Amplicon Metagenomic Sequencing Research Articles

Real-time application of ITS and D1-D3 nanopore amplicon metagenomic sequencing in fungal infections: Enhancing fungal infection diagnostics

While fungal infections cause considerable morbidity and mortality, the performance of the current diagnostic tests for fungal infection is low. Even though fungal metagenomics or targeted next-generation sequencing have been investigated for various clinical samples, the real-time clinical utility of these methods still needs to be elucidated. In this study, we used internal transcribed spacer (ITS) and D1-D3 ribosomal DNA nanopore amplicon metagenomic sequencing to assess its utility in patients with fungal infections. Eighty-four samples from seventy-three patients were included and categorized into ‘Fungal infection,’ ‘Fungal colonization,’ and ‘Fungal contamination’ groups based on the judgement of infectious disease specialists. In the ‘Fungal infection’ group, forty-seven initial samples were obtained from forty-seven patients. Three fungal cases detected not by the sequencing but by conventional fungal assays were excluded from the analysis. In the remaining cases, the conventional fungal assay-negative/sequencing-positive group (n=11) and conventional fungal assay-positive/sequencing-positive group (n=33) were compared. Non-Candida and non-Aspergillus fungi infections were more frequent in the conventional-negative/sequencing-positive group (p-value = 0.031). We demonstrated the presence of rare human pathogens, such as Trichosporon asahii and Phycomyces blakesleeanus. In the ‘Fungal infection’ group and ‘Fungal colonization’ group, sequencing was faster than culturing (mean difference = 4.92 days, p-value < 0.001/ mean difference = 4.67, p-value <0.001). Compared to the conventional diagnostic methods including culture, nanopore amplicon sequencing showed a shorter turnaround time and a higher detection rate for uncommon fungal pathogens.

Evaluation of Rumen Microbes For Milk Fat Yield in Cattle Based on 16S rRNA Amplicon Metagenomic Sequencing

Background: Ruminal microbes support growth, lactation and maintenance of animals by providing energy and protein yielding nutrients. Metagenomics is the study of microbial communities directly in their natural environments such as soil, water, gut samples etc. The 16S rRNA gene sequencing is one of the basic genetic markers for the characterisation of prokaryote microorganism community. Current study exploited complex gut microbial communities present in cattle rumen based on variation in milk fat yield. Methods: The study was conducted in cattle reared at University Livestock Farm, Kerala Veterinary and Animal Sciences University. Twelve healthy cattle in the initial stages of lactation (less than three months) were selected and divided into two groups of six each based on high and low milk fat composition. Rumen samples were collected and DNA was isolated. Amplicons of 16S rRNA has been generated from metagenomic DNA targeting V3 and V4 hyper variable regions. Qiime2 was used for analysis for 16S rRNA amplicon sequencing. Result: Results revealed that a total of 38,28,241 sequences were obtained after demultiplexing. Majority of sequence length ranged around 295 to 301 nucleotides for forward reads and 216 to 300 nucleotides for reverse reads. After denoising, a total of 6,62,498 sequences were obtained. Clustering (de-replication) produced 711 unique sequence features. In kingdom level of taxonomy, for bacterial kingdom, there was no significant difference in abundance of organisms for high milk fat yield and low milk fat yield cows. For archaea, significant difference observed between abundance of high milk fat and low milk fat groups at five per cent level in the current study.

Bacterial diversity, community structure and function in association of potato scabby tubers during storage in northern Thailand.

Potato scab is a common potato tuber disease that affects quality and cost in the marketplace, shortening storage, and increasing the chance for secondary infection. The tubers with disease severity of 1 to 4 are accepted and stored in potato storage for cheap selling in Thailand. However, there are few studies of the bacterial community of the scabby tuber during storage. Thus, we aim to elucidate the diversity, structure, and function of the bacterial community of 30-day storage potato scabby tubers stored in different temperatures using 16S amplicon metagenomic sequencing. Bacterial communities of storage potato scabby tubers (Spuntacultivar) collected from different storage temperatures, 4°C (MEP1) and 6°C (MEP2), were characterized using 16S rRNAamplicon metagenomic sequencing. The alpha-diversity abundance in the bacteriome of the scabby tubers stored at 6 °C was higher than in those stored at 4 °C. Actinobacteria (34.7%) was a dominant phylum in MEP1, while Proteobacteria (39.9%) was predominant in MEP2. The top 10 genera of both communities were Rhizobium group, Streptomyces, Pectobacterium, Ruminococcus, Cellulomonas, Promicromonospora, Prevotella, Enterobacter, Pedobacter, and Paenarthrobacter. Moreover, functional profile prediction of both communities reveals essential genes in the pathosystem: nos, bglA, and cebEFG-msiK for potato scab disease and phc and peh operons for rot disease. Our findings are the first study to explore details of the bacteriome of the accepted potato scabby tubers for selling during storage in Thailand and strongly indicate that although potatoes were stored at low temperatures, diseases still occur by secondary pathogens.

Identification of prosthetic joint infections with 16S amplicon metagenomic sequencing – comparison with standard cultivation approach

Prosthetic joint infections (PJIs) are commonly diagnosed via culture-based methods, which may miss hard-to-grow pathogens. This study contrasts amplicon metagenomic sequencing (16S AS) with traditional culture techniques for enhanced clinical decision-making. We analyzed sonicate fluid from 27 patients undergoing revision arthroplasty using both methods, emphasizing the distinction between contaminants and true positives. Our findings show moderate agreement between the two methods, with a Cohen's kappa of 0.490, varying across bacterial genera (Cohen's kappa -0.059 to 1). The sensitivity of 16S AS compared to culture was 81% (95% CI, 68% to 94%). Sequencing revealed greater microbial diversity, including anaerobic genera like Anaerococcus and Citrobacter. Interestingly, several culture-negative PJI samples showed diverse bacteria via 16S AS. Despite rigorous controls and algorithms to eliminate contaminants, confirming bacteria presence with 16S AS remains a challenge. This highlights the need for improved PJI diagnostic methods, while also pointing out the limitations of next-generation sequencing (NGS) as a clinical diagnostic tool.

Analysis of fungal diversity in the gut feces of wild takin (Budorcas taxicolor).

The composition of the intestinal microbiome correlates significantly with an animal's health status. Hence, this indicator is highly important and sensitive for protecting endangered animals. However, data regarding the fungal diversity of the wild Budorcas taxicolor (takin) gut remain scarce. Therefore, this study analyzes the fungal diversity, community structure, and pathogen composition in the feces of wild B. taxicolor. To ensure comprehensive data analyses, we collected 82 fecal samples from five geographical sites. Amplicon sequencing of the internal transcribed spacer (ITS) rRNA was used to assess fecal core microbiota and potential pathogens to determine whether the microflora composition is related to geographical location or diet. We further validated the ITS rRNA sequencing results via amplicon metagenomic sequencing and culturing of fecal fungi. The fungal diversity in the feces of wild Budorcas taxicolor primarily comprised three phyla (99.69%): Ascomycota (82.19%), Fungi_unclassified (10.37%), and Basidiomycota (7.13%). At the genus level, the predominant fungi included Thelebolus (30.93%), Functional_unclassified (15.35%), and Ascomycota_unclassified (10.37%). Within these genera, certain strains exhibit pathogenic properties, such as Thelebolus, Cryptococcus, Trichosporon, Candida, Zopfiella, and Podospora. Collectively, this study offers valuable information for evaluating the health status of B. taxicolor and formulating protective strategies.

Influence of Soil Type, Land Use, and Rootstock Genotype on Root-Associated Arbuscular Mycorrhizal Fungi Communities and Their Impact on Grapevine Growth and Nutrition

Soil characteristics, land management practices, and plant genotypes influence arbuscular mycorrhizal fungi (AMF) communities, leading to the proliferation of AMF taxa with different growth and nutritional outcomes in their hosts. However, the specific patterns driving these relationships are still not well understood. This study aimed to (1) evaluate the influence of soil characteristics, land use, and rootstock on AMF diversity and community structure and (2) assess the effect of those AMF communities on grapevine growth and nutrition. Soil samples were collected from vineyard and non-agricultural areas in Lisbon and Pegões, Portugal, and trap cultures established using Richter 110 and 1103 Paulsen rootstocks. After 3.5 months growth under greenhouse conditions, root-associated AMF communities were assessed by amplicon metagenomic sequencing using AMF-specific primers. Alpha diversity was only influenced by the soil type, while in β-diversity, an interaction was found between the soil type and land use. Both diversity measures were positively correlated with foliar K and negatively with leaf Mn and Mg. Notably, the concentrations of these nutrients were highly correlated with the relative abundance of operational taxonomic units (OTUs) within the genera Glomus, Rhizophagus, and Claroideoglomus. These results are valuable for supporting AMF selection for improved plant nutrition based on varying soil types and land uses.

Next Generation Sequencing of Bee Gut Microbiota in Urban and Rural Environments

The gut microbiota plays a vital role in the physiological and behavioral processes of organisms, thereby influencing the quality of life of their host. Investigating the microbial diversity of the gut microbiota of Apis mellifera, the ecological organizer, may provide key insights into the ecological and health-related factors affecting host populations. This study aims to investigate the comparative gut microbiota of forager A. mellifera collected from both rural and urban environments in each of the four provinces located in southeastern Türkiye by employing Next Generation Sequencing (NGS) technology, specifically amplicon metagenome sequencing targeting the V3–V4 region of the 16S rRNA gene. In general, the urban samples possessed a higher level of gut microbial diversity when compared with the rural samples. Interestingly, the rural samples, in addition to the majority of previously reported core microbiota members, contained human pathogens such as Salmonella enterica, Klebsiella pneumoniae, Escherichia coli, and Streptococcus pyogenes. Moreover, this study is the first to report the predominant existence of Enterococcus wangshanyuanii, Alkalihalobacillus halodurans, and Vitreoscilla sp. in several samples. This study contributes to the idea that urban apiculture practices can alter bacteria residing in the digestive tracts and discusses possible effects of potential infestation of A. mellifera by human-pathogenic bacteria on different aspects of life traits.

Generation of dysbiotic microbiota in cutaneous leishmaniasis and enhancement of skin inflammation

Cutaneous Leishmaniasis (CL) affects millions of people globally and has a significant impact on morbidity and mortality. Innate immune mediators are likely to influence the clinical phenotype of CL through primary responses that restrict or facilitate parasite spread. The aim of this preliminary study was to bring to attention the significance of microbiota in the development of CL and emphasized the necessity of including the role of microbiota in CL while promoting a One Health approach for managing diseases. To achieve this, we used 16S amplicon metagenome sequencing and QIIME2 pipeline to analyze the microbiome composition of CL-infected patients compared to non-infected, healthy subjects. 16S sequencing analysis showed serum microbiome was dominated by Firmicutes, Proteobacteria, Bacteroidota, and Actinobacteria. CL-infected individuals, Proteobacteria were the most prevalent (27.63 ± 9.79), with the relative abundance (10.73 ± 5.33) of Proteobacteria in control. Bacilli class was found to be the most prevalent in healthy controls (30.71 ± 8.44) while (20.57 ± 9.51) in CL-infected individuals. The class Alphaproteobacteria was found to be more in CL-infected individuals (5.47 ± 2.07) as compared to healthy controls (1.85 ± 0.39). The CL-infected individuals had a significantly lower relative abundance of the Clostridia class (p < 0.0001). An altered serum microbiome of CL infection and higher microbial abundance in the serum of healthy individuals was observed.

The Gut Microbiota of Broilers Reared with and without Antibiotic Treatment.

The aim of this study was to examine the microbiota in broilers reared with and without antibiotics and to investigate differences between the upper, middle and lower sections of the gastrointestinal tract (GIT). One of two commercial flocks was treated with an antibiotic (T) (20 mg trimethoprim and 100 mg sulfamethoxazole per ml in the drinking water for 3 days) and the other was left untreated (UT). The GIT contents of 51 treated and untreated birds were aseptically removed from the upper (U), middle (M) and lower (L) sections. These were pooled in triplicate (n = 17 per section per flock), the DNA extracted and purified, 16S amplicon metagenomic sequencing performed and the resultant data analysed using a range of bioinformatics software. There were significant differences in the microbiota of the upper, middle and lower GIT, and treatment with the antibiotic significantly affected the microbiota in each of these sections. This study provides new data on broiler GIT microbiota and suggests that GIT location is a more important determinant of the constituent bacterial flora rather than the use or otherwise of antimicrobial treatments, at least when applied early in the production cycle.

The layout measures of micro-sprinkler irrigation under plastic film regulate tomato soil bacterial community and root system.

The change in rhizosphere soil bacterial community and root system under new water-saving device is not clear. A completely randomized experimental design was used to explore the effects of different micropore group spacing (L1: 30 cm micropore group spacing, L2: 50 cm micropore group spacing) and capillary arrangement density (C1: one pipe for one row, C2: one pipe for two rows, C3: one pipe for three rows) on tomato rhizosphere soil bacteria community, roots and tomato yield under MSPF. The bacteria in tomato rhizosphere soil were sequenced by 16S rRNA gene amplicon metagenomic sequencing technology, the interaction of bacterial community, root system and yield in tomato rhizosphere soil was quantitatively described based on regression analysis. Results showed that L1 was not only beneficial to the development of tomato root morphology, but also promoted the ACE index of tomato soil bacterial community structure and the abundance of nitrogen and phosphorus metabolism functional genes. The yield and crop water use efficiency (WUE) of spring tomato and autumn tomato in L1 were about 14.15% and 11.27%, 12.64% and 10.35% higher than those in L2. With the decrease of capillary arrangement density, the diversity of bacterial community structure in tomato rhizosphere soil decreased, and the abundance of nitrogen and phosphorus metabolism functional genes of soil bacteria also decreased. The small abundance of soil bacterial functional genes limited the absorption of soil nutrients by tomato roots and roots morphological development. The yield and crop water use efficiency of spring and autumn tomato in C2 were significantly higher than those in C3 about 34.76% and 15.23%, 31.94% and 13.91%, respectively. The positive interaction between soil bacterial community and root morphological development of tomato was promoted by the capillary layout measures of MSPF. The L1C2 treatment had a stable bacterial community structure and good root morphological development, which positively promoted the increase of tomato yield. The interaction between soil microorganisms and roots of tomato was regulated by optimizing the layout measures of MSPF to provide data support for water-saving and yield-increasing of tomato in Northwest China.

Effects of Bile Duct Ligation and Ghrelin Treatment on the Colonic Barrier and Microbiome of Mice

Introduction: Cholestatic liver disease (CLD) is associated with intestinal barrier dysfunction. The peptide hormone ghrelin may exert both hepatoprotective and barrier-strengthening effects. Here, we have evaluated these effects under the conditions of experimental cholestasis. Methods: C57BL/6J mice with bile duct ligation (BDL) or sham surgery were treated with ghrelin or solvent for 9 days. Liver injury was assessed by histological and laboratory analyses. Paracellular macromolecule permeability and transmural electrical resistance (TMER) of colonic tissues were measured using a Ussing chamber. Expression of tight junction (TJ) genes was quantified by real-time PCR. Amplicon metagenomic sequencing was employed to analyze bacterial 16S rRNA from colonic stool samples. Results: Mice with BDL exhibited weight loss and signs of severe liver injury. These changes were unaffected by ghrelin treatment. FITC-4-kDa-dextran flux was increased and TMER decreased after BDL. Treatment with ghrelin tended to reduce these effects. Furthermore, application of ghrelin was associated with higher mRNA levels of claudin-4, occludin, and ZO-1 in colonic tissues of mice with BDL. Reduced alpha-diversity of the microbiome was observed in solvent-treated mice with BDL but not in ghrelin-treated animals. Conclusion: Ghrelin treatment did not improve weight loss and liver damage but increased gene expression of colonic TJ proteins and restored the alpha-diversity of the microbiome. Since protective effects of ghrelin might be masked by the severity of the model, we suggest follow-up studies in models of milder CLD.

Substrate-restricted methanogenesis and limited volatile organic compound degradation in highly diverse and heterogeneous municipal landfill microbial communities

Microbial communities in landfills transform waste and generate methane in an environment unique from other built and natural environments. Landfill microbial diversity has predominantly been observed at the phylum level, without examining the extent of shared organismal diversity across space or time. We used 16S rRNA gene amplicon and shotgun metagenomic sequencing to examine the taxonomic and functional diversity of the microbial communities inhabiting a Southern Ontario landfill. The microbial capacity for volatile organic compound degradation in leachate and groundwater samples was correlated with geochemical conditions. Across the landfill, 25 bacterial and archaeal phyla were present at >1% relative abundance within at least one landfill sample, with Patescibacteria, Bacteroidota, Firmicutes, and Proteobacteria dominating. Methanogens were neither numerous nor particularly abundant, and were predominantly constrained to either acetoclastic or methylotrophic methanogenesis. The landfill microbial community was highly heterogeneous, with 90.7% of organisms present at only one or two sites within this interconnected system. Based on diversity measures, the landfill is a microbial system undergoing a constant state of disturbance and change, driving the extreme heterogeneity observed. Significant differences in geochemistry occurred across the leachate and groundwater wells sampled, with calcium, iron, magnesium, boron, meta and para xylenes, ortho xylenes, and ethylbenzene concentrations contributing most strongly to observed site differences. Predicted microbial degradation capacities indicated a heterogeneous community response to contaminants, including identification of novel proteins implicated in anaerobic degradation of key volatile organic compounds.

Effect of Doxycycline Use in the Early Broiler Production Cycle on the Microbiome.

16S rRNA amplicon sequencing was used to investigate changes in the broiler gastrointestinal tract (GIT) microbiota throughout the rearing period and in combination with antibiotic treatment. Thirty birds (from a commercial flock) were removed at multiple points throughout the rearing period on days 13, 27, and 33, euthanised, and their GIT aseptically removed and divided into upper (the crop, proventriculus, and the gizzard), middle (the duodenum, jejunum, and ileum) and lower (the large intestine, the caeca, and the cloaca) sections. In a separate commercial flock, on the same farm with similar husbandry practices and feed, doxycycline (100 mg/ml per kg body weight) was administered in drinking water between day 8 and 12 (inclusive) of the production cycle. Birds were removed on days, 13, 27, and 33 and GIT samples prepared as above. The contents of three merged samples from each GIT section were pooled (n = 60), the DNA extracted and analysed by 16S rRNA amplicon metagenomic sequencing and analysed. Major changes in the broiler microbiota were observed as the birds aged particularly with the Firmicutes/Bacteroidetes ratio (F:B) of the lower GIT. Moreover, Chao1, ACE, and Shannon indices showed the antibiotic treatment significantly altered the microbiota, and this change persisted throughout the rearing period. Further research is required to investigate the effect of these changes on bird performance, susceptibility to infections and Campylobacter carriage.

Unraveling the Tropaeolum majus L. (Nasturtium) Root-Associated Bacterial Community in Search of Potential Biofertilizers.

Although Tropaeolum majus (nasturtium) is an agriculturally and economically important plant, especially due to the presence of edible flowers and its medicinal properties, its microbiome is quite unexplored. Here, the structure of the total bacterial community associated with the rhizosphere, endosphere and bulk soil of T. majus was determined by 16S rRNA amplicon metagenomic sequencing. A decrease in diversity and richness from bulk soil to the rhizosphere and from the rhizosphere to the endosphere was observed in the alpha diversity analyses. The phylum Proteobacteria was the most dominant in the bacteriome of the three sites evaluated, whereas the genera Pseudomonas and Ralstonia showed a significantly higher relative abundance in the rhizosphere and endosphere communities, respectively. Plant growth-promoting bacteria (236 PGPB) were also isolated from the T. majus endosphere, and 76 strains belonging to 11 different genera, mostly Serratia, Raoultella and Klebsiella, showed positive results for at least four out of six plant growth-promoting tests performed. The selection of PGPB associated with T. majus can result in the development of a biofertilizer with activity against phytopathogens and capable of favoring the development of this important plant.

A mouse model of occult intestinal colonization demonstrating antibiotic-induced outgrowth of carbapenem-resistant Enterobacteriaceae

BackgroundThe human intestinal microbiome is a complex community that contributes to host health and disease. In addition to normal microbiota, pathogens like carbapenem-resistant Enterobacteriaceae may be asymptomatically present. When these bacteria are present at very low levels, they are often undetectable in hospital surveillance cultures, known as occult or subclinical colonization. Through the receipt of antibiotics, these subclinical pathogens can increase to sufficiently high levels to become detectable, in a process called outgrowth. However, little is known about the interaction between gut microbiota and Enterobacteriaceae during occult colonization and outgrowth.ResultsWe developed a clinically relevant mouse model for studying occult colonization. Conventional wild-type mice without antibiotic pre-treatment were exposed to Klebsiella pneumoniae but rapidly tested negative for colonization. This occult colonization was found to perturb the microbiome as detected by both 16S rRNA amplicon and shotgun metagenomic sequencing. Outgrowth of occult K. pneumoniae was induced either by a four-antibiotic cocktail or by individual receipt of ampicillin, vancomycin, or azithromycin, which all reduced overall microbial diversity. Notably, vancomycin was shown to trigger K. pneumoniae outgrowth in only a subset of exposed animals (outgrowth-susceptible). To identify factors that underlie outgrowth susceptibility, we analyzed microbiome-encoded gene functions and were able to classify outgrowth-susceptible microbiomes using pathways associated with mRNA stability. Lastly, an evolutionary approach illuminated the importance of xylose metabolism in K. pneumoniae colonization, supporting xylose abundance as a second susceptibility indicator. We showed that our model is generalizable to other pathogens, including carbapenem-resistant Escherichia coli and Enterobacter cloacae.ConclusionsOur modeling of occult colonization and outgrowth could help the development of strategies to mitigate the risk of subsequent infection and transmission in medical facilities and the wider community. This study suggests that microbiota mRNA and small-molecule metabolites may be used to predict outgrowth-susceptibility.CiXsSNTB417URyJP4k6GoXVideo

Diversity of Rhizo-Bacteriome of Crocus sativus Grown at Various Geographical Locations and Cataloging of Putative PGPRs

Earlier plant growth promoting rhizo-bacteria (PGPRs) were isolated from the plants, by cultivation based techniques and the interaction was mostly thought to be bilateral. The routine bilateral study, with no information on the associated microbiome, could be one of the reasons for the limited success of PGPRs in the field conditions. Keeping in view the role of PGPRs in rhizo-bacteriome on the growth and production of plant, the present study was aimed at studying the diversity of the rhizo-bacteriome of saffron grown across three geographical locations namely Kashmir, Kishtwar and Bengaluru. Variation in the rhizo-bacteriome of saffron growing across 10 different sites from 3 geographical locations was studied using 16S rDNA amplicon metagenomic sequencing. 16 bacterial phyla, 261 genera and 73 bacterial species were cataloged from all the rhizosphere samples. Proteobacteria was a dominant phylum in all the rhizosphere samples. Rhizo-bacteriome of saffron grown in Kishtwar was found to be significantly different from the rhizo-bacteriome of saffron grown in Kashmir and Bengaluru. Interestingly, the rhizo-bacteriome of saffron grown in Bengaluru was very similar to the saffron grown in Kashmir, thereby indicating that the rhizo-bacteriome in saffron is “plant driven” as the corm sown in Bengaluru were from Kashmir. Despite variation in rhizo-bacteriome, core rhizo-bacteriome in saffron was identified that was represented by 53 genera and eight bacterial species belonging to 11 phyla irrespective of their geographical distribution. In addition, 21 PGPRs were reported for the first time from the saffron rhizosphere. The high yielding saffron field Wuyan was found to have the highest number of PGPRs; this indicates that the presence of PGPR is important for yield enhancement than diversity. The two PGPR Rhizobium leguminosarum and Luteibacter rhizovicinus were reported from all the locations except Kishtwar that had escaped isolation in our previous attempts using cultivation based techniques. It is being proposed instead of going for random isolation and screening for PGPRs from plant rhizosphere, an alternate strategy using metagenomic cataloging of the rhizo-bacteriome community and cultivation of the dominant PGPR should be undertaken. This strategy will help in the selection of dominant PGPRs, specific to the plant in question.

Bacterial Diversity in a Sri Lankan Geothermal Spring Assessed by Culture-Dependent and Culture-IndependentApproaches.

Hot springs harbour diverse and interesting groups of microorganisms adapted to extreme conditions. However, due to limitations in the culture-dependent approach, most of such thermophiles remain uncultured and unexplored. Hence, this study was conducted to gain a comprehensive understanding of the bacterial diversity of Mahapelessa hot spring, Sri Lanka using both culture-dependent and culture-independent approaches. Thein situtemperature of the water sample was 44.5°C and thepH was 8.14. 16S rRNA Sanger sequencing of DNA extracted from the 18 bacterial isolates revealed the presence of eight genera belonging to two phyla: Proteobacteria (84%) and Firmicutes (16%) and the most abundant genus being Klebsiella. A total of 23 bacterial phyla representing 80 classes, 43 orders, 123 families, 205 genera and 83 species were detected by 16S rRNA V3-V4 region by amplicon metagenome sequencing of DNA extracted from water samples, where the most abundant phylum was the Proteobacteria (57.39%), followed by Firmicutes (23.7%) and Chloroflexi (4.14%). The three phyla Actinobacteria, Planctomycetes and Bacteroidetes were also detected less than 3% in abundance while 4.48% of bacteria could not be fit into any known phylum. The most abundant genera were Burkholderia (14.87%), Desulfotomaculum (7.23%) and Stenotrophomonas (6.1%). Four strictly anaerobic bacteria, Anaerosolibacter carboniphilus (0.71%), Bellilinea caldifistulae (0.04%), Salimesophilobacter vulgaris (0.1%), Anaerobacterium chartisolvens (0.12%); two potential plant growth-promoting bacteria, Azospirillum halopraeferens (0.04%) and Bradyrhizobium liaoningense (0.16%) and one potential alkali tolerant and sulphate-reducing bacterium,Desulfovibrio alkalitolerans (0.45%) were recorded. Pigmentiphaga sp. was isolated from Mahapelessa hot spring and to the best of our knowledge, this is the first record of this genus from a hot spring. This study gives insight into the vast bacterial diversity present in the Mahapelessa hot spring from the culture-independent approach which could not be identified using standard culturing techniques.

Root rot alters the root-associated microbiome of field pea in commercial crop production systems

Pulses are important crops that have been used to diversify cropping rotations in the Canadian prairies. With increasing cultivation intensity, pulses are becoming more susceptible to root rots. Understanding how root-associated microbiomes are affected by plant health is vital for developing sustainable crop production systems. In this study, we investigated the effect of root rot on rhizosphere and root microbiomes of healthy and root-rot infected field pea (Pisum sativum L.) plants from nine commercial fields in Saskatchewan using amplicon metagenomic sequencing. Bacterial and oomycete α-diversity was higher in diseased root and rhizosphere samples than their healthy counterparts, while fungal diversity was higher in diseased rhizosphere samples. The community structure of root and rhizosphere microbiomes were also affected by the health status of the plants, with root bacterial communities exhibiting the strongest differences. Healthy samples were associated with a higher relative abundance of Rhizobium, Olpidium and Mortierella, and lower abundance of Fusarium and Pythium. Diseased samples had a higher number of indicator genera (42 in diseased vs. 11 in healthy) and more taxa exclusively detected in the diseased core microbiome. Results from this study revealed that root rot generally increased the diversity and altered the community structure and relative abundance of key taxa of the pea root-associated microbiome. This knowledge may aid in the development of microbiome-based disease management strategies, such as identification of key microbial taxa that could help commercial pea growers assess root rot risk.

Современные молекулярно-генетические технологии в диагностике вирусных заболеваний

High contagiousness and rapid spread of viral infections highlight the importance of their timely clinical diagnosis. There is a current need to gain in-depth knowledge of viral agents’ origin, routes, and evolution to predict and prevent viral diseases. The introduction of advanced molecular genetic testing made this possible. Laboratories worldwide develop and manufacture urgently needed test kits for rapidly detecting infections since asymptomatic cases favor further dissemination of these diseases. Over the last year, the active development of diagnostic kits for COVID-19 contributed to the improvement of molecular genetic testing, particularly for mass and screening testing. RT-PCR is widely applied to detect viral RNA. Meanwhile, other tests for nucleic acids, e.g., isothermal amplification, microarray hybridization, amplicon metagenome sequencing, and CRISPR, are now introduced into daily practice. KEYWORDS: viruses, diagnostics, molecular genetic testing, polymerase chain reaction, RNA. FOR CITATION: Arnaudova K.Sh., Yasenyavskaya A.L., Rostoshvili G.A. et al. State-of-the-art molecular genetic testing for the diagnosis of viral infections. Russian Medical Inquiry. 2021;5(7):497–502 (in Russ.). DOI: 10.32364/2587-6821-2021-5-7-497-502.

Microbial Diversity in Commercial Bee Pollen from Europe, Chile, and Mexico, Based on 16S rRNA Gene Amplicon Metagenome Sequencing.

ABSTRACTBee pollen is a highly nutritive natural foodstuff. Because of its use as a comestible, the association of bacteria with bee pollen is commercially and biologically important. We report here the bacterial diversity of seven bee pollen samples (five from Europe, one from Chile, and one from Mexico) based on 16S rRNA gene amplicon metagenome sequencing.