Applied and Environmental Microbiology最新文献

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.

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.

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.

Phosphorus forms and distribution in organic manures vary under different treatment conditions, thereby exerting distinct effects on the soil microbiome and soil phosphorus transformation process. This study examined the effects of a novel manure treated with hyper-thermophilic fermentation combined with Bacillus strain inoculation, compared with raw and composted manure, on the oat rhizosphere microbiome and phosphorus transformation across different soil types in a controlled pot experiment. Our findings demonstrate that hyper-thermophilic fermentation with Bacillus inoculation not only promotes the survival and abundance of the bacterial genus Bacillus but also selectively enriches the hyper-thermophilic bacterial genus Thermobifida in the fermented manure. Notably, the application of hyper-thermophilic fermented manure led to a significant enrichment of keystone species like Bacillus and Thermobifida across both soil types, relative to other manure applications. These genera emerged as key drivers of available phosphorus, phosphatase activity, and differential metabolites in the rhizosphere, exhibiting a synergistic effect on soil phosphorus transformation. Fermented manure exhibited superior performance relative to conventional composted manure, as it increased the phosphorus uptake rate of oats by 35.5% in black soil and 27.9% meadow soil, respectively, over a single growing season. Additionally, among all organic manures, the application of fermented manure significantly enhanced the sequestration of phosphorus from manure in the soils, with 78.0% in black soil and 56.9% in meadow soil. This consequently reduced P loss to 13.6% and 34.4% in the respective soil types.

Importance: Phosphate-solubilizing microorganisms (PSMs) are frequently proposed as catalysts for promoting phosphorus recycling; however, their performance is often inefficient or ineffective in the context of a circular bioeconomy within agricultural systems. This study introduces innovative concepts and methodologies by integrating hyper-thermophilic fermentation with heat-resistant phosphate-solubilizing Bacillus inoculation, thereby enhancing the effective phosphorus recovery and utilization from manure waste in sustainable agricultural practices.

Marine bacteria such as Alteromonas are key players in regulating algal blooms, yet the genomic basis for their strain-specific algicidal activities remains poorly understood. Here, we use comparative genomics to dissect the mechanisms of functional divergence between two closely related Alteromonas macleodii strains: strain FDHY-03, which employs a broad-spectrum strategy, and FDHY-CJ, which has adapted a narrow-spectrum strategy specifically targeting diatoms. We reveal that these distinct predatory strategies are underpinned by divergent genomic architectures. The broad-spectrum strain FDHY-03 leverages a versatile, synergistic enzymatic arsenal rich in polysaccharide lyases to enable its broad-spectrum attack. In contrast, the specialist FDHY-CJ has evolved an integrated, high-precision system comprising: (i) a specialized CAZyme toolkit, uniquely enriched with GH16 isoforms, tailored to breach diatom-specific defenses; (ii) an enhanced chemotaxis system (Tsr) to home in on its algal targets; and (iii) a complex quorum sensing network (AHL/solo-LuxR) to coordinate its behavior in diatom-rich niches. Our findings provide a high-resolution model for microbial microevolution, demonstrating how genomic plasticity enables rapid niche partitioning within a single species. This work illuminates the molecular details of marine microbial warfare and provides a blueprint for the genome-informed selection of targeted biocontrol agents for harmful algal blooms.

Importance: Frequent harmful algal blooms pose a severe threat to global biogeochemical cycles. Algicidal bacteria, acting as natural antagonists, serve as effective biological tools for controlling harmful algal blooms. While extensive research has been conducted on the isolation and identification of algicidal bacteria, the genomic basis for their strain-specific algicidal activity remains unclear. This study employs comparative genomics to analyze the genomic architecture of two closely related Alteromonas macleodii strains, revealing distinctly different algicidal strategies. Our findings offer valuable insights into the molecular basis of microbial warfare in marine environments, ultimately contributing to the advancement of microbial-based approaches for mitigating harmful algal blooms.

A consequence of past acid rain events has been chronic acidification of Nova Scotian forests, leading to a loss of essential nutrients and subsequent decreases in forest productivity and biodiversity. Liming-supplementing forests with crushed limestone-can restore essential nutrients to acidified soils and increase the pH of soils and the carbon capture by forests through the promotion of tree growth. Liming treatments are often assessed through tree growth measurements, although little is known about how microorganisms respond to these changes in pH and nutrient availability. Understanding the impacts of liming on microbial communities will help determine whether liming is a good remediation strategy for Nova Scotia. A pilot study evaluating liming in acidified forests in Nova Scotia began in 2017. Microbiome analysis (prokaryotic 16S and fungal ITS2 rRNA gene amplicon sequencing) of three different horizons (depths; upper forest floor, lower forest floor, and upper B horizon) of soil in a softwood forest area showed significant differences between lime-treated and control soils for the prokaryotic but not fungal communities, particularly in the uppermost soil horizon. Several genera from the Alphaproteobacteria class were significantly higher in abundance in treated than control soils, whereas genera from the Acidobacteriia (previously Acidobacteriae) class were significantly lower in abundance in treated versus control soils. Soil chemistry analysis of the same three horizons showed a significant increase in base cations and pH of the uppermost soil horizon in control versus treatment sites.IMPORTANCEForests are increasingly being managed with an emphasis on understanding how forests function. Lime amendments are used to promote forest health and increase resilience to climate change. To date, only a handful of studies have analyzed the effects of liming on microbial communities in forest soils. Our study combines soil chemistry with prokaryotic and fungal communities of limed and control soils. Shifts in microbial composition that are coincident with liming may provide early indications of the effectiveness of liming and provide insight into the roles of microbes in forest health.

Co-evolution of bacterial defense systems and phage counter-defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. In order to evade nuclease-based mechanisms that target DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits the recognition and restriction by endonucleases. We found that Shewanella phage Thanatos DNA is insensitive to multiple restriction enzymes and also to Cas I-Fv and Cas9 cleavage. Furthermore, with nanopore sequencing, the phage DNA showed severely impaired basecalling. In addition to an adenine methylation, the data indicated an additional, much more substantial nucleobase modification. Using liquid chromatography-mass spectrometry (LC-MS), we identified an unknown configuration of a deoxypentose attached to cytosine as an undiscovered modification of phage DNA, which is present in Thanatos genomic DNA and likely mediates resistance to restriction endonucleases, as well as reducing Cas nuclease activity significantly. To elucidate the underlying enzyme functions, we identified structural homologs of Thanatos proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of the phage protein TH1_063 by in vitro. Inactivation of TH1_060 leads to an almost complete inhibition of phage propagation, indicating an important role of the cytosine modification in phage survival and/or proliferation.

Importance: Several phages extensively decorate their DNA building blocks, providing an effective protection against various host and phage-produced restriction systems. These modifications allow the phages to distinguish between their own DNA and that of the host, significantly increasing the establishment of the phage chromosome upon entry into the host and subsequent phage proliferation. Several different modifications have been previously identified and characterized. Here, we describe a hitherto unknown cytosine modification, consisting of a deoxypentose-putatively xylose-that provides protection against various bacterial restriction systems, including DNA-targeting CRISPR/Cas systems. Our findings expand the range of DNA modifications that phages use for protection.

Antimicrobial resistance is one of the largest and most pressing global health threats. This is not only a huge burden on the global economy but also a growing threat to animal, environmental, plant, and human health, and new strategies are needed to avoid resistance and improve treatment. Novel antimicrobial resistance genes are normally first detected once they cause problems in clinical infections, and we have limited knowledge on their evolutionary trajectories. Current antimicrobial susceptibility testing and research have a limited focus on key environmental factors in pathogen-reservoir-host interactions, possibly leading to inaccurate results that do not reflect the in vivo conditions. Focusing on differences in pH, we determined the MIC of a panel of isogenic strains expressing CTX-M-15 and CMY-2 β-lactamases. We found that pH has a large impact on the activity of β-lactamases, and treatment of these resistant isolates could be possible if the pH of the environment is modified. We verified this using enzyme kinetics, co-cultures, and growth experiments, suggesting that exposure to different environmental conditions may lead to distinct evolutionary trajectories for specific β-lactamases. Exploring the effect of different temperatures, we also observed a differential effect of avian and mammal host temperatures. Environmental factors such as pH and temperature may have a large unnoticed effect on antimicrobial resistance, and we might use this knowledge to renew and extend the use of old antibiotics for certain infections.IMPORTANCEAntimicrobial resistance is a huge burden to global health and economy. We need new options for avoiding selection of resistance and improved treatment. Overlooked aspect: current susceptibility testing does not take pH into account. With this study, we show that pH and temperature can have large and contrasting effects on the activity (and therefore MIC) of specific β-lactamases. This might help to explain the phenomenon of bacteria often harboring multiple β-lactamases seemingly with the same function as well as be utilized to enable treatment of genotypically resistant strains under very specific conditions, that is, treatment of CTX-M-15, the most prevalent ESBL in healthcare, under alkaline conditions.

Pseudomonas putida is a ubiquitous, metabolically versatile microbe that has gained visibility as a chassis for bioengineering. The dbu operon in P. putida, comprised of a D-branched-chain amino acid (D-BCAA) oxidase (DbuA), a Rid2 protein (DbuB), and a transporter (DbuC), was previously characterized for its role in the catabolism of D-branched-chain amino acids. In the present study, we show that the addition of any of three D-BCAAs catabolized by these gene products increases expression of the dbu genes. The presence of D-tyrosine does not increase transcription of the dbu genes, nor is it used as a sole nitrogen source by P. putida. Derepression of the dbu operon allows catabolism of D-tyrosine, thus uncovering an additional capacity of the dbu gene products. Results herein show that the dbuR gene, which is divergently transcribed from the operon, encodes a xenobiotic response element (XRE) family transcription factor that is a transcriptional repressor of the dbu genes. The effect of D-BCAA on dbu expression appears to be at least partially independent of DbuR, suggesting transcriptional regulation of this operon involves multiple components. In total, this work contributes to understanding the complex regulatory and metabolic networks of the environmentally and economically important microbe P. putida.IMPORTANCEPseudomonas putida is a broadly utilized bioengineering chassis, primarily due to the versatile and robust metabolic network of the organism. P. putida, like many microorganisms, employs a complex regulatory network to coordinate its metabolism, allowing it to adapt to changing environments and dynamic nutrient availability. The physiological role of the D-branched-chain amino acid (D-BCAA) utilization pathway (dbu) operon in D-BCAA metabolism by P. putida has been defined, and herein we provide insights into the regulation of the genes in this operon. DbuR is encoded by a gene near the dbu operon, and this protein represses the expression of the dbu genes. A new metabolic capability (catabolism of D-tyrosine) of P. putida was revealed, a capability that increases the potential of this organism as a chassis for bioengineering. This work expands current knowledge of P. putida and contributes insights into the metabolic and regulatory capabilities of this environmentally, industrially, and economically relevant microbe.