16S rRNA Gene Libraries Research Articles

Clinical and historical infection of Tacheng tick virus 2: A retrospective investigation.

Tacheng tick virus 2 (TcTV-2) is an emerging tick-borne virus belonging to the genus Uukuvirus in the family Phenuiviridae. Initially isolated in 2019 from a patient in Xinjiang Uygur Autonomous Region (XUAR), northwestern China, who developed fever and headache after a tick bite, TcTV-2 was concurrently molecularly detected in hard ticks across various countries, including China, Kazakhstan, Romania, and Turkey. This study conducted a retrospective epidemiological investigation of TcTV-2 infection. In this retrospective cohort study, we collected samples from 47 tick-bitten patients, 984 herdsmen, 7 Asian badgers, 13 red foxes, and 168 Hyalomma asiaticum tick egg batches. Patients' samples were primarily analyzed by using high-throughput sequencing, targeting the V3-V4 region of the bacterial 16S rRNA gene and viral cDNA libraries. Typical tick-borne pathogens were further confirmed using RT-PCR and detected in Asian badgers, red foxes and Hy. asiaticum tick egg batches. We also conducted enzyme-linked immunosorbent assay (ELISA) to detected specific IgM and IgG antibodies against TcTV-2 in herdsmen. Phylogenetic analysis was performed to genetically characterize TcTV-2 detected in this study. TcTV-2 was detected in various samples, including blood, urine, and throat swabs from 12.77% (6/47) tick-bitten patients. It was found in blood samples of 14.29% (1/7) of wild badgers, 7.69% (1/13) of red foxes, and 13.69% (23/168) of Hy. asiaticum egg batches. Furthermore, ELISA results revealed that 9.55% (94/984) of the serum samples (34 from males and 60 from females) were tested positive for TcTV-2-specific IgG, while 2.95% (29/984, 7 males and 22 females) showed positivity for TcTV-2-specific IgM. Additionally, 1.02% (10/984, 4 males and 6 females) of the sera tested positive for both TcTV-2-specific IgM and IgG. Phylogenetic analysis indicated that the TcTV-2 strains detected in this study were genetically similar, regardless of their origin and host species. Clinical symptoms of TcTV-2 infection in patients are nonspecific, with common symptoms including headache, fever, asthenia, vomiting, myalgia, rash, and meningitis-like signs. TcTV-2 can be detected in blood, urine, and throat swab samples of infected patients. Among local herdsmen, 9.55% tested positive for TcTV-2-specific IgG and 2.95% for TcTV-2-specific IgM. Importantly, TcTV-2 can be transovarially transmitted in Hy. asiaticum ticks, and the Asian badgers and red foxes are potential reservoirs of TcTV-2.

72 Effect of In-feed fecal microbiota transplant on growth performance and gut microbiota dynamics of piglet subjected to post-weaning stress

Abstract Weaning is one of the most stressful events for piglets, due to diet change from sow milk to grain-based feed. This diet change causes turnover of the gut microbial which can allow proliferation of pathogens leading to diarrhea. Antibiotics are administered to treat or prevent post-weaning diarrhea (PWD). Increased occurrence of antimicrobial resistance has increased incentives to develop viable antibiotic alternatives. PWD is most often caused by enterotoxigenic Escherichia coli. Similarly, in humans, severe diarrhea can be caused by Clostridium difficile colonization. Use of fecal microbiota transplants (FMT) has been shown to resolve C. difficile-induced diarrhea in 80 to 90% of human cases which may suggest FMT as a potential therapeutic option for post weaning diarrhea. We previously have shown that in feed fecal microbiota transplant (FMT) improved body weight (BW) gain in newly weaned piglets but it is not clear if this benefit will be replicated in piglets subjected to weaning stress. We hypothesize that FMT (in-feed) will increase colonization efficiency, improve growth performance and reduce diarrhea incidence compared with control in piglets subjected to post weaning stress. The objective of this study was to determine the effect of In-feed FMT of diarrhea index, growth performance and gut microbiota dynamics. Piglets (n = 240) subjected to physiological and transport weaning stress were allotted to 5 treatments: In-feed FMT (FMT1), doubles dose of In-feed FMT1 (FMT2), Oral gavage (FMT3), Antibiotic (positive control), and no supplementation (negative control). The donor material was screened for enterotoxigenic E. coli (F18 and F4). FMT was carried out for 14 d. Diarrhea index was observed for the first 14 d and animal BW, and fecal microbiota was collected by rectal swab on d 0, 3, 5, 7, 14, 21, 28 and 32 for analysis of microbiota composition. DNA was extracted from fecal samples and 16S rRNA gene libraries (V4 region) were prepared for bacterial community analysis. Data were analyzed with one-way ANOVA in R. Only positive control had significantly decrease in diarrhea index and significant increase BW gain on d 14, 21, and 28 (P < 0.05) but there was no effect of FMT (P > 0.05). In conclusion, antibiotics but not FMT was efficient in alleviating the negative performance effect in piglets subjected to post-weaning stress.

Diversity of endophytic bacterial community in rice roots and their roles in phosphate solubilization and plant growth

Root endophytic bacteria have significant beneficial effects on plant growth and health, while specific coexisting pathogenic bacteria can cause various diseases. In this study, we assessed the diversity and cultivability of the endophytic bacterial community in rice roots with a focus on their capacity for phosphate solubilization and impact on plant growth. Employing PacBio Single Molecule Real-Time (SMRT) and Sanger sequencing, we generated 16S rRNA gene libraries using metagenomic DNA extracted from the roots of three field-grown rice plants, along with genomic DNA from 156 associated bacterial isolates. QIIME 2-based microbiome analysis revealed dominant classified species at diverse taxonomic levels, including Arcobacter marinus, Paraburkholderia kururiensis, Lactobacillus plantarum, and Lactobacillus brevis. Furthermore, we effectively isolated 25.97% of rice root endophytic bacteria, with a notable 33.33% of pure culture endophytic bacteria exhibiting phosphate solubilization abilities. Finally, we revealed the inhibitory role of Burkholderia vietnamiensis and the growth promoting role of Paraburkholderia kururiensis subsp. Kururiensis in both the monocot model Brachypodium distachyon and the dicot Arabidopsis thaliana. Our study provided novel insight into the microbiome structure in rice roots and its potential to benefit rice cultivation and enhance overall crop health.

Diversity of microbial communities in long-term ice-covered water bodies of the Cold Pole region, Yakutia

Water bodies located in the north-east of Yakutia during the period of ice cover were studied. High-throughput sequencing of 16S rRNA gene libraries revealed a high taxonomic diversity of microbial communities in the studied ecotopes. The majority of the under-ice communities was represented by Proteobacteria, Actinobacteriota, Cyanobacteria, Verrucomicrobiota, Bacteroidota, and Planctomycetota. The difference in community diversity between deep-water lakes Labynkyr, Vodorazdelnoye, and Ulu, the source of the Labynkyr River, and shallow-water lakes Myamichi and Mertvoye, the mouth of the Labynkyr River, is shown.

277 Effect of Mode of Delivery of Fecal Microbiota Transplants on Growth Performance and Gut Microbiome in Weaning Piglets

Abstract Weaning is one of the most stressful events for piglets, due to diet change from sow milk to grain-based feed. This diet change causes turnover of the gut microbial which can allow proliferation of pathogens leading to diarrhea. Antibiotics are administered to treat or prevent post-weaning diarrhea (PWD). Increased occurrence of antimicrobial resistance has increased incentives to develop viable antibiotic alternatives. Previous studies have shown that fecal microbiota transplant (FMT) reduced diarrhea incidence and improved body weight gain in piglets. However, one barrier to FMT adoption in swine production is that FMT are generally given via oral gavage, which is difficult and labor intensive for large swine operations. For FMT to be a viable option in swine management, a simple method is needed to administer the transplant. The objective of this study was to determine the effect of three methods of FMT administration (oral, rectal and lyophilized in-feed) on growth performance, diarrhea incidence and gut microbiome. We hypothesize that FMT (in-feed and rectal) will increase colonization efficiency, improve growth performance and reduce diarrhea incidence compared with control. Weaned male piglets (n = 40) were allotted to 4 treatments at 10 piglets per treatment (no FMT - Control, Oral gavage - FMT1, rectal gavage - FMT2 and lyophilized in-feed – FMT3). Feces from 12-week-old pigs, screened for pathogens, was donor material. The study lasted nine days and FMT was for five days. Diarrhea incidences and body weight were observed for the first week. Fecal samples were collected on days 0, 2, 5 and 7. Five piglets per group were sacrificed on day 4 and the remaining on day 8. Cecal and colon contents were collected from the sacrificed piglets. DNA was extracted from fecal and digesta samples and 16S rRNA gene libraries (V4 region) were prepared for bacterial community analysis. Data were analyzed with one-way ANOVA in R. All FMT groups had significantly greater body weight on day 3 (P < 0.05) and average daily gain between day 0 – 3 (P < 0.05) compared with control but there was no difference between FMT groups. There was no effect of FMT on average daily feed intake and diarrhea incidence. Only FMT3 had greater alpha diversity (Observed features and Faith’s phylogenetic diversity) on day 5 (P < 0.05) compared with control, but there was no difference in beta diversity between FMT groups. There was decreased abundance of 27 ASVs (amplicon sequence variant) and increased abundance of 30 ASVs in increased in FMT3 compared with control on day 5. In conclusion, the three FMT groups has similar colonization pattern but there was no difference in diarrhea incidence. FMT3 (lyophilized in-feed) altered overall community structure of piglets and modulated individual taxa.

Optimization of high-throughput 16S rRNA gene amplicon sequencing: an assessment of PCR pooling, mastermix use and contamination.

16S rRNA gene sequencing is widely used to characterize human and environmental microbiomes. Sequencing at scale facilitates better powered studies but is limited by cost and time. We identified two areas in our 16S rRNA gene library preparation protocol where modifications could provide efficiency gains, including (1) pooling of multiple PCR amplifications per sample to reduce PCR drift and (2) manual preparation of mastermix to reduce liquid handling. Using nasal samples from healthy human participants and a serially diluted mock microbial community, we compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. One hundred and fifty-eight 16S rRNA gene sequencing libraries were prepared, including a replicate experiment. Comparing PCR pooling strategies, we found no significant difference in high-quality read counts and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate on principal coordinate analysis (PCoA) and non-metric dimensional scaling (NMDS) analysis. Choice of mastermix had no significant impact on high-quality read and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate in PCoA and NMDS analysis. Importantly, we observed contamination and variability of rare species (<0.01 %) across replicate experiments; the majority of contaminants were accounted for by removal of species present at <0.1 %, or were linked to reagents (including a primer stock). We demonstrate no requirement for pooling of PCR amplifications or manual preparation of PCR mastermix, resulting in a more efficient 16S rRNA gene PCR protocol.

Biological methane production and accumulation under sulfate-rich conditions at Cape Lookout Bight, NC.

Anaerobic oxidation of methane (AOM) is hypothesized to occur through reverse hydrogenotrophic methanogenesis in marine sediments because sulfate reducers pull hydrogen concentrations so low that reverse hydrogenotrophic methanogenesis is exergonic. If true, hydrogenotrophic methanogenesis can theoretically co-occur with sulfate reduction if the organic matter is so labile that fermenters produce more hydrogen than sulfate reducers can consume, causing hydrogen concentrations to rise. Finding accumulation of biologically-produced methane in sulfate-containing organic-rich sediments would therefore support the theory that AOM occurs through reverse hydrogenotrophic methanogenesis since it would signal the absence of net AOM in the presence of sulfate. 16S rRNA gene libraries were compared to geochemistry and incubations in high depth-resolution sediment cores collected from organic-rich Cape Lookout Bight, North Carolina. We found that methane began to accumulate while sulfate is still abundant (6-8 mM). Methane-cycling archaea ANME-1, Methanosarciniales, and Methanomicrobiales also increased at these depths. Incubations showed that methane production in the upper 16 cm in sulfate-rich sediments was biotic since it could be inhibited by 2-bromoethanosulfonoic acid (BES). We conclude that methanogens mediate biological methane production in these organic-rich sediments at sulfate concentrations that inhibit methanogenesis in sediments with less labile organic matter, and that methane accumulation and growth of methanogens can occur under these conditions as well. Our data supports the theory that H2 concentrations, rather than the co-occurrence of sulfate and methane, control whether methanogenesis or AOM via reverse hydrogenotrophic methanogenesis occurs. We hypothesize that the high amount of labile organic matter at this site prevents AOM, allowing methane accumulation when sulfate is low but still present in mM concentrations.

Concatenated 16S rRNA sequence analysis improves bacterial taxonomy.

Background: Microscopic, biochemical, molecular, and computer-based approaches are extensively used to identify and classify bacterial populations. Advances in DNA sequencing and bioinformatics workflows have facilitated sophisticated genome-based methods for microbial taxonomy although sequencing of the 16S rRNA gene is widely employed to identify and classify bacterial communities as a cost-effective and single-gene approach. However, the 16S rRNA sequence-based species identification accuracy is limited because of the occurrence of multiple copies of the 16S rRNA gene and higher sequence identity between closely related species. The availability of the genomes of several bacterial species provided an opportunity to develop comprehensive species-specific 16S rRNA reference libraries. Methods: Sequences of the 16S rRNA genes were retrieved from the whole genomes available in the Genome databases. With defined criteria, four 16S rRNA gene copy variants were concatenated to develop a species-specific reference library. The sequence similarity search was performed with a web-based BLAST program, and MEGA software was used to construct the phylogenetic tree. Results: Using this approach, species-specific 16S rRNA gene libraries were developed for four closely related Streptococcusspecies ( S. gordonii, S. mitis, S. oralis, and S. pneumoniae). Sequence similarity and phylogenetic analysis using concatenated 16S rRNA copies yielded better resolution than single gene copy approaches. Conclusions: The approach is very effective in classifying genetically closely related bacterial species and may reduce misclassification of bacterial species and genome assemblies.

Bacterial Community Shifts during Polyp Bail-Out Induction in Pocillopora Corals.

Polyp bail-out constitutes both a stress response and an asexual reproductive strategy that potentially facilitates dispersal of some scleractinian corals, including several dominant reef-building taxa in the family Pocilloporidae. Recent studies have proposed that microorganisms may be involved in onset and progression of polyp bail-out. However, changes in the coral microbiome during polyp bail-out have not been investigated. In this study, we induced polyp bail-out in Pocillopora corals using hypersaline and hyperthermal methods. Bacterial community dynamics during bail-out induction were examined using the V5-V6 region of the 16S-rRNA gene. From 70 16S-rRNA gene libraries constructed from coral tissues, 1,980 OTUs were identified. Gammaproteobacteria and Alphaproteobacteria consistently constituted the dominant bacterial taxa in all coral tissue samples. Onset of polyp bail-out was characterized by increased relative abundance of Alphaproteobacteria and decreased abundance of Gammaproteobacteria in both induction experiments, with the shift being more prominent in response to elevated temperature than to elevated salinity. Four OTUs, affiliated with Thalassospira, Marisediminitalea, Rhodobacteraceae, and Myxococcales, showed concurrent abundance increases at the onset of polyp bail-out in both experiments, suggesting potential microbial causes of this coral stress response. IMPORTANCE Polyp bail-out represents both a stress response and an asexual reproductive strategy with significant implications for reshaping tropical coral reefs in response to global climate change. Although earlier studies have suggested that coral-associated microbiomes likely contribute to initiation of polyp bail-out in scleractinian corals, there have been no studies of coral microbiome shifts during polyp bail-out. In this study, we present the first investigation of changes in bacterial symbionts during two experiments in which polyp bail-out was induced by different environmental stressors. These results provide a background of coral microbiome dynamics during polyp bail-out development. Increases in abundance of Thalassospira, Marisediminitalea, Rhodobacteraceae, and Myxococcales that occurred in both experiments suggest that these bacteria are potential microbial causes of polyp bail-out, shedding light on the proximal triggering mechanism of this coral stress response.

The mature phyllosphere microbiome of grapevine is associated with resistance against Plasmopara viticola.

Phyllosphere microbiota represents a substantial but hardly explored reservoir for disease resistance mechanisms. The goal of our study was to understand the link between grapevine cultivars susceptibility to Plasmopara viticola, one of the most devastating leaf pathogens in viticulture, and the phyllosphere microbiota. Therefore, we analyzed a 16S rRNA gene library for the dominant phyllosphere bacterial phyla Alphaproteobacteria of seven Vitis genotypes at different developmental stages, i.e., flowering and harvesting, via amplicon sequencing. Young leaves had significantly higher Alphaproteobacterial richness and diversity without significant host-specificity. In contrast, the microbial communities of mature leaves were structurally distinct in accordance with P. viticola resistance levels. This statistically significant link between mature bacterial phyllosphere communities and resistant phenotypes was corroborated by beta diversity metrics and network analysis. Beyond direct host-driven effects via the provision of microhabitats, we found evidence that plants recruit for specific bacterial taxa that were likely playing a fundamental role in mediating microbe-microbe interactions and structuring clusters within mature communities. Our results on grape-microbiota interaction provide insights for targeted biocontrol and breeding strategies.

Biogeographic distribution, ecotype partitioning and controlling factors of Chloroflexi in the sediments of six hadal trenches of the Pacific Ocean

The hadal trenches are “hot spots” for mineralization of organic matter in the deep ocean. Chloroflexi are one of the most dominant and active taxa in trench sediments, serving as important drivers of carbon cycles in hadal trenches. However, current understanding on hadal Chloroflexi is largely restricted to individual trench. This study systematically analyzed the diversity, biogeographic distribution, ecotype partitioning as well as environmental drivers of Chloroflexi in the sediments of hadal trenches, by reanalyzing 16S rRNA gene libraries of 372 samples from 6 trenches around the Pacific Ocean. The results showed that Chloroflexi averagely account for 10.10 % and up to 59.95 % of total microbial communities in the trench sediments. Positive correlations between relative abundance of Chloroflexi and depths down the vertical sediment profiles were observed in all of the sediment cores analyzed, suggesting the increasing significance of Chloroflexi in deeper sediment layers. Overall, trench sediment Chloroflexi were mainly composed of the classes Dehalococcidia, Anaerolineae and JG30-KF-CM66, and four orders i.e. SAR202, Anaerolineales, norank JG30-KF-CM66 and S085, were identified as core taxa that were dominant and prevalent in the hadal trench sediments. A total of 22 subclusters were identified within these core orders, and distinct patterns of ecotype partitioning related with depths down the vertical sediment profiles were observed, suggesting the great diversification of metabolic potentials and environment preference of different Chloroflexi lineages. The spatial distribution of hadal Chloroflexi were found to be significantly related with multiple environmental factors, while depths down the vertical sediment profiles explained the highest proportion of variations. These results provide valuable information for further exploring the roles of Chloroflexi in biogeochemical cycle of the hadal zone, and lay the foundation for understanding the adaptive mechanisms and evolutionary characteristics of microorganisms in hadal trenches.

Concatenated 16S rRNA sequence analysis improves bacterial taxonomy

Background: Microscopic, biochemical, molecular, and computer-based approaches are extensively used to identify and classify bacterial populations. Advances in DNA sequencing and bioinformatics workflows have facilitated sophisticated genome-based methods for microbial taxonomy although sequencing of the 16S rRNA gene is widely employed to identify and classify bacterial communities as a cost-effective and single-gene approach. However, the 16S rRNA sequence-based species identification accuracy is limited because of the occurrence of multiple copies of the 16S rRNA gene and higher sequence identity between closely related species. The availability of the genomes of several bacterial species provided an opportunity to develop comprehensive species-specific 16S rRNA reference libraries. Methods: Sequences of the 16S rRNA genes were retrieved from the whole genomes available in the Genome databases. With defined criteria, four 16S rRNA gene copy variants were concatenated to develop a species-specific reference library. The sequence similarity search was performed with a web-based BLAST program, and MEGA software was used to construct the phylogenetic tree. Results: Using this approach, species-specific 16S rRNA gene libraries were developed for four closely related Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae). Sequence similarity and phylogenetic analysis using concatenated 16S rRNA copies yielded better resolution than single gene copy approaches. Conclusions: The approach is very effective in classifying genetically closely related bacterial species and may reduce misclassification of bacterial species and genome assemblies.

A Review of Biogenic Coalbed Methane Experimental Studies in China

Biogenic coalbed methane (CBM) is an important alternative energy that can help achieve carbon neutrality. Accordingly, its exploration and development have become a research hotspot in the field of fossil energy. In this review, the latest detection technologies for and experimental research on biogenic CBM in China in recent decades are summarized. The factors influencing the generation of biogenic CBM and the identification method of biogenic CBM are systematically analyzed. The technologies to detect biogas and the research methods to study microbial diversity are summarized. The literature shows that biogenic CBM is easily produced in the presence of highly abundant organic matter of low maturity, and the organic matter reaching a certain thickness can compensate for the limitation of biogenic CBM gas production due to the small abundance of organic matter to a certain extent. Biogenic CBM production could be increased in an environment with low salinity, medium alkalinity, and rich Fe2+ and Ni2+ sources. Furthermore, biogenic CBM can be identified by considering three aspects: (1) the presence of gas composition indicators; (2) the content of heavy hydrocarbon; and (3) variation in the abundance of biomarkers. In recent years, research methods to study the microbial community and diversity of CBM-producing environments in China have mainly included 16S rRNA gene library, fluorescence in situ hybridization, and high-throughput sequencing, and the dominant microorganisms have been determined in various basins in China. The results of numerous studies show that the dominant bacterial phyla are commonly Firmicutes and Proteobacteria, while the archaeal fraction mainly includes Methanoculleus, Methanobacterium, Methanocorpusculum, and Methanothrix. This review summarizes and discusses the advances in biogenic CBM production and the associated microbial community in order to promote further development of coal biotransformation and CO2 bio-utilization to meet energy demands under carbon neutrality.

Spatiotemporal variations, assembly processes, and co-occurrence patterns of particle-attached and free-living bacteria in a large drinking water reservoir in China.

Particle-attached (PA) and free-living (FL) bacterial communities are sensitive to pollutant concentrations and play an essential role in biogeochemical processes and water quality maintenance in aquatic ecosystems. However, the spatiotemporal variations, assembly processes, co-occurrence patterns, and environmental interactions of PA and FL bacteria in drinking water reservoirs remain as yet unexplored. To bridge this gap, we collected samples from 10 sites across four seasons in Lake Tianmu, a large drinking water reservoir in China. Analysis of 16S rRNA gene libraries demonstrated spatiotemporal variations in bacterial diversity and identified differences in bacterial community composition (BCC) between PA and FL lifestyles. Capacity for nitrogen respiration, nitrogen fixation, and nitrate denitrification was enriched in the PA lifestyle, while photosynthesis, methylotrophy, and methanol oxidation were enriched in the FL lifestyle. Deterministic processes, including interspecies interactions and environmental filtration, dominated the assembly of both PA and FL bacterial communities. The influence of environmental filtration on the FL community was stronger than that on the PA community, indicating that bacteria in the FL lifestyle were more sensitive to environmental variation. Co-occurrence patterns and keystone taxa differed between PA and FL lifestyles. The ecological functions of keystone taxa in the PA lifestyle were associated with the supply and recycling of nutrients, while those in FL were associated with the degradation of complex pollutants. PA communities were more stable than FL communities in the face of changing environmental conditions. Nutrients (e.g., TDN and NO3 -) and abiotic and biotic factors (e.g., WT and Chl-a) exerted positive and negative effects, respectively, on the co-occurrence networks of both lifestyles. These results improve our understanding of assembly processes, co-occurrence patterns, and environmental interactions within PA and FL communities in a drinking water reservoir.

Concatenated 16S rRNA sequence analysis improves bacterial taxonomy

Background: Microscopic, biochemical, molecular, and computer-based approaches are extensively used to identify and classify bacterial populations. Advances in DNA sequencing and bioinformatics workflows have facilitated sophisticated genome-based methods for microbial taxonomy although sequencing of the 16S rRNA gene is widely employed to identify and classify the bacterial community as a cost-effective and single-gene approach. However, the 16S rRNA sequence-based species identification accuracy is limited by multiple copies of the gene and their higher sequence identity between closely related species. The availability of a large volume of bacterial whole-genome data provided an opportunity to develop comprehensive species-specific 16S rRNA reference libraries. Methods: The 16S rRNA copies were retrieved from the whole genomes in the complete stage at the Genome database. With defined rules, four 16S rRNA gene copy variants were concatenated to develop a species-specific reference library. The sequence similarity search was performed with a web-based BLAST program, and MEGA software was used to construct the phylogenetic tree. Results: Using this approach, species-specific 16S rRNA gene libraries were developed for four closely related Streptococcus species (S. gordonii, S. mitis, S. oralis, and S. pneumoniae). Sequence similarity and phylogenetic analysis using concatenated 16S rRNA copies yielded better resolution than single gene copy approaches. Conclusions: The approach is very effective in classifying genetically related species and may reduce misclassification of bacterial species and genome assemblies.

Characterization of a Methanogenic Microbial Consortium from a Coal Mine in Bogotá Basin

The work studied the methanogenic microbial consortium in a coal mine from the Bogotá basin in Colombia. Ex situ coal-enrichment cultures were established for in vitro growth and de novo gas production. Biogenic gas produced by cultures was analyzed by gas chromatography using thermal conductivity and flame ionization detectors. Cultures were used to isolate microbial specimens and to generate 16S rRNA gene libraries employing bacterial and archaeal primer sets. The gas chromatographic analysis showed methane production at 37 oC, but not at 60 oC, where CO2 was the major component of the biogenic gas. 16S rRNA gene sequence analysis of microbial isolates and clone libraries established that the methanogenic microbial consortium was formed by bacteria species from Bacillus and Gracilibacter genera plus archaea from the Methanothermobacter genus. This methanogenic microbial consortium was potentially responsible for biogenic gas generation in La Ciscuda coal mine. The results suggested that these methanogens produced methane by hydrogenotrophic or CO2 reduction pathways.

Microbiome composition of disturbed soils from sandy-gravel mining complexes with different reclamation approaches

Activities connected to mineral mining disrupt the soil layer and bring parent rock material to the surface. It leads to altering the environmental conditions and leaves behind vast areas of disturbed lands. Returning these lands to natural ecosystems is an important contemporary challenge, which can be acquired by reclamation practices. Soil microbiome composition reflects changes happening to disturbed lands; thus, its analysis is a powerful tool for evaluating the disturbance degree and estimating the effect of the implementation of reclamation techniques. Additionally, factors connected to the characteristics of a particular geographical region have a certain impact on the microbiome and should be taken into account. Thereby, studies of soil microbiomes of disturbed soils of different origins are essential in understanding the dynamics of soil restoration. Here, we focus on soil microbiomes from two sandy-gravel mining complexes in mountainous areas with a moderate continental climate of the Central Caucasus. These quarries share the same parent rock material, but differ in benchmark soil type and reclamation approach - one was left for passive recovery and the other was technically reclaimed with overburden material. Comparative analysis of microbiome composition, based on sequencing of 16S rRNA gene libraries, showed that region and disturbance are the key factors explaining microbiome variation, which surpass the influence of local factors. However, the application of reclamation techniques greatly reduces the dissimilarity of soil microbiomes caused by disturbance. Linking of soil chemical parameters to microbiome composition showed that the disturbance factor correlates with a lack of organic carbon. Other chemical parameters, like pH, ammonium, nitrates and total carbon explain microbiome variability on a smaller scale between sampling sites. Thus, while regional and disturbance factors reflected differentiation of soil microbiomes, soil chemical parameters explained local variation of certain groups of microorganisms.

A high-throughput DNA sequencing study of fecal bacteria of seven Mexican horse breeds.

Horses are non-ruminant, herbivorous mammals, been used through history for various purposes, with a gut microbiota from cecum to the colon, possessing remarkable fermentative capacity. We studied the fecal microbiota of Azteca, Criollo, Frisian, Iberian, Pinto, Quarter and Spanish horse breeds living in Mexico by next-generation DNA sequencing of 16S rRNA gene libraries. Dominant phyla Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, Fibrobacteres, Actinobacteria and Verrucomicrobia have different relative abundances among breeds, with contrasted alpha and beta diversities as well. Heatmap analysis revealed that Ruminococcaceae, Lachnospiraceae, Mogibacteriaceae families, and order Clostridiales are more abundant in Spanish, Azteca, Quarter and Criollo breeds. The LEfSe analysis displayed higher abundance of order Bacteroidales, family BS11, and genera Faecalibacterium, Comamonas, Collinsella, Acetobacter, and Treponema in Criollo, Azteca, Iberian, Spanish, Frisian, Pinto, and Quarter horse breeds. The conclusion is that dominant bacterial taxa, found in fecal samples of horse breeds living in Mexico, have different relative abundances.

Feed Insects as a Reservoir of Granadaene-Producing Lactococci.

Insects are a component of the diet of different animal species and have been suggested as the major source of human dietary protein for the future. However, insects are also carriers of potentially pathogenic microbes that constitute a risk to food and feed safety. In this study, we reported the occurrence of a hemolytic orange pigmented producing phenotype of Lactococcus garvieae/petauri/formosensis in the fecal microbiota of golden lion tamarins (Leontopithecus rosalia) and feed larvae (Zophobas atratus). Feed insects were identified as a regular source of L. garvieae/petauri/formosensis based on a reanalysis of available 16S rRNA gene libraries. Pan-genome analysis suggested the existence of four clusters within the L. garvieae/petauri/formosensis group. The presence of cyl cluster indicated that some strains of the L. garvieae/petauri/formosensis group produced a pigment similar to granadaene, an orange cytotoxic lipid produced by group B streptococci, including Streptococcus agalactiae. Pigment production by L. garvieae/petauri/formosensis strains was dependent on the presence of the fermentable sugars, with no pigment being observed at pH <4.7. The addition of buffering compounds or arginine, which can be metabolized to ammonium, restored pigment formation. In addition, pigment formation might be related to the source of peptone. These data suggest that edible insects are a possible source of granadaene-producing lactococci, which can be considered a pathogenic risk with zoonotic potential.

Analysis of Intestinal Microbial Diversity of Four Species of Grasshoppers and Determination of Cellulose Digestibility.

Simple SummaryGrasshoppers are typical phytophagous pests, which prefer eating monocotyledons with more cellulose and hemicellulose. Due to its large appetite and high utilization rate, the intestinal contents of grasshoppers have the potential to be developed into a bioreactor, which can be applied to improve straw utilization efficiency in the future. The digestive tract of grasshoppers is a complex ecosystem, inhabited by a large number of microorganisms. The existence of these microorganisms enables grasshoppers to have high decomposition and utilization of plant fibers. However, there are few reports on the microflora structure and diversity of the digestive tract of grasshoppers. In this study, the diversity of symbiotic bacteria in the intestinal tract of four species of grasshoppers, namely Acrida cinerea, Trilophidia annulata, Atractomorpha sinensis and Sphingonotus mongolicus, was studied by using the method of constructing a 16S rRNA gene library and Illumina Miseq sequencing technology. At the same time, the digestibility of cellulose and hemicellulose of the four species of grasshoppers were determined and the relationship between digestibility and intestinal microbial diversity was analyzed. This study provided basic data for the development of the digestible bioreactor of cellulose and hemicellulose, which may provide a new idea for degrading straw.Grasshoppers (Insecta, Orthoptera, Acridoidea) are a large group of agricultural and animal husbandry pests. They have a large food intake with high utilization of plants fibers. However, the composition of the grasshopper gut microbial community, especially the relationship between gut microbial community and cellulose digestibility, remains unclear. In this research, 16S rRNA gene sequences were used to determine the intestinal microbial diversity of Acrida cinerea, Trilophidia annulata, Atractomorpha sinensis and Sphingonotus mongolicus, and Spearman correlation analysis was performed between the intestinal microbes of grasshoppers and the digestibility of cellulose and hemicellulose. The results showed that Proteobacteria was the dominant phylum and Klebsiella was the dominant genus in the guts of the four species of grasshoppers; there was no significant difference in the species composition of the gut microbes of the four species of grasshoppers. Spearman correlation analysis showed that Brevibacterium and Stenotrophomonas were significantly correlated with cellulose digestibility. Brevibacterium, Clavibacter, Microbacterium and Stenotrophomonas were significantly associated with hemicellulose digestibility. Our results confirmed that the gut microbes of grasshoppers were correlated with the digestibility of cellulose and hemicellulose, and indicated that grasshoppers may have the potential to develop into bioreactors, which can be applied to improve straw utilization efficiency in the future.