination to Mobile Bay, Alabama, an urbanized estuary in the southern United States. We utilized a multi-species qPCR source tracking approach targeting total Bacteroides and human-, pig-, dog-, cow-, and fowl-associated Bacteroides. Water samples and environmental parameters were collected over one year from 13 sites that represent a range of potential fecal inputs common to coastal waters. Human-associated feces were a consistent major contributor to the system (positive in 82% of samples), with localized inputs from pig (wild or domestic) and dog. Freshwater discharge and nitrogen constituents were good predictors of Bacteroides concentrations in this system, while areas with impervious surfaces and developed land cover may be contributing elevated fecal indicator levels. The multi-species qPCR source tracking approach described in this study can be implemented in other watersheds to support the development of better detection, identification, and fecal management strategies for community health of coastal areas.

Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen capable of adapting to diverse and challenging environments, including the oxygen- and nutrient-limited conditions encountered during chronic infections. Its ability to utilize a broad range of carbon sources and to switch from aerobic to anaerobic respiration contributes to its persistence within the host and its resilience to therapeutic interventions. In particular, denitrification enables P. aeruginosa to grow under oxygen-limited conditions by using nitrate and nitrite as alternative electron acceptors, a process particularly relevant in biofilms and chronic infections such as cystic fibrosis. Ubiquinone (UQ) is essential for respiratory metabolism in P. aeruginosa, which possesses two distinct UQ biosynthetic pathways that ensure UQ production across a broad range of oxygen concentrations. Recent studies have identified the O2-independent UQ biosynthetic pathway as a key determinant of chronic infection. Because this pathway is restricted to a limited group of bacteria, we hypothesized that its selective inhibition could represent a strategy to specifically target P. aeruginosa under anaerobic conditions. To test this hypothesis, we screened analogues of 4-hydroxybenzoic acid (4-HB), the earliest precursor of the UQ biosynthetic pathway, and identified 4-aminobenzoic acid, also known as para-aminobenzoic acid (pABA), as a potential inhibitor. pABA primarily affects the anaerobic metabolism of P. aeruginosa by acting as a competing substrate for UQ biosynthetic enzymes, thereby interfering with UQ biosynthesis. This inhibitory effect positions pABA as a valuable tool for investigating, and potentially targeting, anaerobic metabolism in P. aeruginosa.