Applied and Environmental Microbiology最新文献

Oyster aquaculture is essential for ensuring a sustainable food source. Despite stringent controls, cases of oyster-related illnesses linked to pathogenic Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) persist. This study investigated the impact of aquaculture practices on the oyster microbiome and pathogen levels, focusing on two common systems: on-bottom and floating cages. From June to November 2019, monthly samples were collected from the Chesapeake Bay, including oysters and water from each aquaculture system. Oyster samples included both fresh and temperature-abused oysters. The study utilized the most probable number and real-time PCR (MPN-qPCR) method to quantify total and pathogenic Vp and Vv in water and oyster samples. DNA was extracted from oyster homogenates and filtered water samples for shotgun metagenomic sequencing. The results revealed significant impacts of aquaculture practices on the diversity of the oyster microbiome, particularly affecting the distribution of phages, antibiotic resistance, and virulence factor genes. Shotgun metagenomic sequencing consistently showed higher genetic representation of Vibrio in floating cages for both fresh and temperature-abused oyster samples. MPN-qPCR results differed between practices, showing higher Vibrio levels in bottom cages for fresh oysters and higher levels in floating cages under temperature abuse. These discrepancies are likely explained by the stable conditions in bottom cages, the effects of temperature abuse, and the growth bias inherent to the MPN method. These results underscore the need for a holistic, time-sensitive approach, taking into account microbial states and the dynamic aspects of the oyster environment to understand the complex relationship between aquaculture practices and the oyster microbiome.IMPORTANCEThis study holds great importance for food safety, antibiotic resistance surveillance, aquaculture management, and environmental health. Unraveling the population dynamics of microbial communities in oysters and their responses to different aquaculture practices enhances our ability to ensure safer seafood, monitor antibiotic resistance, optimize aquaculture methods, and mitigate potential public health challenges. Moreover, it demonstrates the applicability of advanced metagenomic tools for future research. Furthermore, this research addresses critical aspects of food safety, food security, public health, and sustainable aquaculture practices, making it highly relevant in today's context.

Understanding the assembly mechanisms of soil microbial communities is critical for maintaining nitrogen cycling in agricultural ecosystems, which underpins soil fertility and sustains crop productivity. While environmental filtering and biotic interactions shape these communities, our understanding of how functional taxa interact with soil properties across extensive agricultural landscapes remains limited. Here, we investigated the influence of environmental factors on the Chinese agricultural soil microbiome, integrating assessments of microbiota diversity, composition, and assembly process. The results indicated that soil pH and moisture were among the strongest abiotic factors explaining the agricultural soil microbiota compositional variation at a continental scale, surpassing the examined geographical and climatic effects. Stochastic processes dominated the assembly of microbial communities in large-scale agricultural soils, whereas the relative importance of deterministic processes increased with rising pH from acidic to alkaline soils. Phylogenetic turnover, as indicated by the beta nearest taxon index (βNTI), revealed determinism peaked under nitrogen-limited conditions but weakened with moderate precipitation, suggesting that both extreme aridity and rainfall amplify environmental filtering. We also found that divergent environmental preferences were displayed by ammonia-oxidizing microorganisms, including four archaeal genera belonging to the Nitrososphaeria class. Their significant correlations with βNTI as well as soil pH, nitrate, and moisture suggested that soil properties likely influenced prokaryotic community assembly primarily through modulating these functional taxa. This study highlights the vital role of ammonia-oxidizing-related soil properties in shaping the functional groups and assembly mechanisms of soil microbial communities, while enhancing our understanding of how ecological niche modifications by ammonia-oxidizers influence community interactions and nutrient dynamics in agricultural soils.

Importance: Agricultural soil microbiomes are essential for element cycling, fertility maintenance, and crop productivity, yet how key functional taxa interact with environmental factors to shape community assembly remains poorly understood. In this transcontinental study spanning diverse vegetation types, we demonstrate that ammonia-oxidizing archaea mediate soil microbial community assembly in response to pH and nitrate levels, with evidence of nonlinear threshold effects driven by nitrate. These findings underscore the pivotal role of keystone taxa in structuring soil biodiversity and ecological functions. Our study offers valuable insights into microbially mediated carbon and nitrogen cycling under climate change and supports crop-specific soil management strategies for sustainable agriculture.

To ensure safe, long-term use, topical products should be investigated to understand how they interact with the resident skin microbiota to mitigate potential risk. Sunscreens are essential for protecting skin from UV damage, but their effects on skin-resident microbes have not been well characterized. We examined the impact of two sunscreen formulations (containing titanium dioxide or zinc oxide) on both cultured skin bacteria and the skin microbiomes of human volunteers. No loss of viability was observed after a 2 h exposure to either sunscreen in cultures of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, Micrococcus luteus, and Corynebacterium tuberculostearicum. The effects of the sunscreens were then studied across the skin microbiomes of 20 human participants. Skin swabs were collected before application and at 1, 6, and 24 h afterward. DNA was extracted and sequenced at the 16S rRNA V4 region, and sequences were denoised and taxonomically assigned using the nf-core/ampliseq pipeline. Across all time points, alpha diversity (Shannon index, Friedman test) and beta diversity (permutational multivariate analysis of variance) remained stable, with no significant differences in beta dispersion. Differential abundance analysis revealed minor fluctuations in some low-abundance genera, identified as likely transient due to their low prevalence, but overall resident community composition was not significantly altered. These findings suggest that short-term sunscreen application does not disrupt the skin microbiome, supporting their safe use from a microbial standpoint. Outcomes from both in vitro and in vivo experimentation point to the compositional resilience of the skin microbiota to sunscreens.

Importance: Understanding how sunscreens affect the skin microbiome is important, given their widespread use and the role of the microbiome in skin health. This study demonstrates that common sunscreens do not significantly alter skin microbiome diversity or viability, including that of the core skin microbiome genera, Staphylococcus, Micrococcus, Kocuria, Cutibacterium, and Corynebacterium. This highlights the resilience of the skin microbiota and supports the microbiome-safe profile of these products.

Piscirickettsia salmonis is the causative agent of salmonid rickettsial septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous reporter (sfGFP) and an endogenous regulator (Fur) that controls intracellular iron homeostasis in bacteria. The inducible expression of dCas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains when the expression of the fur gRNA was induced with IPTG. By RNA-seq, we detected a significant increase in the expression of genes encoding the Fe²⁺ and Fe³⁺ acquisition systems and iron mobilization in the fur1 knockdown after IPTG induction. All the genes with over 2-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The implementation of the Mobile-CRISPRi system in P. salmonis has been demonstrated to be effective, thus providing a tool with potential application for the analysis of gene function in this pathogen. It is anticipated that these analyses will be valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis.

Importance: Salmonid rickettsial septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacterium's chromosome to ensure their constitutive and inducible expression and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Fur's relevance to the iron metabolism in the pathogen.

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.

Dehalococcoides mccartyi (D. mccartyi) plays a critical role in the dechlorination of halogenated organic pollutants, yet its performance on mixed chlorinated organophosphate esters (Cl-OPEs) remains poorly understood. In this study, a mixed microbial culture containing D. mccartyi was developed to dechlorinate commercial V6, a mixture comprising 80.2% 2,2-bis(chloromethyl)trimethylene bis(bis(2-chloroethyl) phosphate) (V6), 9.4% tris(2-chloroethyl) phosphate (TCEP), and other Cl-OPE impurities. Within 10 days, 99.5% of TCEP and 95.4% of V6 were dechlorinated. The presence of TCEP slightly enhanced the dechlorination of V6 compared with V6 as a purified compound. Both compounds underwent cleavage of C-O and C-Cl bonds in their chloroethoxy groups (Cl-CH₂-CH₂-O-), yielding different phosphate de-esterification products and ethene. D. mccartyi exhibited significant growth, with two reductive dehalogenase homologous (rdhA) genes co-transcribed during the dechlorination of commercial V6, purified V6, or TCEP. Metaproteomic analysis revealed that the enzymes encoded by these two genes were significantly expressed, suggesting that they may be the key enzymes mediating the dechlorination of mixed Cl-OPEs. Overall, this study provides insights into the role of D. mccartyi and its reductive dehalogenases in the natural attenuation of mixed Cl-OPEs in contaminated environments.

Importance: Commercial V6, a chlorinated organophosphate esters mixture, is widely used in polyurethane foam and has been detected in various environmental matrices. This study is the first to elucidate the microbial transformation pathways and mechanisms of commercial V6. A mixed culture containing Dehalococcoides mccartyi was found to dechlorinate V6 and tris(2-chloroethyl) phosphate (TCEP) into phosphate de-esterification products, chloride ion, and ethene. Notably, two reductive dehalogenase genes were simultaneously transcribed and their corresponding enzymes co-expressed, indicating a key role of D. mccartyi in the natural attenuation of commercial V6 in the environment.

Antimicrobial resistance (AMR) is a growing global public health concern affecting animals, humans, and the environment. Given its ubiquity, Escherichia coli may play a key role in the dissemination of AMR across these domains. Peri-urban regions where urban and rural systems intersect present unique challenges for controlling AMR. Despite this, limited data are available on AMR dissemination across the One Health continuum in peri-urban settings such as the Fraser Valley region of Canada. This study adopted a One Health approach to assess associations in AMR traits between E. coli from dairy production systems, nearby natural environments, and peri-urban communities within the same geographic location and timeframe. Over 1,000 isolates were recovered, and 421 were whole-genome sequenced to determine the presence of antimicrobial resistance genes (ARGs), plasmids, and virulence genes and to assess genomic relatedness. Findings revealed that AMR was not widespread: 17.8% of isolates carried at least one ARG, 10.2% were classified as multidrug-resistant, and 9.5% carried beta-lactamase genes. Phylogenomic analysis revealed high genomic diversity, with isolates belonging to 174 different sequence types (STs), including clinically important ST131 and ST10. Pairwise single-nucleotide polymorphism (SNP) comparisons identified 207 isolate pairs differing by ≤100 SNPs, indicating early-stage cross-domain movement of AMR. Overall, the findings from this study show that the prevalence of AMR E. coli is low, but there is evidence of transmission between animals and the environment, highlighting the importance of proactive, integrated surveillance and mitigation strategies to reduce the risk of future AMR dissemination across the One Health continuum.

Importance: Antimicrobial resistance (AMR) is a global public health concern that spans all three One Health domains (humans, animals, and the environment). Escherichia coli is present in humans, animals, and environmental sources-its ubiquity makes it an ideal organism to study AMR hotspots and transmission pathways across the One Health continuum. While surveillance of AMR in agricultural settings is increasing globally, little is known about transmission pathways in peri-urban agriculture areas where there is a high density of livestock farming in close proximity to residential communities. To identify potential AMR hotspots and transmission routes, this study investigated the occurrence and genomic relatedness of generic E. coli in the Fraser Valley region of British Columbia, a highly diverse agricultural region in western Canada. Our findings expand current knowledge by suggesting that early-stage transmission of AMR is occurring between the human, animal, and environmental sectors of the One Health triad, highlighting areas for improved resistance mitigation to prevent widespread dissemination.

Engineering of highly thermostable keratinase is of great theoretical interest in understanding protein stability mechanisms and practical significance for processing keratinous wastes such as feathers and wool. The thermostable subtilisin-like protease C2 is the major keratinase secreted by Thermoactinomyces vulgaris CDF but is rapidly inactivated at temperatures above 90°C. Here, we employed various methods to further stabilize protease C2. Four of the 35 selected single-point mutations designed by automated computational tools (PROSS, FireProt, ProteinMPNN, HyperMPNN, and ThermoMPNN) retained higher residual activity (~72%-84%) than protease C2 (~54%) after 1-h incubation at 85°C. The rational design of surface ion pairs and proline substitutions in β-turns generated two single-point variants with increased thermostability. Although three single-point aspartate substitutions appeared to be neutral, they could synergistically or cumulatively improve enzyme stability. The combination of these nine stabilizing mutations yielded the variant SM9 with a half-life of ~4 h at 100°C. The molecular dynamics simulations of protease C2 revealed several relatively flexible regions, including two Ca²⁺-binding sites (Ca1 and Ca2). Empirically modifying the Ca1 site and incorporating an additional two Ca²⁺-binding sites (Ca3 and Ca4) into the flexible regions yielded the variant CM1 with enhanced thermostability. By combining the mutations in SM9 and CM1, the variant CM16 was generated with a half-life of more than 9 h at 100°C. SM9 and CM16 are also highly resistant to high alkalinity, high salinity, urea, sodium dodecyl sulfate (SDS), organic solvents, and reductants, enabling them to efficiently degrade chicken feathers at temperatures near the boiling point.IMPORTANCEThe boiling-resistant enzymes are especially valuable not only for probing the molecular basis that allows proteins to function at the maximum temperature capable of supporting life but also offer the opportunity to greatly expand the enzymatic reaction conditions. Besides exploring naturally occurring boiling-resistant enzymes from hyperthermophiles, artificial engineering of enzymes with boiling resistance remains an important challenge. Our results demonstrate that the thermostability of the subtilisin-like protease C2 with keratinolytic activity can be largely improved by the combined use of automated computational design, structure-based rational design, and empirical engineering. The resulting variants are not only stable and functional at temperatures near or above 100°C but also show improved resistance to polyextreme conditions, providing new clues about the stabilization mechanism of subtilases. Moreover, by virtue of their hyperthermostability, the boiling-resistant variants could be repeatedly used for processing keratin substrates at high temperatures and find practical applications in feed, food, and leather industries.

The widespread persistence of antimicrobial resistance (AMR) plasmids presents a fundamental challenge to microbial evolution, known as the "plasmid paradox": if these plasmids cause fitness cost, why are they not eliminated by selection? The classical view, which imposed a fixed generic fitness cost, is insufficient to explain their epidemiological success. Here, we propose a new paradigm-the plasmid-host fitness landscape-a multi-dimensional model that takes into account the complex interplay between ecology and genetics. This landscape unfolds into three main axes. First, the host axis reveals that fitness costs often arise from host-dependent genetic conflicts, not a generic burden. Second, the time axis demonstrates that the fitness cost of any plasmid can be negated over time through plasmid or chromosome compensations, which leads to ameliorating initial costs and locking in resistance. Third, the environmental axis shows that the fitness cost of any plasmid can be affected by external factors like temperature and sub-inhibitory concentrations of antibiotics. These factors dynamically modulate the benefits and costs of plasmid carriage. By integrating the complex interplay between these dimensions, we argue that the plasmid fitness costs are not a fixed generic measurement, but rather a contingent trajectory across this landscape. This paradigm shifts the focus from static measurements to a dynamic, predictive science, providing a new foundation for assessing and managing the threat of mobile resistance.