Applied and Environmental Microbiology最新文献

Vibrio cholerae, the etiological agent of cholera, is ubiquitous in environmental brackish waters. Exposure to low water temperatures induces the bacterium to enter a viable but non-culturable (VBNC) state. In this study, a stepwise decrease in water temperature to 4°C was found to delay the transition to the non-culturable state compared to an abrupt temperature drop, suggesting that V. cholerae cells partially adapt to low temperatures. V. cholerae VBNC cells maintained at 4°C gradually lost their ability to revert to a culturable state. However, VBNC cells in the early stage of dormancy were efficiently resuscitated following treatment with proteolytic enzymes, including proteinase K. The abundance of culturable V. cholerae cells in brackish estuarine waters was quantified using the most probable number (MPN)-quantitative polymerase chain reaction (qPCR) method. Although culturable cells were undetectable in samples treated with bovine serum albumin, they were estimated at 93 and 1,500 MPN/mL in two water samples collected on different days and pre-incubated with proteinase K. Similarly, the abundance of Vibrio species increased markedly following treatment with this enzyme. Additionally, cells of Vibrio species were enumerated by the plating method using CHROMagar Vibrio plates. Consistent with the results of the MPN-qPCR method, treatment with proteinase K resulted in over a 100-fold increase in colony formation. Collectively, these findings suggest that treatment with proteinase K is effective for resuscitating and quantifying V. cholerae VBNC cells in environmental water samples.

Importance: V. cholerae enters into a viable but non-culturable (VBNC) state when exposed to low water temperatures. Contamination of food and drinking water with VBNC cells poses a critical public health risk, as these cells retain their virulence but cannot be detected by conventional methods. In this study, we demonstrated that VBNC cells of V. cholerae could be efficiently resuscitated by treatment with proteolytic enzymes such as proteinase K, enabling their detection through standard culture-based assays. Environmental brackish water samples were analyzed for V. cholerae density using the most probable number (MPN)-quantitative polymerase chain reaction (qPCR) method. While V. cholerae was not detected in untreated samples, proteinase K treatment revealed densities of 93 or 1,500 MPN/mL. Therefore, the combination of proteinase K treatment with the MPN-qPCR method offers a promising approach for detecting VBNC bacterial contamination in food, drinking water, and environmental water.

Hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. The zoonotic HEV genotype 3 (HEV-3) is present in various animal species, including pigs, wild boars, and other game animals. Foodborne transmission with the consumption of raw or undercooked pork products is the major transmission route of HEV-3. HEV RNA has been detected in various types of food, but particularly in pork liver-based food products. High hydrostatic pressure processing (HPP) can be used for the inactivation of pathogens in food. In the present study, the impact of HPP treatments was evaluated on HEV-3 infectivity in raw pork liver. Different pressure/time combinations (500 MPa for 1 or 5 min, 600 MPa for 1, 5, or 10 min) were applied to raw pork livers, artificially contaminated with HEV-3 (8.3 log₁₀ HEV ge/g). Residual HEV infectivity was evaluated using the HepaRG cell culture model in p-24 well plates. The results obtained have shown the absence of residual infectious HEV particles in pork liver after a treatment of 600 MPa, during 1 min in a refrigerated room at +8°C. Then, liver sausages were prepared with pork liver treated at 600 MPa for 1 min. Technological measurements showed that the treatment had a significant impact on brightness, firmness, red hue, and cohesiveness. Nevertheless, these differences have not been perceived after food testing, which highlighted no major difference in taste or color. Thus, inactivation of HEV-3 in raw pork liver by HPP is a possible treatment to limit the risk of HEV exposure through food consumption.

Importance: The hepatitis E virus (HEV) is the leading cause of enterically transmitted acute hepatitis worldwide. It can have a zoonotic origin through the consumption of infected meat. Pigs are the main reservoir of zoonotic HEV, and pork livers are frequently contaminated by HEV. In the present study, we investigated the use of high-pressure processing (HPP) to inactivate HEV-3 in pork liver. This study is the first to identify HPP treatment parameters that can be applied to pork liver to reduce HEV infectivity. Additionally, it is the first study to demonstrate the feasibility of processing HPP-treated pork liver into food products, such as dry liver sausage.

The prevailing dogma in microbial ecology holds that sulfate-reducing microorganisms (SRMs) outcompete methanogenic archaea for common substrates (e.g., H₂/formate and acetate), leading to the mutual exclusion of sulfate reduction and methanogenesis in sulfate-rich anaerobic environments. This principle underpins models of organic carbon flow to sulfate-respiration-derived CO₂ in ecosystems like oil reservoirs, where seawater injection introduces high concentrations of sulfate. In an Applied and Environmental Microbiology article by S. Beilig, L. Voskuhl, I. Geydirici, L. K. Tintrop, T. C. Schmidt, and R. U. Meckenstock (91:e00141-25, 2025, https://doi.org/10.1128/aem.00141-25), the authors challenge this view by demonstrating coexistence of methanogenesis and sulfate reduction in a sulfate-adapted enrichment culture from an oil reservoir. The authors employ incubation experiments and microbial activity assessment via the reverse stable isotope labeling (RSIL) method to argue for metabolic coexistence, even under conditions thought to favor complete competitive exclusion. This commentary discusses the mechanistic reasons underlying the coexistence and explores the broader implications for predicting microbial activities and interactions. The study compellingly argues that thermodynamic and kinetic arguments alone are insufficient to predict microbial community function, necessitating a more nuanced understanding of microbial interactions in complex environments.

This study investigates the corrosion inhibition behavior of Bacillus subtilis on B30 copper-nickel alloy in seawater, focusing on its biomass components in regulating biomineralization. Results show that B. subtilis formed a protective biofilm and induced the precipitation of a uniform biomineral layer, mainly composed of Ca-Mg carbonates. This layer acted as a physical barrier, resulting in a low corrosion current of (5.85 ± 0.08) × 10⁻⁷ A/cm² and reducing the maximum pit depth from 44.74 to 18.54 µm. Furthermore, the roles of different biomass components, such as bacterial cells, extracellular polymeric substances (EPS), and soluble microbial products (SMPs), were also investigated. It was found that all components could initiate mineralization, but with distinct outcomes: bacterial cells primarily served as structural templates; EPS facilitated the formation of highly crystalline and stable Mg-calcite, providing the most durable protection, while SMPs promoted the formation of well-crystallized calcite with comparatively lower protective efficacy.IMPORTANCECorrosion is a critical issue prevalent across various industries, where traditional corrosion control technologies are often limited by high costs, complex implementation, and potential environmental hazards. Biomineralization, as an emerging green anti-corrosion strategy, is not only environmentally friendly but also enables long-term effective protection, reducing reliance on toxic chemical agents and lowering economic costs. However, due to the complexity of microbial systems, the mechanisms underlying biomineralization are not yet fully understood. In this study, different biomass components-including bacterial cells, extracellular polymeric substances, and secreted metabolites-were isolated from Bacillus subtilis cultures using a series of separation techniques, and their impacts on the mineralization process were systematically evaluated. This work elucidates the corrosion inhibition mechanism of biomineralization and provides valuable insights into the relationship between specific microbial components and biomineral formation, which holds significant implications for developing eco-friendly corrosion inhibition technologies.

The Kuroshio-Oyashio Extension (KOE) region is a highly variable region in the North Pacific Ocean, characterized by strong environmental gradients and multi-scale oceanographic processes. However, the fine-scale impact of these currents and associated water masses on microbial communities remains poorly understood. Here, high-resolution samples from 18 to 24 layers were collected along a transect in the KOE region in 2021, with 16S rRNA gene amplicon sequencing and environmental parameter measurements conducted to investigate the Kuroshio-Oyashio influence on microbial communities. Strong regional and vertical variations in environmental parameters and microbial communities were observed, with main horizontal regional differentiations confined to the upper 500 m. Photoautotrophic and oligotrophic taxa (e.g., SAR11 clade and Cyanobacteria) were enriched in warm, oligotrophic Kuroshio region, whereas the cold nutrient-rich Oyashio and confluence regions supported higher microbial abundance, diversity, and complex microbial interactions. Consistently, heterotrophic bacteria (1.00 × 10⁶-1.17 × 10⁹ cells L⁻¹) were more abundant in the upper 55 m of the Oyashio and confluence regions than in the Kuroshio region. Below the thermocline (~500 m), community composition was primarily structured by depth, indicating a diminishing Kuroshio-Oyashio current influence. Three main water masses (subtropical mode water [STMW], central mode water, and North Pacific intermediate water [NPIW]) with distinct microbial communities were identified, explaining ~11% of microbial variation beyond depth and geography, with biomarker taxa identified (e.g., Actinomarinales for STMW, Nitrosopumilales for NPIW). This study reveals the extent of Kuroshio-Oyashio influence on microbial communities and highlights the integrated impacts of large-scale currents and fine-scale water masses on shaping microbial biogeography in the KOE region.IMPORTANCEThe convergence of the Kuroshio and Oyashio currents shapes high microbial diversity, as well as complex microbial-mediated biogeochemical processes. However, investigations into the microbial distribution patterns in relation to these current systems remain limited in spatial resolution. This study with high-resolution samples reveals the extent of Kuroshio-Oyashio influence on microbial communities and advances the understanding of how multi-scale oceanographic processes influence microbial biogeographical patterns. It provides a fine-scale perspective for exploring microbial distribution and assembly in highly dynamic oceanic environments.

Marine environments are frequently oligotrophic, characterized by low amount of bioassimilable nitrogen sources. At the global scale, the microbial fixation of N₂, or diazotrophy, represents the primary source of fixed nitrogen in pelagic marine ecosystems, playing a key role in supporting primary production and driving the export of organic matter to the deep ocean. However, given the high energetic cost of N₂ fixation, the active release of fixed nitrogen by diazotrophs appears counterintuitive, suggesting the existence of alternative passive release pathways that remain understudied to date. Here, we show that the marine non-cyanobacterial diazotroph Vibrio diazotrophicus is endowed with a prophage belonging to the Myoviridae family, whose expression is induced under anoxic and biofilm-forming conditions. We demonstrate that this prophage can spontaneously excise from the genome of its host and that it forms intact and infective phage particles. Moreover, phage-mediated host cell lysis leads to increased biofilm production compared with a prophage-free derivative mutant and to increased release of dissolved organic carbon and ammonium. Altogether, the results suggest that viruses may play a previously unrecognized role in oceanic ecosystem dynamics by structuring microhabitats suitable for diazotrophy and by contributing to the recycling of (in)organic matter.

Importance: Diazotrophs are key players in ocean functioning by providing fixed nitrogen to ecosystems and fueling primary production. However, from a physiological point of view, the active release of nitrogenous compounds by diazotrophs is paradoxical, since they would invest in an energy-intensive process and supply nutrient to non-sibling cells, with the risk of being outcompeted. Therefore, alternative ways leading to the release of fixed nitrogen must exist. Here, we show that the marine non-cyanobacterial diazotroph Vibrio diazotrophicus possesses one prophage, whose activation leads to cell death, increased biofilm production, and the release of dissolved organic compounds and ammonium. Taken together, our results provide evidence that marine phage-diazotroph interplay leads to the creation of microhabitats suitable for diazotrophy, such as biofilm, and to nutrient cycling, and contributes to better understanding of the role of viruses in marine ecosystems.

The increasing use of metagenomic sequencing (MGS) for microbiome analysis has significantly advanced our understanding of microbial communities and their roles in various biological processes, including human health, environmental cycling, and disease. However, the inherent compositionality of MGS data, where the relative abundance of each taxon depends on the abundance of all other taxa, complicates the measurement of individual taxa and the interpretation of microbiome data. Here, we describe an experimental design that incorporates exogenous internal standards in routine MGS analyses to correct for compositional distortions. A mathematical framework was developed for using the observed internal standard relative abundance to calculate "Scaled Abundances" for native taxa that were (i) independent of sample composition and (ii) directly proportional to actual biological abundances. Through analysis of mock community and human gut microbiome samples, we demonstrate that Scaled Abundances outperformed traditional relative abundance measurements in both precision and accuracy and enabled reliable, quantitative comparisons of individual microbiome taxa across varied sample compositions and across a wide range of taxon abundances. By providing a pathway to accurate taxon quantification, this approach holds significant potential for advancing microbiome research, particularly in clinical and environmental health applications where precise microbial profiling is critical.IMPORTANCEMetagenomic sequencing (MGS) analysis has become central to modern characterizations of microbiome samples. However, the inherent compositionality of these analyses, where the relative abundance of each taxon depends on the abundance of all other taxa, often complicates interpretations of results. We present here an experimental design and corresponding mathematical framework that uses internal standards with routine MGS methods to correct for compositional distortions. We validate this approach for both amplicon and shotgun MGS analysis of mock communities and human gut microbiome (fecal) samples. By using internal standards to remove compositionality, we demonstrate significantly improved measurement accuracy and precision for quantification of taxon abundances. This approach is broadly applicable across a wide range of microbiome research applications.

Bifidobacterium is a key member of the human gut microbiota, and many strains are widely used as probiotics due to their health-promoting properties. Despite growing interest, genetic studies in Bifidobacterium have been relatively limited, primarily due to the lack of available genome editing tools. Recent advances in genomics and CRISPR-Cas systems provide opportunities for targeted genome modification in this genus. In this review, we provide an overview of the occurrence, diversity, and distribution of CRISPR-Cas systems across Bifidobacterium species and examine the editing tools developed and implemented to date. We also highlight practical challenges such as strain variability and low transformation efficiency and introduce future avenues of research such as large-payload insertion and in situ editing. Expanding the genetic toolbox for Bifidobacterium will broaden our understanding of this important genus and enable the development of next-generation probiotics.

Coastal lagoon project is a common strategy for enhancing flood control capability and ecosystem services, yet its impact on microbiota, especially archaea, remains unclear. Using 16S rRNA gene and transcript sequencing, we compared archaeal diversity, community assembly processes, and potential activity in an artificial lagoon and adjacent seaward waters through monthly sampling over an annual cycle. The lagoon has created a distinct water environment with reduced salinity and turbidity, along with unique dissolved organic matter profiles. The lagoon's influence overrode seasonal variability in archaeal alpha-diversity, yielding overall higher levels within the lagoon. Despite pronounced seasonal shifts-Nitrosopumilaceae dominating in cooler seasons and Poseidoniales prevailing in warmer months-the lagoon's influence on archaeal community composition across taxonomic scales remained evident, particularly in the spatial niche partitioning of Poseidoniales populations. Lagoon archaeal communities exhibited higher turnover rates and accelerated seasonal recurrence compared with those in the seaward zone. Although archaeal community assembly was primarily driven by water-mass effects, selection occasionally exerted a stronger influence in seaward waters. Analyses involving the 50 most abundant zero-radius operational taxonomic units (ZOTUs) revealed that the lagoon project had a stronger and more widespread effect on the distribution of key archaeal taxa than on their potential activity, consistent with the trend observed at the genus level, except for two Nitrosopumilaceae genera: Nitrosopumilus often exhibited lower activity, while Nitrosopelagicus occasionally showed higher activity in the lagoon. Our findings highlight that the lagoon project variably altered archaeal diversity, community assembly, and potential activity, underscoring microbial consequences and potential ecological impacts of nearshore restoration projects.

Importance: Coastal lagoon projects are widely employed to enhance ecosystem services, such as water quality, yet their impacts on microbial communities-particularly archaea-remain poorly understood. This year-long study reveals that artificial lagoon environments significantly reshape archaeal communities by increasing alpha-diversity, accelerating seasonal turnover, and shifting dominant taxa, especially among ammonia-oxidizing archaea and Poseidoniales. Community assembly was primarily governed by water-mass effects introduced through lagoon maintenance, while archaeal potential activity exhibited taxon-specific patterns. These findings uncover critical, previously overlooked microbial consequences of lagoon engineering and emphasize the importance of incorporating microbial dynamics into the planning and evaluation of nearshore restoration projects.