Applied and Environmental Microbiology最新文献

The rising atmospheric concentration of CO₂ is a major concern to society due to its global warming potential. In soils, CO₂-fixing microorganisms are preventing some of the CO₂ from entering the atmosphere. Yet, the controls of dark CO₂ fixation are rarely studied in situ. Here, we examined the gene and transcript abundance of key genes involved in microbial CO₂ fixation along major environmental gradients within estuarine wetlands. A combined multi-omics approach incorporating metabarcoding, deep metagenomic, and metatranscriptomic analyses confirmed that wetland microbiota harbor four out of seven known CO₂ fixation pathways, namely, the Calvin cycle, reverse tricarboxylic acid cycle, Wood-Ljungdahl pathway, and reverse glycine pathway. These pathways are transcribed at high frequencies along several environmental gradients, albeit at different levels depending on the environmental niche. Notably, the transcription of the key genes for the reverse tricarboxylic acid cycle was associated with high nitrate concentration, while the transcription of key genes for the Wood-Ljungdahl pathway was favored by reducing, O₂-poor conditions. The transcript abundance of the Calvin cycle was favored by niches high in organic matter. Taxonomic assignment of transcripts implied that dark CO₂ fixation was mainly linked to a few bacterial phyla, namely, Desulfobacterota, Methylomirabilota, Nitrospirota, Chloroflexota, and Pseudomonadota.

Importance: The increasing concentration of atmospheric CO₂ has been identified as the primary driver of climate change and poses a major threat to human society. This work explores the mostly overlooked potential of light-independent CO₂ fixation by soil microbes (a.k.a. dark CO₂ fixation) in climate change mitigation efforts. Applying a combination of molecular microbial tools, our research provides new insights into the ecological niches where CO₂-fixing pathways are most active. By identifying how environmental factors, like oxygen, salinity and organic matter availability, influence these pathways in an estuarine wetland environment, potential strategies for enhancing natural carbon sinks can be developed. The importance of our research is in advancing the understanding of microbial CO₂ fixation and its potential role in the global climate system.

Diarrheal diseases attributable to multidrug-resistant F4+ enterotoxigenic Escherichia coli (ETEC) are escalating in severity, posing significant risks to the health and safety of both humans and animals. This study used Saccharomyces cerevisiae EBY100 to display the FaeG subunit of F4 colonizing factor as an oral vaccine against F4+ ETEC infection. Mice were orally immunized twice with 10⁸ CFU of EBY100/pYD1-FaeG, followed by a challenge with F4+ ETEC EC6 on day 7 post-immunization. The results showed that the recombinant strain EBY100/pYD1-FaeG orally enhanced the growth of the small intestine villi, significantly boosted the expression of tight junction proteins (ZO-1, Occludin, MUC2, and Claudin) (P < 0.05), and modulated the gut microbiota composition. Additionally, immunization with EBY100/pYD1-FaeG also upregulated the levels of IL-2, IL-4, and IFN-γ in the intestines of mice (P < 0.01), while serum IgG and fecal sIgA titer significantly increased (P < 0.05). These immune responses enhanced the capacity to fight against ETEC, leading to an increased survival rate of mice and relieved damage to tissues and organs of mice infection. In summary, the study suggested that the recombinant Saccharomyces cerevisiae EBY100/pYD1-FaeG could effectively stimulate the immune response and generate specific antibodies against F4+ ETEC, showing its potential to serve as a subunit oral vaccine candidate for preventing F4+ ETEC infection.IMPORTANCEThe multidrug-resistant F4+ enterotoxigenic Escherichia coli (ETEC) strains are the primary clinical pathogens responsible for post-weaning diarrhea in piglets, resulting in substantial economic losses in the pig farming industry. In the study, we developed an oral vaccine candidate, Saccharomyces cerevisiae EBY100/pYD1-FaeG, to prevent diarrhea caused by multidrug-resistant F4+ ETEC. Oral administration of EBY100/pYD1-FaeG significantly enhanced immune responses, improved intestinal health, and provided protection against F4+ ETEC infection in mice. This approach offers a potential application prospect for preventing F4+ ETEC infections that lead to post-weaning diarrhea in clinical settings and provides a promising solution for addressing the growing threat of antibiotic resistance in bacterial pathogens.

Previous work reported that deletion of the Enzyme IIAB subunits (EIIAB^Man and manL) of the glucose phosphotransferase system (PTS) (glucose-PTS, manLMNO) in Streptococcus sanguinis impacted carbon catabolite repression and bacterial fitness. Here, a single-nucleotide polymorphism in ManN, ManNA91E, produced the unusual phenotype of increased excretion of organic acids and H₂O₂ yet elevated PTS activities. To characterize the contributions of each component of the glucose-PTS to bacterial fitness, we performed genetic analyses by deleting from S. sanguinis SK36 the entire operon and each EII^Man subunit individually; and genes encoding the catabolite control protein A (ΔccpA) and the redox regulator Rex (Δrex) for comparison. Deletion of each subunit incurred a growth defect on glucose partly due to elevated excretion of H₂O₂; when supplemented with catalase, this defect was rescued, instead resulting in a significantly higher yield than the parent. All glucose-PTS deletion mutants presented an increased antagonism against the oral pathobiont Streptococcus mutans, a phenotype absent in ΔccpA despite increased H₂O₂ output. A shift in the pyruvate node toward mixed acid fermentation and increased arginine deiminase activity enhanced pH homeostasis in glucose-PTS mutants but not ΔccpA. Despite the purported ability of Rex to regulate central carbon metabolism, deletion of rex had no significant impact on most of the phenotypes discussed here. These findings place glucose-PTS in the pivotal position of controlling central carbon flux in streptococci, with critical outcomes impacting acidogenicity, aciduricity, pH homeostasis, and antagonism, highlighting its potential as a therapeutic target for treating diseases with a dysbiotic microbiome.

Importance: Management of carbohydrate metabolism and environmental stress is key to the survival of oral commensal species such as S. sanguinis. Antagonism of oral pathobionts and modulation of the environmental pH and oxidative potential by commensals are crucial to the maintenance of microbial homeostasis and prevention of oral diseases including dental caries. It is therefore vital to understand how these species regulate sugar fermentation, production of acids and ammonia, and stress management in an environment known for a feast-and-famine cycle of carbohydrates and similar fluctuations in pH and oxygen tension. Here, we detail that genetic alterations of the glucose-PTS transporter in S. sanguinis can significantly affect the regulation of factors required for bacterial fitness and homeostatic ability independent of known catabolic regulators. It is then discussed how these changes may impact the survival of streptococcal species and affect caries onset.

During the coronavirus disease 2019 epidemic, excessive chlorine disinfectants have been used to block the spread of severe acute respiratory syndrome-coronavirus 2, resulting in large amounts of residual disinfectants entering wastewater treatment plants (WWTPs) through sewage systems. So far, no relevant research has been conducted on the impact of chlorine disinfectants on microfauna, an important microbial component in activated sludge treatment systems. This study comprehensively investigated the changes in microfauna habitat, community structure, and colonization mode under the chlorine stress by combining the full-scale WWTP survey and laboratory-scale sequencing batch reactor experiments. The results showed that chlorine disinfectants significantly changed the community structure of microfauna, including decrease in sedentary ciliates and increase in free-living ciliates, amoebas, and flagellates. Besides the disinfection effect of chlorine disinfectants, the microfauna community was also influenced by changes in habitat and bacterial community. The loose structure and excessive extracellular polymeric substance (EPS) of activated sludge caused by chlorination would impact the colonization of sedentary ciliates, while it was conducive to the survival of free-living ciliates due to their predation on EPS as the nutrients. Bacteria in the activated sludge had strong interactions with protozoa, and their changes under chlorine stress directly affected the protozoan community and even indirectly affected the micro-metazoa community through the food chain.

Importance: This study revealed that chlorine disinfectant significantly changed microfauna habitat, community structure, and colonization mode in wastewater treatment plants during the coronavirus disease 2019 pandemic. Chlorine disinfectant could destroy the structure and stability of sludge flocs, reduce the abundance of beneficial microfauna in activated sludge, and even affect the colonization of sedentary ciliates on sludge. In addition, chlorine disinfectants might induce environmental and ecological risks related to microfauna, such as elevated suspended solids and release of bacteria and microfauna in the effluents.

Creating and maintaining an appropriate chemical environment is essential for biomineralization, the process by which organisms precipitate minerals to form their shells or skeletons, yet the mechanisms involved in maintaining calcifying fluid chemistry are not fully defined. In particular, the role of microorganisms in facilitating or hindering animal biomineralization is poorly understood. Here, we investigated the taxonomic diversity and functional potential of microbial communities inhabiting oyster calcifying fluid. We used shotgun metagenomics to survey calcifying fluid microbial communities from three different oyster harvesting sites. There was a striking consistency in taxonomic composition across the three collection sites. We also observed archaea and viruses that had not been previously identified in oyster calcifying fluid. Furthermore, we identified microbial energy-conserving metabolisms that could influence the host's calcification, including genes involved in sulfate reduction and denitrification that are thought to play pivotal roles in inorganic carbon chemistry and calcification in microbial biofilms. These findings provide new insights into the taxonomy and functional capacity of oyster calcifying fluid microbiomes, highlighting their potential contributions to shell biomineralization, and contribute to a deeper understanding of the interplay between microbial ecology and biogeochemistry that could potentially bolster oyster calcification.

Importance: Previous research has underscored the influence of microbial metabolisms in carbonate deposition throughout the geological record. Despite the ecological importance of microbes to animals and inorganic carbon transformations, there have been limited studies characterizing the potential role of microbiomes in calcification by animals such as bivalves. Here, we use metagenomics to investigate the taxonomic diversity and functional potential of microbial communities in calcifying fluids from oysters collected at three different locations. We show a diverse microbial community that includes bacteria, archaea, and viruses, and we discuss their functional potential to influence calcifying fluid chemistry via reactions like sulfate reduction and denitrification. We also report the presence of carbonic anhydrase and urease, both of which are critical in microbial biofilm calcification. Our findings have broader implications in understanding what regulates calcifying fluid chemistry and consequentially the resilience of calcifying organisms to 21st century acidifying oceans.

Ultraviolet (UV) C light emitted by a krypton chloride (KrCl) lamp consists of mainly less harmful 222-nm Far-UVC (unfiltered 222-mm Far-UVC) compared with conventionally used 254-nm UVC. It also contains wavelengths that are harmful to mammalian cells. By contrast, UVC from a KrCl lamp with optical filter (filtered 222-nm Far-UVC) consists of much less harmful 222-nm Far-UVC and is available for sterilization of dwelling spaces. The germicidal mechanisms of the 254-nm UVC and unfiltered 222-nm Far-UVC have been partially elucidated; however, the mechanism of action of filtered 222-nm Far-UVC remains unknown. It is known that 254 nm UVC induces cyclobutene pyrimidine dimers (CPDs), which are DNA lesions in Escherichia coli (E. coli); however, the CPDs are repaired by photoreactivation. In the present study, it was demonstrated that filtered 222-nm Far-UVC also generated CPDs, which were not repaired by photoreactivation. Therefore, a germicidal mechanism of filtered 222-nm Far-UVC may be different from a 254-nm UVC. It was reported that unfiltered 222-nm Far-UVC induced reactive oxygen species (ROS) in E. coli. In the present study, filtered 222-nm Far-UVC also induced ROS production. In accordance with increased ROS production, the levels of carbonylated proteins were increased, and morphological alteration was observed in E. coli. From these results, it was suggested that ROS generated by filtered 222-nm Far-UVC inactivated ROS scavenger enzymes and the enzyme photolyase that is involved in photoreactivation. The increased ROS levels and unrepaired CPDs impaired photoreactivation in E. coli and may be involved in the germicidal mechanism of action of the filtered 222-nm Far-UVC.IMPORTANCEThe 222 nm Far-ultraviolet (UV) C light (UVC) emitted from a krypton chloride lamp with an optical filter is currently available for the sterilization of dwelling spaces. To use the filtered 222-nm Far-UVC more effectively and safely for sterilization, it is necessary to understand its germicidal mechanism. The present study suggests that the germicidal effect of filtered 222-nm Far-UVC on E. coli may not only involve CPD but also ROS. These results could be useful in establishing more effective preventive methods in dwelling spaces for infectious diseases by UVC irradiation.

An air sanitizer was evaluated using an aerobiology protocol, compliant with the U.S. Environmental Protection Agency's Air Sanitizer Guidelines, for virucidal activity against bacteriophages Phi6 and MS2 (used as surrogates for enveloped and non-enveloped human pathogenic viruses). The phages were suspended in a medium containing a tripartite soil load simulating body fluids and aerosolized using a six-jet Collison nebulizer in an enclosed 25 m³ aerobiology chamber at 22 ± 2°C and 50 ± 10% relative humidity. The air sanitizer was sprayed into the chamber for 30 s. Viable phages in the air were captured directly, in real time, on host bacterial lawns using a slit-to-agar sampler. Reductions in viable phage concentration ≥3.0 log₁₀ (99.9%) were observed after a mean exposure of 3.6 min for Phi6, suggesting efficacy against enveloped viruses (e.g., SARS-CoV-2, influenza, and RSV), and ~10.6 min for MS2, suggesting virucidal efficacy for non-enveloped viruses (e.g., noroviruses and rhinoviruses). This targeted air sanitization approach represents an important non-pharmaceutical public health intervention with virucidal efficacy against airborne viral pathogens.IMPORTANCEAirborne viruses are implicated in the transmission indoors of respiratory and enteric viral infections. Air sanitizers represent a non-pharmaceutical intervention to mitigate the risk of such viral transmission. We have developed a method that is now an ASTM International standard (ASTM E3273-21) as well as a test protocol approved by the U.S. EPA to evaluate the efficacy of air sanitizing sprays for inactivating airborne MS2 and Phi6 bacteriophage (used as surrogates for non-enveloped and enveloped human pathogenic viruses, respectively). The test phages were individually suspended in a soil load and aerosolized into a room-sized aerobiology chamber maintained at ambient temperature and relative humidity. Reductions in viable phage concentration ≥3.0 log₁₀ (99.9%) were observed after a mean exposure of 3.6 min for Phi6, suggesting efficacy against enveloped viruses (e.g., SARS-CoV-2; influenza; RSV), and ~10.6 min for MS2, suggesting virucidal efficacy for non-enveloped viruses (e.g., noroviruses and rhinoviruses). The data suggest the utility of the air sanitizer for mitigating the risk of indoor viral transmission during viral pandemics and outbreaks.

Viral detection methodologies used for wastewater-based epidemiology (WBE) studies have a broad range of efficacies. The complex matrix and low viral particle load in wastewater emphasize the importance of the concentration method. This study focused on comparing three commonly used virus concentration methods: polyethylene glycol precipitation (PEG), immuno-magnetic nanoparticles (IMNP), and electronegative membrane filtration (EMF). Influent and effluent wastewater samples were processed by the methods and analyzed by DNA/RNA quantification and sequencing for the detection of human viruses. SARS-COV-2, Astrovirus, and Hepatitis C virus were detected by all the methods in both sample types. PEG precipitation resulted in the detection of 20 types of viruses in influent and 16 types in effluent samples. The corresponding number of virus types detected was 21 and 11 for IMNP, and 16 and 8 for EMF. Certain viruses were unique to only one concentration method. For example, PEG detected three types of viruses in influent and six types in effluent compared to IMNP, which detected seven types in influent and one type in effluent samples. However, the EMF method appeared to be the least effective, detecting three types in influent and none in effluent samples. Rotavirus was detected in influent sample using IMNP method, whereas EMF and PEG methods failed to yield a similar outcome. Consequently, the potential false negative results pose a risk to the credibility of WBE applications. Therefore, implementation of a proper concentration technique is critical to minimize method biases and ensure accurate viral profiling in WBE studies.IMPORTANCEIn recent years, significant research efforts have been focused on the development of viral detection methodology for wastewater-based epidemiology studies, showing a range of variability in detection efficacies. A proper methodology is essential for an appropriate evaluation of disease prevalence and community health in such studies and necessitates designing a concentration method based on the target pathogenic virus. There remains a need for comparative performance evaluations of methods in the context of detection efficiencies. This study highlights the significant impact of sample matrix, viral structure, and nucleic acid composition on the efficacy of viral concentration methods. Assessing WBE techniques to ensure accurate detection and understanding of viral presence within wastewater samples is critical for revealing viral profiles in municipality wastewater samples.