rRNA Libraries Research Articles

Aptima Combo2-avoiding variants detected in cervical and endometrial specimens from a cohort of sexually active cisgender women during 16S microbiome profiling.

Performance of a 16S rRNA analysis of the cervicovaginal microbiome of 220 participants recruited into the T Cell Response against Chlamydia (TRAC) cohort between February 2011 and August 2014 in Allegheny County, Pennsylvania USA surprisingly detected DNA encoding chlamydial 16S rRNA in samples from seven participants who had tested negatively for Chlamydia trachomatis (CT) and DNA encoding gonococcal 16S rRNA from five participants who had tested negatively for Neisseria gonorrhoeae (NG) infection with the Aptima Combo2 assay (Hologic). We used targeted PCR amplification followed by sequencing to characterize the chlamydial 23S rRNA locus and qPCR to detect gonococcal DNA in residual diagnostic swab eluates or DNA used to generate 16S rRNA libraries. Discrepant specimens that contained chlamydial DNA carried a diagnostic-avoidant, G1526A variant in the 23S rRNA locus identical to variants previously detected in Finland, Denmark, and the UK. PCR validation of gonococcal DNA was confirmed for all participants who had tested negatively, with stochastic effects consistent with infection levels close to the limit of detection by the diagnostic assay. These data indicate that this probe-avoidant CT mutant, and possibly others, were circulating in the northeastern US prior to their detection and characterization in 2019 and subsequently. Although infrequent, documentation of false negative results for CT indicates a need for clinicians to consider performance of a second test that uses alternate PCR probes if patients have persistent symptoms or have known contact to an infected sex partner and their initial NAAT is negative.

Salivary microbiome signatures of Poles and Serbians and its potential for prediction of biogeographic ancestry

Biogeographical ancestry analysis is valuable in forensic investigations, especially in missing person cases or crimes without eyewitnesses, as it helps to infer geographic origins from genetic markers. This approach enhances forensic efforts by providing essential clues for identifying individuals with limited direct evidence. Slavic-speaking populations are poorly distinguishable based on human genome variability. However, recent studies show that even populations with close biogeographic origin could be differentiated based on salivary microbiomes. Nevertheless, the salivary microbiomes of Slavs have not been characterized yet. Therefore, this study aimed to compare the composition of the salivary microbiomes of Western and Southern Slavs’ representatives. 16S rRNA libraries from salivary microbiomes of 40 Poles (Western Slavs) and 40 Serbians (Southern Slavs) were prepared via PCR and sequenced on the MiSeq FGx platform (Illumina), giving approximately 100,000 reads per sample. Bioinformatic and statistical analyses were performed to assess the alpha and beta diversity of microbiomes and determine the differences in the abundance of bacterial genera between the groups studied. Analyses of alpha (ACE, Chao1, Shannon, and Simpson) and beta (Jaccard and Bray-Curtis dissimilarity) diversities in the salivary microbiomes clearly distinguished between Poles and Serbians. Alpha and beta diversity metrics were significantly higher in the Serbian population. Fusobacterium, Lautropia, Porphyromonas, Actinobacillus, Capnocytophaga, and Kingella were the most significantly increased genera in Serbians, whereas Veillonella, Selenomonas, Megasphaera, and Atopobium were more prevalent in Poles. In summary, our study identified significant differences in the salivary microbiomes of Poles and Serbians, with distinct microbial signatures associated with each population. These findings highlight the potential of salivary microbiome analysis as a tool for predicting biogeographic ancestry. Nevertheless, further analysis extended to other Slavic-speaking populations is necessary to clarify this issue.

Levilactobacillus brevis SG031 modulates mood-related behaviors and attenuates stress-related sleep disturbance and autonomic dysfunction via gut microbiota modulation in Wistar–Kyoto rats

AimsThe probiotic bacterium Levilactobacillus brevis (L. brevis) has been proposed as a potential solution to manage mood disorders and alleviate stress-related sleep disturbances. However, the underlying mechanisms of its effects have not been fully elucidated. The aim of this study was to explore the impact and potential mechanisms of L. brevis SG031 supplementation on anxiety/depression-like behaviors and stress-induced changes in sleep patterns and sleep-related autonomic function. Main methodsMale Wistar–Kyoto rats were administered low, medium, or high doses of L. brevis SG031 or a vehicle for 4 weeks, followed by behavioral tests to evaluate anxiety and depression. After an additional 2 weeks of SG031 or vehicle administration, a cage-exchange paradigm was performed with 24-hour physiological signal measurements under different stress conditions. Fecal samples were collected to construct a 16S rRNA library and assess fecal short-chain fatty acids (SCFAs). Key findingsHigh-dose SG031 administration yielded reduced depression-like responses and enhanced social interaction in behavioral tests. It also exhibited a protective effect against stress-induced sleep disturbance characterized by decreased sleep time, increased awake time, and autonomic dysfunction during sleep. Fecal examination indicated that high-dose SG031 administration exerted beneficial effects on gut health by maintaining the gut microbial abundance, preserving stability of the microbial composition, and enriching the gut with SCFAs, which were associated with improvements in sleep and autonomic function. SignificanceThese findings collectively underscore the multifaceted potential of SG031 in addressing mental health and stress-related sleep challenges through the modulation of the gut microbiota.

Influence of Compost Amendments on Soil and Human Gastrointestinal Bacterial Communities during a Single Gardening Season.

To measure associations between gardening with different compost amendments and the human gut microbiota composition, gardeners (n = 25) were provided with one of three types of compost: chicken manure (CM), dairy manure and plant material (DMP), or plant-based (P). Stool samples were collected before gardening (T1), after compost amendment (T2), and at peak garden harvest (T3). Compost and soil samples were collected. DNA was extracted, 16S rRNA libraries were established, and libraries were sequenced by Illumina MiSeq. Sequences were processed using mothur, and data were analyzed in R software version 4.2.2. Fast expectation-maximization microbial source tracking analysis was used to determine stool bacteria sources. At T2/T3, the gut microbiotas of P participants had the lowest Shannon alpha diversity, which was also the trend at T1. In stool from T2, Ruminococcus 1 were less abundant in the microbiotas of those using P compost as compared to those using CM or DMP. At T2, Prevotella 9 had the highest abundance in the microbiotas of those using CM compost. In participants who used CM compost to amend their gardening plots, a larger proportion of the human stool bacteria were sourced from CM compared to soil. Soil exposure through gardening was associated with a small but detectable change in the gardeners' gut microbiota composition. These results suggest that human interactions with soil through gardening could potentially impact health through alterations to the gut microbiota.

Characterization of bacterial species and antibiotic resistance observed in Seoul, South Korea's popular Gangnam-gu area

Public transportation facilities, especially road crossings, which raise the pathogenic potential of urban environments, are the most conducive places for the transfer of germs between people and the environment. It is necessary to study the variety of the microbiome and describe its unique characteristics to comprehend these relationships. In this investigation, we used 16 S rRNA gene sample sequencing to examine the biological constituents and inhalable, thoracic, and alveolar particles in aerosol samples collected from busy areas in the Gangnam-gu district of the Seoul metropolitan area using a mobile vehicle. We also conducted a comparison analysis of these findings with the previously published data and tested for antibiotic resistance to determine the distribution of bacteria related to the human microbiome and the environment. Actinobacteria, Cyanobacteria, Bacteriodetes, Proteobacteria, and Firmicutes were the top five phyla in the bacterial 16 S rRNA libraries, accounting for >90 % of all readings across all examined locations. The most prevalent classes among the 12 found bacterial classes were Bacilli (45.812 %), Gammaproteobacteria (25.238 %), Tissierellia (13.078 %), Clostridia (5.697 %), and Alphaproteobacteria (5.142 %). The data acquired offer useful information on the variety of bacterial communities and their resistance to antibiotic drugs on the streets of Gangnam-gu, one of the most significant social centers in the Seoul metropolitan area. This work emphasizes the relevance of biological particles and particulate matter in the air, and it suggests more research is needed to perform biological characterization of the ambient particulate matter.

Influence of Elution Characteristics of Steelmaking Slags on Major Bacterial Communities in Biofilms

Steelmaking slags are prospective base materials for seaweed beds, resulting from a continuous process of biofouling, starting from biofilm formation and leading to growing algae. While focusing on biofilm formation, we investigated specific features of steelmaking slags when utilized as a base for seaweed beds by comparing the bacterial communities in marine biofilms between steelmaking slags and artificially produced ones. Genomic DNA was extracted from the biofilms collected on days 3 and 7, and partial 16S rRNA libraries were generated and sequenced by second-generation next-generation sequencing. The read sequences were analyzed using QIIME 2™, then heatmaps and non-metric multidimensional scaling based on the Bray–Curtis dissimilarity index in the R program. Rhodobacteraceae and Flavobacteriaceae were the most dominant family members in all samples on both days 3 and 7. However, Mariprofundus, comprising iron-oxidative bacteria, was predominantly detected in the samples of steelmaking slags on day 7. This suggested that the growth of Mariprofundus was dependent on Fe(II) ion concentration and that steelmaking slags eluted Fe(II) ions more easily than artificial slags. In contrast, Sulfurovaceae, sulfur-oxidizing bacteria, were dominantly present in all samples on day 3, but decreased by day 7, regardless of the sulfur content. It was supposed that engine oil-derived sulfur compounds strongly influenced Sulfurovaceae growth, whereas slag-derived sulfur compounds did not. Heatmap analysis indicated that the submersion period significantly influenced the bacterial communities, regardless of the differences in the main slag content ratios. Summarizing these results, the elution characteristics of steelmaking slags have the potential to influence the formation of marine biofilms, and this formation is significantly influenced by environmental conditions.

Isolation of Bacteria from Freeze-Dried Samples and the Functional Characterization of Species-Specific Lactic Acid Bacteria with a Comparison of Wild and Captive Proboscis Monkeys.

Previously, we isolated a novel lactic acid bacteria species (Lactobacillus nasalidis) from the fresh forestomach contents of a captive proboscis monkey (Nasalis larvatus) in a Japanese zoo. In this study, we isolated two strains of L. nasalidis from the freeze-dried forestomach contents of a wild proboscis monkey inhabiting a riverine forest in Malaysia. The samples had been stored for more than six years. Phenotypic analysis showed that strains isolated from the wild individual had more diverse sugar utilization and lower salt tolerance than strains previously isolated from the captive counterpart. These phenotypic differences are most likely induced by feeding conditions; wild individuals consume a wide variety of natural food, unlike their zoo-raised counterparts that consume formula feed with sodium sufficiency. Since 16s rRNA sequences of L. nasalidis were detected in the previously created 16S rRNA libraries of wild, provisioned, and captive proboscis monkeys in Malaysia and Japan, L. nasalidis may be an essential bacterium of the foregut microbial community of the proboscis monkey. The currently established method for the isolation of gut bacteria from freeze-dried samples under storage will be applicable to many already-stored precious samples.

7. Does Cutibacterium acnes (C. acnes, formerly Propionibacterium acnes) play a role in degenerative disc disease?

<h3>BACKGROUND CONTEXT</h3> Degenerative disc disease (DDD) is a major burden to society, leading to disability and loss in productivity. In 2005, an estimated $86 billion was spent in the United States for management of neck and back problems. Recent evidence suggests that Cutibacterium acnes (C. acnes), an anaerobic bacterium, may latently reside in intervertebral discs (IVDs) and contribute to DDD. <h3>PURPOSE</h3> To determine the presence of C. acnes in IVD specimens and to correlate this with Pfirrmann grading on MRI. <h3>STUDY DESIGN/SETTING</h3> The intervertebral discs of 63 DDD patients and 5 control patients were collected prospectively. <h3>PATIENT SAMPLE</h3> Sixty-eight patients presenting to a single academic center for spine surgery were enrolled. <h3>OUTCOME MEASURES</h3> Pfirrmann grading, presence of C. acnes based on taxonomic identification. <h3>METHODS</h3> We collected human IVD specimens from surgical patients undergoing discectomy and interbody fusion from a single institution from 8/2019 to 9/2020. Disc degeneration was graded according to the 5-level Pfirrmann grading system on MRI imaging taken prior to surgery. IVD specimens from trauma patients (Pfirrmann grades I-II) were included in the control group, and IVD specimens were included in the degenerative group with Pfirrmann grades III-V. IVD specimens were processed for DNA extraction, and 16S rRNA libraries were constructed and analyzed for taxonomic data. <h3>RESULTS</h3> There were 63 patients included within the DDD group and 5 patients within the control group. Within the degenerative group, IVD specimens were taken from patients who had undergone spine surgery within the lumbar spine (n = 42; 66.7%), cervical spine (n = 19; 30.2%), and thoracic spine (n = 2; 3.2%). The average age of the DDD group was 54.3 ±13.9 years old, 57.1% (36/63) were male, and 85.7% (54/63) were Caucasian. A total of 31.7% (20/63) of DDD specimens were identified with the presence of C. acnes. There were 28.6% (12/42) of lumbar spine IVD specimens, 42.1% (8/19) of cervical spine IVD specimens, 0% (0/2) of thoracic spine IVD specimens that were identified with C. acnes. There was no difference in the presence of C. acnes when comparing the cervical, thoracic, and lumbar spine specimens (p = 0.36). When stratifying the DDD specimens by MRI Pfirmann grading, there were 31.3% (5/16) of specimens with a Pfirmann grade of 3, 31.3% (10/32) of specimens with a Pfirrmann grade of 4, and 33.3% (5/15) of specimens with a Pfirrmann grade of 5 that were identified with C. acnes. There was no difference in the presence of C. acnes when comparing the DDD specimens based on Pfirrmann grading (p = 0.99). There were 5 IVD specimens included within the control group with an average age of 39.8 ±20.5 years old, and 80% (4/5) were Caucasian males. The control group included IVD specimens taken from two lumbar and three cervical spine surgerie. Four of five (80%) of the specimens had a Pfirrmann grade of 2 on MRI, and 1/5 (20%) had a Pfirrmann grade of 1. No control specimens were identified with the presence of C. acnes. <h3>CONCLUSIONS</h3> Of the DDD specimines, 31.7% (20/63) were identified with the presence of C. acnes, while none of the specimens of the control group were identified with this bacterium. These results suggest that indolent C. acnes infection may be one of the factors contributing to degenerative disc disease. <h3>FDA DEVICE/DRUG STATUS</h3> This abstract does not discuss or include any applicable devices or drugs.

Impact of Compost Amendments on the Human Gut Microbiota During Gardening

ObjectivesTo measure associations between gardening with three different types of compost amendments and the human gut microbiota composition.MethodsGardeners (n = 25) were provided with one of three types of compost: chicken manure (CM), compost with dairy manure and plant material (DMP), or plant-based compost (P). Stool samples were collected before gardening (T1), after amending soil with compost (T2), and at the peak of garden harvest (T3). Compost and soil samples were also collected. DNA was extracted from the samples, 16S rRNA libraries were made, and libraries were sequenced by Illumina MiSeq. Sequences were processed using mothur, and data were analyzed in R. Kruskal-Wallis with post hoc Dunn test was performed to compare Shannon Alpha diversity. Individual taxa were compared between time points using a negative binomial test. Fast expectation-maximization microbial source tracking (FEAST) analysis was used to determine stool bacteria sources. Proportions were compared by Chi-Square.ResultsAt T2 and T3, gut bacterial communities of participants who used P compost had lower Shannon alpha diversity compared to the gut bacterial communities of participants using the other two compost types, and this was also the trend at T1. In T2 stool, Ruminococcus 1 and Ruminococcus 2 were less abundant in the microbiotas of those using P compost as compared to those using CM or DMP. However, at T1, Ruminococcus 2 was less abundant in the microbiotas of individuals that later used P as well as those that later used CM compared to those who later used DMP. During T2, Prevotella 9 had the highest abundance in the microbiotas of those using P compost. In participants who used CM compost to amend their gardening plots, a larger proportion of the human stool bacteria were sourced from CM compared to soil.ConclusionsSoil exposure through gardening was associated with a small but detectable change in the gardeners’ gut microbiota composition. These results suggest that human interactions with soil through gardening could potentially impact health through alterations to the gut microbiota.Funding SourcesUSDA NIFA AFRI.

Rhizosphere bacteria community shift from vegetative to reproductive stage of Indonesia aromatic rice var. Pulut mandoti EMAS

The popular Indonesian aromatic rice, var. Pulut Mandoti Emas (PME) is produced with high economic value in Salukanan Village, Enrekang Regency, however, the best aroma quality of mandoti rice is restricted to the village. Therefore, this study aims to analyze the rhizosphere microbial group and the nutrients of the Mandoti rice paddy field in Salukanan Village at the vegetative and reproductive phases. Metagenome analysis was carried out with 16S rRNA amplification and Library preparation to examine the macro and micronutrients. The results showed that the most dominant taxa in the rhizosphere soil of vegetative mandoti rice was phylum Acidobacteria (35%), Proteobacteria (30%), and Firmicutes (12%), while in the reproductive phase, it was Phylum Firmicutes (42%), Proteobacteria (29%), and Planctomycetes (14%), respectively. The most dominant genus in vegetative phase was Candidatus koribacter, while at the reproductive phase was Aquisphaera. The analysis of macronutrients P, K, Mg, Ca, and micronutrients Fe, Mn, Cu, Na, Zn showed a reduction in the nutrient concentration at the reproductive phase compared to the vegetative phase excluding S element. Furthermore, the concentration of S element affects the abundance of bacteria in the reproductive phase, while the other elements of nutrients affect the vegetative phase. Based on these results, further strategies can be formulated to increase yield and guide breeding of mandoti aromatic rice.

Green Manures Alter Taxonomic and Functional Characteristics of Soil Bacterial Communities.

Incorporation of plant biomass into soil as green manures can reduce soilborne diseases and improve crop and soil health in agricultural ecosystems. Soil microbial communities can mediate beneficial effects of these amendments, but their response to different types of green manures is poorly understood. This study tested the effect of green manures from broccoli, marigold, and sudangrass on taxonomic and functional characteristics of soil bacterial communities. Green manures were amended to field soil and maintained in microcosms artificially infested with the soilborne plant pathogen Verticillium dahliae. Lettuce seedlings were transplanted into green manure amended and fallow soil and maintained under growth chamber conditions for 12 weeks. Bacterial communities in bulk and rhizosphere soils were characterized using nanopore sequencing of 16S rRNA and shotgun metagenome libraries. Under microcosm conditions, all green manures reduced the abundance of the soilborne plant pathogen V. dahliae and altered the taxonomic composition of bacterial communities. Twelve weeks following amendment, green manures had differential effects on lettuce yield as well as the taxonomic diversity and composition of soil bacterial communities. In addition, multiple green manures increased the abundance of bacterial functional traits in rhizosphere soil related to iron and polysaccharide acquisition and decreased the abundance of functional traits related to bacterial protein secretion systems. This study demonstrates green manures alter the taxonomic composition and functional traits in soil bacterial communities suggesting these changes may impact beneficial effects of green manures on plant and soil health.

Distribution Characteristics and Species Diversity of Bacteria in Hepatocellular Carcinoma Tissues

This study was to explore the differences in the distribution and species diversity of bacteria between hepatocellular carcinoma (HCC) tissues and normal liver tissues. 28 HCC patients treated with surgery were selected as the research objects (HCC group), and 19 healthy volunteers with normal physical examinations were included in the control group (Normal group). The tumor specimens were obtained by intraoperative and biopsy puncture, and a 16S ribosomal ribonucleic acid (rRNA) library was constructed. Based on the sequencing data obtained by the IlluminaHi Seq sequencing platform, the differences of bacteria in the liver tissues of the HCC group and the Normal group were analyzed at the level of phyla, family, and genus. The Ace, Chao1, and Shannon of the two groups were compared. The results showed that IlluminaHi Seq sequencing obtained a total of 11,714,659 valid sequences, with an average of 131,625 sequences per sample. The proportions of Bacteroidetes, Firmicutes, and Proteobacteria in HCC group and Normal group were 48.75% versus 34.16%, 37.44% versus 18.02%, and 10.85% versus 39.26%, respectively. The Bacteroidaceae, Prevotellaceae, Lachnospiraceae, and Ruminococcaceae accounted for 22.49%, 20.62%, 16.54%, and 19.93% in Normal group; while those in the HCC tissues accounted for 26.83%, 14.22%, 11.14%, and 13.18%, respectively. The dominant bacteria at the genus level in HCC group and Normal group were Bacteroides and Prevotella-9, with the proportions of 24.19% versus 26.04% and 14.19% versus 8.44%, respectively. The difference in operational taxonomic unit (OTU) numbers of HCC and Normal group were compared and analyzed, which were 1,266 and 1,082, respectively, and the number of common OTU in the two tissues was 875. The Ace in HCC tissue and normal liver tissue were 1063.8±66.79 and 1003.6±52.19, respectively. The Ace in HCC tissue was greater than that in normal liver tissue (P &lt; 0.05). The Chao1 and Shannon in HCC tissue were 1022.9±67.74 and 5.4269±0.3608, respectively; while those in normal liver tissue were 1003.6±66.79 and 5.2842±0.9714, respectively. The Chao1 and Shannon in HCC tissues were much higher than those in Normal group (P &lt; 0.05). It showed that there was no difference in the types of bacterial species in HCC tissues, but the proportions of their flora at the level of phyla, family, and genus changed greatly, which may be related to the occurrence of HCC. This study could provide a reference for the diagnosis and treatment of HCC.

Comparative analysis of the intestinal tract microbiota and feeding habits of five sympatric flycatchers

Gut microbiota and host interactions co-evolve and develop into stably adapted microbial communities and play vital roles in maintaining the health of organisms. Diet is supposed to be an important driver of differences in gut microbiota, but previous studies would commonly use literature depictions, which are essential but inaccurate, to explain the effects of diet on the gut microbiota of wild birds. In this study, we collected intestinal samples from five sympatric flycatchers to compare the gut microbial differences using bacterial 16S rRNA genes from Illumina MiSeq platform. Over 1,642,482 quality-filtered sequences from 18 16S rRNA libraries were obtained and distinct compositions and diversities of gut microbiota were found in five flycatchers. Their gut microbiota is mainly from the four bacterial phyla of Proteobacteria, Firmicutes, Actinomycetes, and Bacteroidetes, but at the genus level showed a significant difference. Functional predictions revealed that the metabolic capacity of the gut microbiota of five flycatchers is greatly distinguished at KEGG level 3. And multiple food fragments showed a significant correlation with gut microbiota. Besides, the significant differences in the specific composition of the diets of the five insectivorous flycatchers indicated the differentiation of dietary niches. The study of the gut microbiota and feeding habits of sympatric flycatchers would increase the understanding of the gut microbial diversity of wild birds, and also improve our cognition of the co-evolution and co-adaptation within the host gut microbiota relations.

Three phylogenetically distinct and culturable diazotrophs are perennial symbionts of leaf-cutting ants.

The obligate mutualistic basidiomycete fungus, Leucocoprinus gongylophorus, mediates nutrition of leaf‐cutting ants with carbons from vegetal matter. In addition, diazotrophic Enterobacteriales in the fungus garden and intestinal Rhizobiales supposedly mediate assimilation of atmospheric nitrogen, and Entomoplasmatales in the genus Mesoplasma, as well as other yet unidentified strains, supposedly mediate ant assimilation of other compounds from vegetal matter, such as citrate, fructose, and amino acids. Together, these nutritional partners would support the production of high yields of leafcutter biomass. In the present investigation, we propose that three phylogenetically distinct and culturable diazotrophs in the genera Ralstonia, Methylobacterium, and Pseudomonas integrate this symbiotic nutrition network, facilitating ant nutrition on nitrogen. Strains in these genera were often isolated and directly sequenced in 16S rRNA libraries from the ant abdomen, together with the nondiazotrophs Acinetobacter and Brachybacterium. These five isolates were underrepresented in libraries, suggesting that none of them is dominant in vivo. Libraries have been dominated by four uncultured Rhizobiales strains in the genera Liberibacter, Terasakiella, and Bartonella and, only in Acromyrmex ants, by the Entomoplasmatales in the genus Mesoplasma. Acromyrmex also presented small amounts of two other uncultured Entomoplasmatales strains, Entomoplasma and Spiroplasma. The absence of Entomoplasmatales in Atta workers implicates that the association with these bacteria is not mandatory for ant biomass production. Most of the strains that we detected in South American ants were genetically similar with strains previously described in association with leafcutters from Central and North America, indicating wide geographic dispersion, and suggesting fixed ecological services.

First report of grapevine virus H (GVH) in grapevine in Greece.

Grapevine virus H (GVH) is a member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae, order Tymovirales) that infects grapevine (Candresse et al., 2018). GVH was first identified in a symptomless grapevine of an unknown cultivar from Portugal in 2018 (Candresse et al. 2018), and since then the virus has been reported only from California (Diaz‑Lara et al. 2019). Several vitiviruses have been detected in Greek vineyards (Avgelis and Roubos 2000; Dovas and Katis 2003a; 2003b; Panailidou et al. 2019; Lotos et al. 2020), but no information was available on the presence of GVH. In the fall of 2020, in order to investigate the virome of a commercial vineyard of the cultivar Assyrtiko in northern Greece, a composite sample was made of leaves and petioles from nine vines exhibiting leafroll disease symptoms. Total RNA was extracted from the composite sample according to the protocol of White et al. (2008) and subjected to rRNA depletion, library construction (TruSeq Stranded Total RNA kit), and high-throughput sequencing (HTS) in a NovaSeq6000 platform (Illumina Inc.) at Macrogen (Korea). The resulting ~42 million 101-nt paired-end reads were analyzed in Geneious Prime 2020, and the assembled de novo contigs were subjected to a local BLASTn search, which revealed the presence of 18 grapevine infecting viruses and viroids, among which also a GVH-like contig (GeA-9). GeA-9 was 7,404 nucleotides (nt) long, covering 99.4% of the full virus genome and shared 98.2 % nt identity with a GVH isolate from the USA (MN716768). To confirm the presence of GVH, the nine samples of the cultivar Assyrtiko, used initially to produce the composite sample for HTS analysis, were tested individually by RT-PCR, using the primers GVH_F_2504 (5'-CTGCTTCGCTGAACATATGC-3') and GVH_R_2835 (5'-ATCATTRTGATCGAGAGAGTAGTG-3') that amplify a 331-nt segment of ORF1. GVH was detected in five out of the nine tested samples and one of these was reamplified and subjected to Sanger sequencing. The fragment of ORF1 obtained by Sanger sequencing (MW460005) was 97.5% identical to the nucleotide sequence of the corresponding GVH-like de novo contig (GeA-9) from HTS analysis and it shared 97.2% nt identity with GVH sequences reported from Portugal and USA, respectively (NC_040545 and MN716768), confirming the presence of GVH in the tested samples. This is the first report of GVH in grapevine in Greece, thus further increasing the number of vitiviruses known to infect Greek vineyards and also expanding the number of geographic locations in which GVH is recorded so far.

Strength Lies in Diversity: How Community Diversity Limits Salmonella Abundance in the Chicken Intestine.

The transfer of the intestinal microbiota from adult to juvenile animals reduces Salmonella prevalence and abundance. The mechanism behind this exclusion is unknown, however, certain member species may exclude or promote pathogen colonization and Salmonella abundance in chickens correlates with intestinal community composition. In this study, newly hatched chicks were colonized with Salmonella Typhimurium and 16S rRNA libraries were generated from the cecal bacterial community at 21, 28, 35, and 42 days of age. Salmonella was quantified by real-time PCR. Operational taxonomic units (OTUs) were assigned, and taxonomic assignments were made, using the Ribosomal Database Project. Bacterial diversity was inversely proportional to the Salmonella abundance in the chicken cecum (p < 0.01). In addition, cecal communities with no detectable Salmonella (exclusive community) displayed an increase in the abundance of OTUs related to specific clostridial families (Ruminococcaceae, Eubacteriaceae, and Oscillospiraceae), genera (Faecalibacterium and Turicibacter) and member species (Ethanoligenens harbinense, Oscillibacter ruminantium, and Faecalibacterium prausnitzii). For cecal communities with high Salmonella abundance (permissive community), there was a positive correlation with the presence of unclassified Lachnospiraceae, clostridial genera Blautia and clostridial species Roseburia hominis, Eubacterium biforme, and Robinsoniella peoriensis. These findings strongly support the link between the intestinal bacterial species diversity and the presence of specific member species with Salmonella abundance in the chicken ceca. Exclusive bacterial species could prove effective as direct-fed microbials for reducing Salmonella in poultry while permissive species could be used to predict which birds will be super-shedders.

The Influence of Cayenne Pepper on the Human Gastrointestinal Microbiota and Intestinal Inflammation Among Overweight or Obese Adults

ObjectivesTo identify the impact of cayenne pepper on the diversity of the gut microbiome and inflammatory biomarkers in the stool of adults with overweight or obesity. Methods31 individuals participated. All participants had a BMI > 25. Most participants were female (64.5%) and averaged 28 ± 8 years of age. Participants consumed two 250 mL servings of tomato juice or tomato juice plus 1.9 grams (0.8 g per dose) of cayenne pepper each day for one week before crossing over to the other study arm. The study design allowed participants to continue eating a mixed, complex diet but sources of capsaicin, the pungent component of cayenne, were to be avoided. Stool samples were collected in the home at the end of each treatment period. DNA was extracted from the stools, 16S rRNA libraries were made, and libraries were sequenced using an Illumina MiSeq. Sequences were processed using mothur, and data were analyzed in R using the vegan package. ResultsThe spice intervention did not increase the richness of gut microbiota nor alter the overall gut microbiota composition at the genus level. However, at the single taxa at the genus level, participants who consumed tomato juice without cayenne had higher abundances of Prevotella and Bacteroides in their stool. Cayenne treatment did not affect either lipocalin or calprotectin levels in the stool. Calprotectin and lipocalin concentrations were positively correlated with each other, but only when participants were consuming cayenne pepper. Neither lipocalin nor calprotectin levels in stool of participants were related to gut microbiota richness or composition. ConclusionsThere was no detectable impact of the spice treatment on gut microbiota composition or intestinal inflammation in adults with overweight or obesity at the time of treatment. Though these results were unanticipated, given prior results in animal models, these results suggest that research in free-living humans must continue as treatments that are only effective under tightly controlled conditions will be of little use to most individuals. Funding SourcesAcademy of Nutrition and Dietetics Foundation/McCormick Science Institute as well as by startup funds provided by MSU and the MSU Department of Food Science and Human Nutrition.

Students in a Course-Based Undergraduate Research Experience Course Discovered Dramatic Changes in the Bacterial Community Composition Between Summer and Winter Lake Samples

Course-based undergraduate research experience (CURE) courses incorporate high-impact pedagogies that have been shown to increase undergraduate retention among underrepresented minorities and women. As part of the Building Infrastructure Leading to Diversity program at the University of Detroit Mercy, a CURE metagenomics course was established in the winter of 2019. Students investigated the bacterial community composition in a eutrophic cove in Lake Saint Clair (Harrison Township, MI, United States) from water samples taken in the summer and winter. The students created 16S rRNA libraries that were sequenced using next-generation sequencing technology. They used a public web-based supercomputing resource to process their raw sequencing data and web-based tools to perform advanced statistical analysis. The students discovered that the most common operational taxonomic unit, representing 31% of the prokaryotic sequences in both summer and winter samples, corresponded to an organism that belongs to a previously unidentified phylum. This result showed the students the power of metagenomics because the approach was able to detect unclassified organisms. Principal Coordinates Analysis of Bray–Curtis dissimilarity index data showed that the winter community was distinct from the summer community [Analysis of Similarities (ANOSIM) r = 0.59829, n = 18, and p < 0.001]. Dendrograms based on hierarchically clustered Pearson correlation coefficients of phyla were divided into a winter clade and a summer clade. The conclusion is that the winter bacterial population was fundamentally different from the summer population, even though the samples were taken from the same locations in a protected cove. Because of the small class sizes, qualitative as well as statistical methods were used to evaluate the course’s impact on student attitudes. Results from the Laboratory Course Assessment Survey showed that most of the respondents felt they were contributing to scientific knowledge and the course fostered student collaboration. The majority of respondents agreed or strongly agreed that the course incorporated iteration aspects of scientific investigations, such as repeating procedures to fix problems. In summary, the metagenomics CURE course was able to add to scientific knowledge and allowed students to participate in authentic research.

Effect of different fertilizers on the bacterial community diversity in rhizosperic soil of broomcorn millet (Panicum miliaceum L.)

ABSTRACT The bacterial community structure and diversity in the rhizosphere of broomcorn millet treated with different fertilizers were analyzed. The crops were grown under one of the four fertilization treatments: M1 (compound fertilizer), M2 (farmyard manure), M3 (biological bacterial fertilizer), and M4 (no fertilizer). The soil DNA was isolated, and the 16S rRNA library was sequenced using Illumina MiSeq sequencers. The bacterial diversity and the differential bacterial genera in four groups were analyzed. Compound fertilizers had the most significant effect on plant growth, followed by farmyard manure and then biological bacterial fertilizer (p < 0.05). Actinobacteria and Pseudomonas sp. were dominant in all soil samples. The abundance of Actinobacteria was higher in M1 and M4, and the abundance of Pseudomonas sp. was higher in M3 and M4. The number of bacterial genera (208) was the highest in M1. Eight bacterial genera with low abundance were common in M1 vs. M3 and M2 vs. M3. Four bacterial genera with high richness (Phormidium sp., Cellulosimicrobium sp., Yaniella sp., and Cloacibacterium sp.) overlapped in M1 vs. M3 and in M2 vs. M3. Compound fertilizer was the most effective at encouraging broomcorn millet growth (plant height, stem diameter, panicle weight, grain weight, and yield).

Strain-Level Analysis of Bifidobacterium spp. from Gut Microbiomes of Adults with Differing Lactase Persistence Genotypes.

One of the strongest associations between human genetics and the gut microbiome is a greater relative abundance of Bifidobacterium in adults with lactase gene (LCT) single nucleotide polymorphisms (SNPs) associated with lactase nonpersistence (GG genotypes), versus lactase persistence (AA/AG genotypes). To gain a finer-grained phylogenetic resolution of this association, we interrogated 1,680 16S rRNA libraries and 245 metagenomes from gut microbiomes of adults with various lactase persistence genotypes. We further employed a novel genome-capture-based enrichment of Bifidobacterium DNA from a subset of these metagenomes, including monozygotic (MZ) twin pairs, each sampled 2 or 3 times. B. adolescentis and B. longum were the most abundant Bifidobacterium species regardless of host LCT genotype. LCT genotypes could not be discriminated based on relative abundances of Bifidobacterium species or Bifidobacterium community structure. Three distinct metagenomic analysis methods of Bifidobacterium-enriched DNA revealed intraindividual temporal stability of B. longum, B. adolescentis, and B. bifidum strains against the background of a changeable microbiome. Two of our three methods also observed greater strain sharing within MZ twin pairs than within unrelated individuals for B. adolescentis, while no method revealed an effect of host LCT genotype on Bifidobacterium strain composition. Our results support a "rising tide lifts all boats" model for the dominant bifidobacteria in the adult gut: their higher abundance in lactase-nonpersistent than in lactase-persistent individuals results from an expansion at the genus level. Bifidobacterium species are known to be transmitted from mother to child and stable within individuals in infancy and childhood: our results extend this stability into adulthood.IMPORTANCE When humans domesticated animals, some adapted genetically to digest milk into adulthood (lactase persistence). The gut microbiomes of people with lactase-persistent genotypes (AA or AG) differ from those with lactase-nonpersistent genotypes (GG) by containing fewer bacteria belonging to the bifidobacteria, a group which contains beneficial species. Here, we asked if the gut microbiomes of adults with GG and AA/AG genotypes differ in the species of bifidobacteria present. In particular, we used a novel technique which allowed us to compare bifidobacteria in adults at the strain level, without the traditional need for culturing. Our results show that the GG genotype enhances the abundance of bifidobacteria regardless of species. We also noted that a person's specific strains are recoverable several years later, and twins can share the same ones. Given that bifidobacteria are inherited from mother to child, strain stability over time in adulthood suggests long-term, multigenerational inheritance.