High-throughput Amplicon Sequencing Research Articles

Impact of a Potent Strain of Plant Growth-Promoting Bacteria (PGPB), Bacillus subtilis S1 on Bacterial Community Composition, Enzymatic Activity, and Nitrogen Content in Cucumber Rhizosphere Soils.

Antagonistic bacterial strains from Bacillus spp. have been widely studied and utilized in the biocontrol of phytopathogens and the promotion of plant growth, but their impacts on the rhizosphere microecology when applied to crop plants are unclear. Herein, the effects of applying the antagonistic bacterium Bacillus subtilis S1 as a biofertilizer on the rhizosphere microecology of cucumbers were investigated. In a pot experiment on cucumber seedlings inoculated with S1, 3124 bacterial operational taxonomic units (OTUs) were obtained from the rhizosphere soils using high-throughput sequencing of 16S rRNA gene amplicons, and the most abundant phylum was Proteobacteria that accounted for 49.48% in the bacterial community. S1 treatment significantly reduced the abundances of soil bacterial taxa during a period of approximately 30days but did not affect bacterial diversity in the rhizosphere soils of cucumbers. The enzymatic activities of soil nitrite reductase (S-Nir) and dehydrogenase (S-DHA) were significantly increased after S1 fertilization. However, the activities of soil urease (S-UE), cellulase (S-CL), and sucrase (S-SC) were significantly reduced compared to the control group. Additionally, the ammonium- and nitrate-nitrogen contents of S1-treated soil samples were significantly lower than those of the control group. S1 fertilization reshaped the rhizosphere soil bacterial community of cucumber plants. The S-CL activity and nitrate-nitrogen content in rhizosphere soil affected by S1 inoculation play important roles in altering the abundance of rhizosphere soil microbiota.

Exploring the Rhizospheric Microbial Communities under Long-Term Precipitation Regime in Norway Spruce Seed Orchard.

The rhizosphere is the hotspot for microbial enzyme activities and contributes to carbon cycling. Precipitation is an important component of global climate change that can profoundly alter belowground microbial communities. However, the impact of precipitation on conifer rhizospheric microbial populations has not been investigated in detail. In the present study, using high-throughput amplicon sequencing, we investigated the impact of precipitation on the rhizospheric soil microbial communities in two Norway Spruce clonal seed orchards, Lipová Lhota (L-site) and Prenet (P-site). P-site has received nearly double the precipitation than L-site for the last three decades. P-site documented higher soil water content with a significantly higher abundance of Aluminium (Al), Iron (Fe), Phosphorous (P), and Sulphur (S) than L-site. Rhizospheric soil metabolite profiling revealed an increased abundance of acids, carbohydrates, fatty acids, and alcohols in P-site. There was variance in the relative abundance of distinct microbiomes between the sites. A higher abundance of Proteobacteria, Acidobacteriota, Ascomycota, and Mortiellomycota was observed in P-site receiving high precipitation, while Bacteroidota, Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadota, and Basidiomycota were prevalent in L-site. The higher clustering coefficient of the microbial network in P-site suggested that the microbial community structure is highly interconnected and tends to cluster closely. The current study unveils the impact of precipitation variations on the spruce rhizospheric microbial association and opens new avenues for understanding the impact of global change on conifer rizospheric microbial associations.

Adaptation of a microbial consortium to pelagic Sargassum modifies its taxonomic and functional profile that improves biomethane potential.

In recent years, pelagic Sargassum has invaded the Caribbean coasts, and anaerobic digestion has been proposed as a sustainable management option. However, the complex composition of these macroalgae acts as a barrier to microbial degradation, thereby limiting methane production. Microbial adaptation is a promising strategy to improve substrate utilization and stress tolerance. This study aimed to investigate the adaptation of a microbial consortium to enhance methane production from the pelagic Sargassum. Microbial adaptation was performed in a fed-batch mode for 100days by progressive feeding of Sargassum. The evolution of the microbial community was analyzed by high-throughput sequencing of 16S rRNA amplicons. Additionally, 16S rRNA data were used to predict functional profiles using the iVikodak platform. The results showed that, after adaptation, the consortium was dominated by the bacterial phyla Bacteroidota, Firmicutes, and Atribacterota, as well as methanogens of the families Methanotrichaceae and Methanoregulaceae. The abundance of predicted genes related to different metabolic functions was affected during the adaptation stage when Sargassum concentration was increased. At the end of the adaptation stage, the abundance of the predicted genes increased again. The adapted microbial consortium demonstrated a 60% increase in both biomethane potential and biodegradability index. This work offers valuable insights into the development of treatment technologies and the effective management of pelagic Sargassum in coastal regions, emphasizing the importance of microbial adaptation in this context.

Metagenomic analysis of soil from landfill site reveals a diverse microbial community involved in plastic degradation

In this study, we have investigated microbial communities structure and function using high throughput amplicon sequencing and whole metagenomic sequencing of DNA extracted from different depths of a plastic-laden landfill site. With diverse taxonomic groups inhabiting the plastic-rich soil, our study demonstrates the remarkable adaptability of microbes to use this new substrate as a carbon source. FTIR spectroscopic analysis of soil indicated degradation of plastic as perceived from the carbonyl index of 0.16, 0.72, and 0.44 at 0.6, 0.9 and 1.2 m depth, respectively. Similarly, water contact angles of 108.7 degree, 99.7 degree, 62.7 degree, and 77.8 degree of plastic pieces collected at 0.3, 0.6, 0.9, and 1.2 m depths respectively showed increased wettability and hydrophilicity of the plastic. Amplicon analysis of 16S and 18 S rRNA revealed a high abundance of several plastic-degrading bacterial groups, including Pseudomonas, Rhizobiales, Micrococcaceae, Chaetomium, Methylocaldum, Micromonosporaceae, Rhodothermaceae and fungi, including Trichoderma, Aspergillus, Candida at 0.9 m. The co-existence of specific microbial groups at different depths of landfill site indicates importance of bacterial and fungal interactions for plastic. Whole metagenome analysis of soil sample at 0.9 m depth revealed a high abundance of genes encoding enzymes that participate in the biodegradation of PVC, polyethylene, PET, and polyurethane. Curation of the pathways related to the degradation of these materials provided a blueprint for plastic biodegradation in this ecosystem. Altogether, our study has highlighted the importance of microbial cooperation for the biodegradation of pollutants. Our metagenome-based investigation supports the current perception that consortia of fungi-bacteria are preferable to axenic cultures for effective bioremediation of the environment.

Deep insights into the assembly mechanisms, co-occurrence patterns, and functional roles of microbial community in wastewater treatment plants

The understanding of activated sludge microbial status and roles is imperative for improving and enhancing the performance of wastewater treatment plants (WWTPs). In this study, we conducted a deep analysis of activated sludge microbial communities across five compartments (inflow, effluent, and aerobic, anoxic, anaerobic tanks) over temporal scales, employing high-throughput sequencing of 16S rRNA amplicons and metagenome data. Clearly discernible seasonal patterns, exhibiting cyclic variations, were observed in microbial diversity, assembly, co-occurrence network, and metabolic functions. Notably, summer samples exhibited higher α-diversity and were distinctly separated from winter samples. Our analysis revealed that microbial community assembly is influenced by both stochastic processes (66%) and deterministic processes (34%), with winter samples demonstrating more random assembly compared to summer. Co-occurrence patterns were predominantly mutualistic, with over 96% positive correlations, and summer networks were more organized than those in winter. These variations were significantly correlated with temperature, total phosphorus and sludge volume index. However, no significant differences were found among microbial community across five compartments in terms of β diversity. A core community of keystone taxa was identified, playing key roles in eight nitrogen and eleven phosphorus cycling pathways. Understanding the assembly mechanisms, co-occurrence patterns, and functional roles of microbial communities is essential for the design and optimization of biotechnological treatment processes in WWTPs.

Fungal Secondary Metabolite Gliotoxin Enhances Enzymatic Activity in Soils by Reshaping their Microbiome

Gliotoxin (GT) is a sulfur-containing epidithiodioxopiperazine produced by various filamentous fungi, including those used in biological plant protection (Trichoderma virens). The pronounced antimicrobial effect of GT on a variety of fungi and bacteria makes it a promising agent for controlling phytopathogens in agricultural systems. In this study, we aim to investigate the microbiological properties of the soil microbiome after the introduction of GT. GT was applied at doses of 10, 25, 50, 100 and 500 μM kg-1 soil. Soil sampling was carried out after 1, 7, 14, 30, 60 and 90 days of incubation. It was found that GT significantly stimulated the respiratory activity of soil microorganisms and maintained this activity throughout the experiment. Carbon of microbial biomass, on the contrary, decreases under the influence of GT and is restored at the end of the experiment only in microcosms with 10 and 25 μM GT. Separate estimates of bacterial and fungal biomass showed that the bacterial community increased in biomass on day 14, while fungal biomass increased on day 30 after the treatment. Under the influence of GT, the activity of soil enzymes involved in the carbon (CB, βG, βX), nitrogen (NAG, LAP) and phosphate (AP) cycles significantly increased. High-throughput amplicon sequencing of the ITS and 16S rDNA markers revealed that the soil fungal community is more susceptible to GT than the bacterial community. This was reflected in changes in alpha-diversity indices and in the pattern of changes in the abundance of some microbial genera. Thus, on the one hand, the data obtained provides insight into the biological effects of GT on the soil microbial community. On the other hand, it sets the direction for further research into the ecological role of antibiotics produced by soil and rhizosphere microorganisms.

Climate influences the gut eukaryome of wild rodents in the Great Rift Valley of Jordan

BackgroundThe mammalian gut microbiome includes a community of eukaryotes with significant taxonomic and functional diversity termed the eukaryome. The molecular analysis of eukaryotic diversity in microbiomes of wild mammals is still in its early stages due to the recent emergence of interest in this field. This study aimed to fill this knowledge gap by collecting data on eukaryotic species found in the intestines of wild rodents. Because little is known about the influence of climate on the gut eukaryome, we compared the composition of the gut eukaryotes in two rodent species, Mus musculus domesticus and Acomys cahirinus, which inhabit a transect crossing a temperate and tropical zone on the Jordanian side of the Great Rift Valley (GRV).MethodsWe used high-throughput amplicon sequencing targeting the 18S rRNA gene in fecal samples from rodents to identify eukaryotic organisms, their relative abundance, and their potential for pathogenicity.ResultsNematodes and protozoa were the most prevalent species in the eukaryome communities, whereas fungi made up 6.5% of the total. Sixty percent of the eukaryotic ASVs belonged to taxa that included known pathogens. Eighty percent of the rodents were infected with pinworms, specifically Syphacia obvelata. Eukaryotic species diversity differed significantly between bioclimatic zones (p = 0.001). Nippostrongylus brasiliensis and Aspiculuris tetraptera were found to be present exclusively in the Sudanian zone rodents. This area has not reported any cases of Trichuris infections. Yet, Capillaria infestations were unique to the Mediterranean region, while Trichuris vulpis infestations were also prevalent in the Mediterranean and Irano-Turanian regions.ConclusionsThis study highlights the importance of considering host species diversity and environmental factors when studying eukaryome composition in wild mammals. These data will be valuable as a reference to eukaryome study.Graphical

Microbial assessment of the ecological linkage between a red tide of Karenia brevis and bottom water anoxia off the coast of Fort Myers and Sanibel Island, Florida

Coastal ecosystem management necessitates a comprehensive understanding of the physical, chemical, and biological components and their interplay with socio-economic activities. Red tides of Karenia brevis reoccur almost annually on the West Florida Shelf and cause significant human and environmental health problems that often result in negative economic impacts. It is crucial that we understand the processes and dynamics of both phytoplankton and microbial communities as conditions necessary for the formation of hypoxia and anoxia in the coastal ecosystem. With the aim of identifying key microbial communities responsible for anoxia, we investigated the 2017–2018 Karenia bloom. Various water quality parameters, phytoplankton, and microbial community compositions were studied at eight sites on the West Florida Shelf. K. brevis densities were highest at the site of the plume of the Caloosahatchee River. At the site with the highest K. brevis abundance (6.3 ×106 cells/L), dissolved oxygen (DO) was supersaturated (303 %) at the surface, and the chlorophyll a concentration was 118 µg/L. The highest bacterial cell count (9.0 ×106 cells/mL) and the lowest bacterial diversity were also observed at this site. The bacterial community determined by high-throughput amplicon sequencing at this bloom site was unique and dominated by Bacteroidetes, which occupied 65 % of the relative abundance of the microbial community. The bottom water was generally hypoxic (mean ± SD: 0.98 ± 1.13 mg/L), with select stations having DO as low as 0.06 mg/L (anoxic). The anoxic bottom water harbored unique microbial taxa that were associated with the low oxygen conditions. Significant negative correlations were found between DO and the relative abundance of Deltaproteobacteria, Firmicutes, Spirochaetes, and unclassified Proteobacteria. Our investigation lays the groundwork for subsequent studies delving into the responses of phytoplankton and microbial communities to disturbances caused by Karenia blooms, as well as future strategies for mitigation and ecosystem restoration.

Pyrene biodegradation by bacterial consortia enriched from coastal and marine sediments: Insights into bacterial community structures and functions

As a representative polycyclic aromatic hydrocarbon (PAH), pyrene poses a significant threat to the environment and public health due to its persistence and potential toxicity. Extensive research has been conducted on biodegradation of pyrene, highlighting the crucial need to unravel its degradation mechanisms. Coastal and marine sediments are promising sources for discovering microbial candidates capable of degrading pyrene. In this study, three bacterial consortia, designated as BS, YS, and SS, were enriched from sediments collected from the Bohai Sea, Yellow Sea, and Liao River estuarine wetland, respectively. The bacterial consortia BS, YS, and SS demonstrated high pyrene degradation efficiencies, achieving 86.61 %, 80.39 %, and 75.70 % degradation at the concentration of 50 mg/L within 8 days, respectively. High-throughput sequencing of 16S rRNA gene amplicons revealed Pandoraea, Delftia, and Pseudomonas as the dominant populations in bacterial consortia BS, YS, and SS, respectively, with some species potentially being novel pyrene degraders. Further analysis included identifying pyrene degradation metabolites by LC-Q-TOF-MS (high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry) and predicting functional genes through PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved Stats). Based on these results, the metabolic pathways for pyrene degradation by the bacterial consortia were proposed, which predominantly involved the dioxygenation pathway. Overall, these findings should offer valuable bacterial resources for facilitating the bioremediation of PAH-contaminated environments.

Analysis of nasopharyngeal microbiota revealing microbial disturbance associated with ovine respiratory complex

The ovine respiratory complex (ORC) is a complex respiratory disease process in ovine causing a significant health concern with substantial morbidity and mortality. It poses a significant threat of impacting animal health, leading to severe health consequences and considerable economic loss. Research on the upper respiratory tract (URT) microbiota is critical for offering insights into pathogenesis, prevention, and treatment strategies of ORC. The goal of this study is to compare the nasopharyngeal microbiota of clinically healthy Hu sheep (Group H) and sheep with ORC (Group P). The 16S rDNA gene amplicon high-throughput sequencing technique was applied to identify the microbial composition in the nasopharyngeal samples. The results showed that there was no significant difference in the microbiota diversity and richness between ORC and healthy sheep. However, there were significant differences in microbial composition, such as the relative abundance of Enterobacteriaceae, Moraxellaceae, Pasteurellaceae, and Streptococcaceae between the two groups. The abundance of aerobes in sheep with ORC increased significantly, while the abundance of anaerobes and facultative anaerobes decreased significantly. There were also differences in the taxa phenotypes associated with biofilm forming, mobile element-containing, oxidative stress-tolerance, and potential pathogens between the two groups. Our study showed the nasopharyngeal microbiota composition and its associated shifts between clinically healthy sheep and ORC sheep in China. These findings suggest that shifts in the nasopharyngeal microbiota could be a contributing factor to the pathogenesis of ORC, offering a potential avenue for the development of targeted interventions and treatments for this condition in sheep.

Integrative analysis of microbiome and metabolome revealed the effect of microbial inoculant on microbial community diversity and function in rhizospheric soil under tobacco monoculture.

Over-application of chemical fertilizers and continuous cropping obstacles seriously restrict the sustainable development of tobacco production. Localized fertilization of beneficial microbes has potential advantages in achieving higher productivity, but the underlying biological mechanisms of interactions between rhizospheric microorganisms and the related metabolic cycle remain poorly characterized. Here, an integrative analysis of microbiomes with non-targeted metabolomics was performed on 30 soil samples of rhizosphere, root surrounding, and bulk soils from flue-cured tobacco under continuous and non-continuous monocropping systems. The analysis was conducted using UPLC-MS/MS platforms and high-throughput amplicon sequencing targeting the bacterial 16S rRNA gene and fungal ITS gene. The microbial inoculant consisted of Bacillus subtilis, B. velezensis, and B. licheniformis at the ratio of 1:1:1 in effective microbial counts, improved the cured leaf yield and disease resistance of tobacco, and enhanced nicotine and nitrogen contents of tobacco leaves. The bacterial taxa Rhizobium, Pseudomonas, Sphingomonadaceae, and Burkholderiaceae of the phylum Proteobacteria accumulated in high relative abundance and were identified as biomarkers following the application of the microbial inoculant. Under continuous monocropping, metabolomics demonstrated that the application of the microbial inoculant significantly affected the soil metabolite spectrum, and the differential metabolites were significantly enriched to the synthesis and degradation of nicotine (nicotinate and nicotinamide metabolism and biosynthesis of alkaloids derived from nicotinic acid). In addition, microbes were closely related to the accumulation of metabolites through correlation analysis. The interactions between plant roots and rhizospheric microorganisms provide valuable information for understanding how these beneficial microbes affect complex biological processes and the adaption capacity of plants to environments.IMPORTANCEThis study elaborated on how the microbial fertilizer significantly changed overall community structures and metabolite spectrum of rhizospheric microbes, which provide insights into the process of rhizosphere microbial remolding in response to continuous monocropping. we verified the hypothesis that the application of the microbial inoculant in continuous cropping would lead to the selection of distinct microbiota communities by establishing models to correlate biomarkers. Through correlation analysis of the microbiome and metabolome, we proved that rhizospheric microbes were closely related to the accumulation of metabolites, including the synthesis and degradation of nicotine. The interactions between plant roots and rhizospheric microorganisms provide valuable information for understanding how these beneficial microbes affect complex biological processes and the adaption capacity of plants to environments.

Individuality and generality of intratumoral microbiome in the three most prevalent gynecological malignancies: an observational study.

Growing evidence have indicated the crucial role of intratumor microbiome in a variety of solid tumor. However, the intratumoral microbiome in gynecological malignancies is largely unknown. In the present study, a total of 90 Han patients, including 30 patients with cancer in cervix, ovary, and endometrium each were enrolled, the composition of intratumoral microbiome was assessed by 16S rDNA amplicon high throughput sequencing. We found that the diversity and metabolic potential of intratumoral microbiome in all three cancer types were very similar. Furthermore, all three cancer types shared a few taxa that collectively take up high relative abundance and positive rate, including Pseudomonas sp., Comamonadaceae gen. sp., Bradyrhizobium sp., Saccharomonospora sp., Cutibacterium acnes, Rubrobacter sp., Dialister micraerophilus, and Escherichia coli. Additionally, Haemophilus parainfluenzae and Paracoccus sp. in cervical cancer, Pelomonas sp. in ovarian cancer, and Enterococcus faecalis in endometrial cancer were identified by LDA to be a representative bacterial strain. In addition, in cervical cancer patients, alpha-fetoprotein (AFP) (correlation coefficient = -0.3714) was negatively correlated (r = 0.4, 95% CI: 0.03 to 0.7) with Rubrobacter sp. and CA199 (correlation coefficient = 0.3955) was positively associated (r = 0.4, 95% CI: 0.03 to 0.7) with Saccharomonospora sp.. In ovarian cancer patients, CA125 (correlation coefficient = -0.4451) was negatively correlated (r = -0.4, 95% CI: -0.7 to -0.09) with Porphyromonas sp.. In endometrial cancer patients, CEA (correlation coefficient = -0.3868) was negatively correlated (r = -0.4, 95% CI: -0.7 to -0.02) with Cutibacterium acnes. This study promoted our understanding of the intratumoral microbiome in gynecological malignancies.IMPORTANCEIn this study, we found the compositional spectrum of tumor microbes among gynecological malignancies were largely similar by sharing a few taxa and differentiated by substantial species owned uniquely. Certain species, mostly unreported, were identified to be associated with clinical characteristics. This study prompted our understanding of gynecological malignancies and offered evidence for tumor microbes affecting tumor biology among cancers in the female reproductive system.

Effects of Mint Oils on the Human Oral Microbiome: A Pilot Study.

The oral microbiome is a diverse and complex ecosystem essential for maintaining oral and systemic health. Our study is the first to define the oral microbial community in Egyptian young adults and investigate the effects of natural antimicrobials on the oral microbiome. SuperMint (SM) is a proprietary blend of peppermint, Japanese mint, bergamot mint, and spearmint essential oils encapsulated in a tiny soft beadlet. This work aimed to evaluate the effects of SM beadlets on the oral microbiome. This study recruited twenty healthy participants. A baseline investigation of the oral microbiome of the selected participants was performed by collecting saliva and swab samples before treatment. Treatment included chewing four SM beadlets twice a day for 7 days, and then, post-administration saliva and swab samples were collected at the end of treatment. The oral microbiome samples were analyzed by the high-throughput amplicon sequencing of 16S rRNA gene fragments, and the community composition was determined. The results showed that the abundance of some microbial genera and families decreased after using SM, including Prevotella, Streptococcus, Neisseria, and Haemophilus. However, some genera showed inconsistent patterns. We also found that the subject's gender and SM usage were significantly associated with diverse microbial composition. The results suggest that SM treatment decreased the abundance of several bacteria associated with halitosis and periodontal diseases, such as Actinomyces and Streptococcus. Furthermore, Corynebacterium species increased and Streptococcus decreased after SM usage. More research is needed to fully understand the antimicrobial effects of mint oils and their potential applications in maintaining good oral health.

Bacterial genera associated with nitrogen cycle in microbial communities of chernozems

BACKGROUND: Reactions associated with the geochemical nitrogen cycle occurring in the soil have a decisive effect on the level of nitrogen availability for green plants. Microorganisms that catalyze them are of interest from the point of view of developing mechanisms for managing soil fertility. In this paper, we focused on bacterial genera associated with nitrogen cycle processes in chernozem soils of the Belgorod Region of Russia. AIM: To determine the list and quantitative ratios of bacterial genera whose representatives are able to participate in the fixation of atmospheric nitrogen, nitrification and denitrification in the chernozems of the forest-steppe zone of the European part of Russia. MATERIALS AND METHODS: Samples of arable and non-arable chernozems of three subtypes were collected in June and August 2022. Microbiological profiling based on high-throughput sequencing of amplicons of V3 and V4 region of the 16S rRNA gene with subsequent computational processing of the results was used in the work. RESULTS: 6 genera of nitrogen-fixing bacteria were found, as well as a group undifferentiated by means of the method used, including genus Rhizobium. 8 genera involved in the first and 5 genera involved in the second stage of nitrification were also found. 7 genera have been found whose representatives are capable of denitrification, without taking into account nitrogen-fixing bacteria, which are also capable of carrying out this process. CONCLUSIONS: Among the bacteria capable of fixing atmospheric nitrogen in the chernozems of the Belgorod region, the genus Bradyrhizobium dominates. Among the nitrifiers performing the first stage of nitrification, Ellin6067 and MND1 from the family Nitrosomonadaceae predominate. The second stage of nitrification is carried out mainly by bacteria of the genus Nitrospira. Of the potential denitrifies, along with nitrogen-fixing genera possessing this ability, representatives of the genera Rubrobacter and Pseudomonas are the most numerous. The study does not claim to give a complete list of organisms that carry out key chemical transformations of nitrogen compounds in chernozems, however, it allows us to name their important participants.

16S rRNA amplicon sequencing and antimicrobial resistance profile of intensive care units environment in 41 Brazilian hospitals.

Infections acquired during healthcare setting stay pose significant public health threats. These infections are known as Healthcare-Associated Infections (HAI), mostly caused by pathogenic bacteria, which exhibit a wide range of antimicrobial resistance. Currently, there is no knowledge about the global cleaning process of hospitals and the bacterial diversity found in ICUs of Brazilian hospitals contributing to HAI. Characterize the microbiome and common antimicrobial resistance genes present in high-touch Intensive Care Unit (ICU) surfaces, and to identify the potential contamination of the sanitizers/processes used to clean hospital surfaces. In this national, multicenter, observational, and prospective cohort, bacterial profiles and several antimicrobial resistance genes from 41 hospitals across 16 Brazilian states were evaluated. Using high-throughput 16S rRNA amplicon sequencing and real-time PCR, the bacterial abundance and resistance genes presence were analyzed in both ICU environments and cleaning products. We identified a wide diversity of microbial populations with a recurring presence of HAI-related bacteria among most of the hospitals. The median bacterial positivity rate in surface samples was high (88.24%), varying from 21.62 to 100% in different hospitals. Hospitals with the highest bacterial load in samples were also the ones with highest HAI-related abundances. Streptococcus spp., Corynebacterium spp., Staphylococcus spp., Bacillus spp., Acinetobacter spp., and bacteria from the Flavobacteriaceae family were the microorganisms most found across all hospitals. Despite each hospital particularities in bacterial composition, clustering profiles were found for surfaces and locations in the ICU. Antimicrobial resistance genes mecA, bla KPC-like, bla NDM-like, and bla OXA-23-like were the most frequently detected in surface samples. A wide variety of sanitizers were collected, with 19 different active principles in-use, and 21% of the solutions collected showed viable bacterial growth with antimicrobial resistance genes detected. This study demonstrated a diverse and spread pattern of bacteria and antimicrobial resistance genes covering a large part of the national territory in ICU surface samples and in sanitizers solutions. This data should contribute to the adoption of surveillance programs to improve HAI control strategies and demonstrate that large-scale epidemiology studies must be performed to further understand the implications of bacterial contamination in hospital surfaces and sanitizer solutions.

Halophilomyces hongkongensis, a Novel Species and Genus in the Lulworthiaceae with Antibacterial Potential, Colonizing the Roots and Rhizomes of the Seagrass Halophila ovalis.

Seagrass serves as a quintessential reservoir for obligate marine Lulworthiaceae fungi. Our current knowledge of the mycological diversity associated with seagrass in Hong Kong remains poor. We analyzed the diversity of fungi associated with the most widely distributed seagrass species in Hong Kong Halophila ovalis (Hydrocharitaceae), using a combination of culture-based methods and high-throughput amplicon sequencing. Halophilomyces hongkongensis, a novel fungal species in a newly proposed genus within the Lulworthiaceae family, was isolated from H. ovalis roots and rhizomes. The novel fungus showed distinct morphological characteristics, while both combined 18S-28S and internal transcribed spacer (ITS) phylogenetic trees based on maximum likelihood and Bayesian methods supported its discrimination from other existing Lulworthiaceae members. The ITS2 region in the Illumina sequencing results of multiple H. ovalis compartments, water, and adjacent non-seagrass sediments revealed continuous recruitment of H. hongkongensis by H. ovalis throughout the year despite dramatically fluctuating environmental conditions, with remarkably high proportions of this taxon found in root and rhizome internal tissues, possibly indicating a strong and specialized relationship established between the Lulworthiaceae fungal partner and its seagrass host. The inhibitory abilities exhibited by H. hongkongensis against Staphylococcus aureus SA29213 and ATCC 43300 (methicillin-resistant) may imply its capacity in producing (novel) antibacterial compounds. The discovery of H. hongkongensis as the first novel Lulworthiaceae taxon in Hong Kong, along with its distributional pattern in the seagrass meadow, provides valuable insights into the systematics and ecology of this strictly marine fungal family.

Temporal changes in the morphological and microbial diversity of biofilms on the surface of a submerged stone in the Danube River.

Epilithic biofilms are ubiquitous in large river environments and are crucial for biogeochemical processes, but their community structures and functions remain poorly understood. In this paper, the seasonal succession in the morphological structure and the taxonomic composition of an epilithic bacterial biofilm community at a polluted site of the Danube River were followed using electron microscopy, high-throughput 16S rRNA gene amplicon sequencing and multiplex/taxon-specific PCRs. The biofilm samples were collected from the same submerged stone and carried out bimonthly in the littoral zone of the Danube River, downstream of a large urban area. Scanning electron microscopy showed that the biofilm was composed of diatoms and a variety of bacteria with different morphologies. Based on amplicon sequencing, the bacterial communities were dominated by the phyla Pseudomonadota and Bacteroidota, while the most abundant archaea belonged to the phyla Nitrososphaerota and Nanoarchaeota. The changing environmental factors had an effect on the composition of the epilithic microbial community. Critical levels of faecal pollution in the water were associated with increased relative abundance of Sphaerotilus, a typical indicator of "sewage fungus", but the composition and diversity of the epilithic biofilms were also influenced by several other environmental factors such as temperature, water discharge and total suspended solids (TSS). The specific PCRs showed opportunistic pathogenic bacteria (e.g. Pseudomonas spp., Legionella spp., P. aeruginosa, L. pneumophila, Stenotrophomonas maltophilia) in some biofilm samples, but extended spectrum β-lactamase (ESBL) genes and macrolide resistance genes could not be detected.

Simultaneous Biogas Upgrading and Valuable Chemical Production Using Homoacetogens in a Membrane Biofilm Reactor.

Biogas produced from anaerobic digestion usually contains impurities, particularly with a high content of CO2 (15-60%), thus decreasing its caloric value and limiting its application as an energy source. H2-driven biogas upgrading using homoacetogens is a promising approach for upgrading biogas to biomethane and converting CO2 to acetate simultaneously. Herein, we developed a novel membrane biofilm reactor (MBfR) with H2 and biogas separately supplied via bubbleless hollow fiber membranes. The gas-permeable hollow fibers of the MBfR enabled high H2 and CO2 utilization efficiencies (∼98% and ∼97%, respectively) and achieved concurrent biomethane (∼94%) and acetate (∼450 mg/L/d) production. High-throughput 16S rRNA gene amplicon sequencing suggested that enriched microbial communities were dominated by Acetobacterium (38-48% relative abundance). In addition, reverse transcription quantitative PCR of the functional marker gene formyltetrahydrofolate synthetase showed that its expression level increased with increasing H2 and CO2 utilization efficiencies. These results indicate that Acetobacterium plays a key role in CO2 to acetate conversion. These findings are expected to facilitate energy-positive wastewater treatment and contribute to the development of a new solution to biogas upgrading.

Biogeography and community assembly of soil fungi from alpine meadows in southwestern China show the importance of climatic selection

Soil fungi are pivotal in alpine and arctic ecosystems that are vulnerable to climate changes. Previous studies have shown broad connections between soil fungi in the arctic and alpine regions, but most of these studies are mainly from Europe and North America, with more sporadic studies from East Asia. Currently, little is known about the biogeographic relationships between soil fungi in alpine meadows of southwestern China (AMSC) and other regions of the world. In addition, the regional-scale spatial patterns of fungal communities in the AMSC, as well as their driving factors and ecological processes, are also poorly understood. In this study, we collected roots and surrounding soils of two dominant ectomycorrhizal plants, Bistorta vivipara and B. macrophylla from the AMSC, and performed bioinformatic and statistical analyses based on high-throughput sequencing of ITS2 amplicons. We found that: (1) fungi from the AMSC were closely related with those from boreal forests and tundra, and saprotrophic fungi had higher dispersal potential than ectomycorrhizal fungi; (2) community compositions exhibited clear divergences among geographic regions and between root and soil samples; (3) climate was the predominant factor driving regional-scale spatial patterns but had less explanatory power for saprotrophic and total fungi from roots than those from soils; (4) homogeneous selection and drift were the key ecological processes governing community assembly, but in communities of saprotrophic and total fungi from soil samples, drift contributed less and its role was partially replaced by dispersal limitation. This study highlights the importance of climatic selection and stochastic processes on fungal community assembly in alpine regions, and emphasizes the significance of simultaneously investigating fungi with different trophic modes and from both roots and soils.

Mutation-Selected Amplification droplet digital PCR: A new single nucleotide variant detection assay for [formula omitted] mutant in tumor and plasma samples

The early detection of gene mutations in physiological and pathological processes is a powerful approach to guide decisions in precision medicine. However, detecting low-copy mutant DNA from clinical samples poses a challenge due to the enrichment of wild-type DNA backgrounds. In this study, we devised a novel strategy, named Mutation-Selected Amplification droplet digital PCR (MSA-ddPCR), to quantitatively analyze single nucleotide variants (SNVs) at low variant allele frequencies (VAFs). Using TP53R249S (a hotspot mutation associated with hepatocellular carcinoma) as a model, we optimized the concentration ratio of primers, the annealing temperature and nucleic acid amplification modifiers. Subsequently, we evaluated the linear range and precision of MSA-ddPCR by detecting TP53R249S and TP53wild-type (TP53WT) plasmid DNA, respectively. MSA-ddPCR demonstrated superior ability to discriminate between mutant DNA and wild-type DNA compared to traditional TaqMan-MGB PCR. We further applied MSA-ddPCR to analyze the TP53R249S mutation in 20 plasma samples and 15 formalin-fixed paraffin-embedded (FFPE) tissue samples, and assessed the agreement rates between MSA-ddPCR and amplicon high-throughput sequencing. The results showed that the limit of blanks of MSA-ddPCR are 0.449 copies μL−1 in the FAM channel and 0.452 copies μL−1 in the VIC channel. MSA-ddPCR could accurately quantify VAFs as low as 0.01 %, surpassing existing PCR and next-generation sequencing (NGS) methods. In the detection of clinical samples, a high correlation was found between MSA-ddPCR and amplicon high-throughput sequencing. Additionally, MSA-ddPCR outperformed sequencing methods in terms of detection time and simplicity of data analysis. MSA-ddPCR can be easily implemented into clinical practice and serve as a robust tool for detecting mutant genes due to its high sensitivity and accuracy.