16S rRNA Hypervariable Region Research Articles

Microbial Communities Associated with the Intestinal Tract of Grey Mullets from a Mediterranean Aquatic Environment

Introduction: Grey mullets comprise different species that represent the most ubiquitous teleost families in the planet’s coastal waters. They are an important proportion of the Mediterranean lagoon’s production and have been recently considered cultivated marine fish. This study aimed to explore the intestinal microbial communities of grey mullets to understand their possible ecological role for fish and the aquatic environment. Methods: Thirty-four wild-caught mullets were sampled from a Mediterranean lagoon during four seasons and the V3-V4 hypervariable regions of 16S rRNA (Illumina MiSeq) of the fish gut were sequenced. Parameters of the aquatic environment were detected: temperature, salinity, DO, PO4, NH4, NO3, NO2, SiO, DIN, and Chla. Results: The results indicated a high bacterial diversity (mean Shannon index: 4.74 ± 1.12; Simpson index: 0.93 ± 0.08) and variations among seasons. Sixty prokaryotic phyla were identified and the most abundant ones were: Proteobacteria (mean relative abundance 35.4% ± 17.9), Actinobacteriota (mean relative abundance 16.4% ± 9.9), and Firmicutes (mean relative abundance 10.1% ± 10.9). Bacteria belonging to the phylum Chloroflexi were relevant in autumn, Spirochaetota, Verrucomicrobiota, Fusobacteriota, and Cyanobacteria were particularly abundant in winter while Bacteroidota characterized summer fish. A total of 332 prokaryotic families were identified with 26 most abundant ones; Rhodociclaceae (Proteobacteria) were dominant in autumn, Brevinemataceae (Spirocheaetota) and Fusobacteriaceae (Fusobacteriota) were especially present in winter and the Staphylococcaceae (Firmicutes) prevailed in spring. Conclusion: This study sheds light on the variation in the complex gut microbial community structure of Mediterranean grey mullets and their potential ecological role in protecting fish and preserving the aquatic environment.

Testing 16S Primers for Proper Identification of Cyanobacterial Communities in Small Water Bodies

The majority of investigations on microbial communities from various environments are presently built on culture-independent methods. Many studies point to the pivotal, selective role of primers targeting hypervariable regions of 16S rRNA in the metabarcoding of bacteria, including cyanobacterial communities. The selectivity of primers designed to amplify targeted regions of the 16S rRNA gene, which has been highlighted by many authors, limited effective amplification. Moreover, the type and specificity of the studied material can also negatively influence the results of 16S metabarcoding. Most of the studies of cyanobacterial communities have been performed for planktonic microbial communities that are often represented by common, well-studied species. In this study, we present the results of 16S metabarcoding analysis using three primer pairs—two already well-known and a third designed in this study—that amplify divergent regions of the 16S rRNA gene (V3–V4, V4–V6, V6) for benthic, microbial mat-forming cyanobacteria communities. Such communities can be a source of toxigenic cyanobacterial taxa and should be monitored with adequate primers. The comparison of three primer pairs suggested that those designed within the present study describe the structure and composition of highly heterogeneous cyanobacterial mats’ communities better than the others.

Analysis of the gut microbiota profile targeted to multiple hypervariable regions of 16S rRNA in a hypertensive heart failure rat model.

The gut microbiota, comprising a diverse community of microorganisms, significantly influences various aspects of health. Changes in the composition of the gut microbiota are implicated in adverse effects on host physiology, contributing to the pathogenesis of cardiovascular diseases, among others pathological conditions. Understanding the role of the gut microbiota in the context of heart failure is particularly important. In this regard, the spontaneously hypertensive heart failure (SHHF) rat is an adequate experimental model since exhibits many features in common with heart failure (HF) in humans. Recent advancements in next-generation sequencing (NGS) have greatly improved microbiome analysis. However, standardization and the adoption of best practices are essential to mitigate experimental variations across studies. This manuscript outlines a straightforward methodology for analyzing gut microbiota composition in SHHF rat fecal samples using 16S rRNA sequencing, emphasizing the relevance of gut microbiota in heart failure.

Unraveling the impact of primary immunodeficiency disorders on the microbiota of dental caries in children through 16S rRNA gene-based metagenomic analysis.

Dental caries is a frequently occurring and multifactorial chronic disease in children resulting from the interaction of cariogenic bacteria and host susceptibility. The aim of this study was to elucidate the impacts of primary immunodeficiency disorders (PIDs) on microbiota of dental caries in children by 16S rRNA gene-based metagenomic analysis. Enrolled in this study were 15 children with primary PID with caries (PID group) and 15 healthy children with caries as a control (CG). The DMFT index, saliva flow rate, and buffering capacity of each participant were assessed before the metagenomic analyses were conducted. For taxonomic profiling, the reads were obtained by high-throughput sequencing of the V3-V4 hypervariable region of 16S rRNA. The DMFT score, saliva flow rate, and buffering capacity of the groups were similar. The flow rate and buffering capacity had no correlation with the number of species with 95% confidence. The metagenomic analysis resulted in the identification of 2440 bacterial species in all of the samples. Among the 50 most prevalent species present at ≥1% relative abundance, Prevotella melaninogenica and Prevotella salivae were differentially more abundant in the PID group. The PID group and CG showed similar species richness and evenness, but 4 of the 5 samples with the highest Shannon-Weiner and Inverse Simpson indices belonged to the PID group. The Spearman test results for correlation of the species in the PID subgroups showed that Prevotella oris had a positively correlated relationship with both Scardovia wiggsiae and Saccharibacteria genera incertae sedis. This study provided insight into the caries microbiota of children with immunodeficiency diseases. Differentially abundant species, novel bacterial associations, and unique bacterial species were disclosed in the PID samples, indicating the role of the immune system in altering the caries microbiota. The prominent bacterial species and associations in the PID group should be suspected in regard to their link with present or future diseases.

Decoding Microbial Networks: An Insight into 16S rRNA and Whole Genome Sequencing Approaches in Metagenomic Studies

The exploration of microbial communities is pivotal in understanding environmental, human, and animal ecosystem dynamics. Advances in high-throughput sequencing technologies have significantly enriched our insights into microbiomes, with 16S rRNA and Whole Genome Sequencing (WGS) being central methodologies. This review delineates a comparative analysis of these sequencing techniques, particularly focusing on different hypervariable regions of 16S rRNA (V3-V4 and V2-3-4-6-7-8-9) and WGS. The 16S rRNA sequencing, despite being cost-effective and less computationally demanding, often limits identification to the genus level. In contrast, WGS, while being resource-intensive, provides a broader spectrum of microbial identification including bacteria, viruses, fungi, and parasites, alongside a deeper insight into microbial functional attributes. The challenges associated with sequencing depth in WGS are discussed, alongside emerging mitigating strategies like host DNA depletion. The choice between these methodologies hinges on the project objectives and available resources. This comparative assessment aims to guide researchers in selecting the apt sequencing approach for their metagenomic studies, thereby facilitating a more extensive understanding of microbial ecosystems.

Associations of gut microbiome with endogenous estrogen levels in healthy postmenopausal women.

The gut microbiome is a potentially important contributor to endogenous estrogen levels after menopause. In healthy postmenopausal women, we examined associations of fecal microbiome composition with levels of urinary estrogens, their metabolites, and relevant metabolic pathway ratios implicated in breast cancer risk. Eligible postmenopausal women (n = 164) had a body mass index (BMI) ≤ 35kg/m2 and no history of hormone use (previous 6months) or cancer/metabolic disorders. Estrogens were quantified in spot urine samples with liquid chromatography-high resolution mass spectrometry (corrected for creatinine). Bacterial DNA was isolated from fecal samples and the V1-V2 hypervariable regions of 16S rRNA were sequenced on the Illumina MiSeq platform. We examined associations of gut microbiome's indices of within-sample (alpha) diversity (i.e., Shannon, Chao1, and Inverse Simpson), phylogenetic diversity, and the ratio of the two main phyla (Firmicutes and Bacteroidetes; F/B ratio) with individual estrogens and metabolic ratios, adjusted for age and BMI. In this sample of 164 healthy postmenopausal women, the mean age was 62.9years (range 47.0-86.0). We found significant inverse associations of observed species with 4-pathway:total estrogens (p = 0.04) and 4-pathway:2-pathway (p = 0.01). Shannon index was positively associated with 2-catechols: methylated 2-catechols (p = 0.04). Chao1 was inversely associated with E1:total estrogens (p = 0.04), and 4-pathway:2-pathway (p = 0.02) and positively associated with 2-pathway:parent estrogens (p = 0.01). Phylogenetic diversity was inversely associated with 4-pathway:total estrogens (p = 0.02), 4-pathway:parent estrogens (p = 0.03), 4-pathway:2-pathway (p = 0.01), and 4-pathway:16-pathway (p = 0.03) and positively associated with 2-pathway:parent estrogens (p = 0.01). F/B ratio was not associated with any of the estrogen measures. Microbial diversity was associated with several estrogen metabolism ratios implicated in breast cancer risk. Further studies are warranted to confirm these findings in a larger and more representative sample of postmenopausal women, particularly with enrichment of minority participants.

Microbial Community Diversity of Coral Reef Sediments on Liuqiu Island, Southwestern Taiwan

Microbes in coral reef sediments are thought to play an important role in organic matter remineralization and nutrient recycling. Microbial communities also reflect the environmental conditions, such as nutrient status, of an ecosystem. This study investigates the relationship between microbial community diversity in the reef sediments and environmental conditions at Liuqiu Island. We sampled sediments seasonally from four sites around the island, Beauty Cave, Geban Bay, Houshi Fringing Reef, and Lobster Cave, from 2015–2020. The V5–V6 hypervariable region of 16S rRNA was amplified and sequenced using the Illumina MiSeq platform to identify the microbial communities. The results showed that the high abundance of Pseudomonadota, Planctomycetota, and Bacteroidota might reflect the eutrophic environments of the sediments on Liuqiu Island. Second, the identification of putative pathogens and human-related genera suggests that human activities have affected the marine environment of Liuqiu Island. Third, the insignificant spatial differences and the significant temporal differences in the microbial communities of Liuqiu Island indicate that annual or periodical events, such as the Kuroshio Branch Current and South China Sea Surface Current, could shape the microbial communities of Liuqiu Island. Furthermore, the abundance of human-related genera—Cutibacterium, Herbaspirillum, Corynebacterium 1, Escherichia-Shigella, and Kocuria—increased dramatically in the Lobster Cave site in September 2015 and may have been induced by a strong climate event, such as a typhoon or heavy rainfall. Our results revealed that the microbial communities of Liuqiu Island are dynamic and sensitive to adjacent environmental conditions. The sedimented microbial communities could monitor the bacteria and pathogens related to human activities and even reveal the putative events that could affect the ecological environments.

Molecular detection of pathogenic bacteria in Rhipicephalus sanguineus (sensu lato) ticks from Bitung, North Sulawesi, Indonesia

Abstract. Tahulending J, Tulung M, Pelealu J, Doda DV. 2022. Molecular detection of pathogenic bacteria in Rhipicephalus sanguineus (sensu lato) ticks from Bitung, North Sulawesi, Indonesia. Biodiversitas 23: 6164-6170. Domesticated dog populations have grown throughout Indonesia, notably in Bitung City. Dogs are frequently kept as home guardians and reside with their owners. Dogs are typically attacked by ectoparasites, parasites that adhere to the skin's surface and potentially spread the disease microorganisms to humans, animals, and the environment. As a specific host, ticks are frequently found in dogs. This research aims to identify and analyze the molecular character bacterial of Rhipicephalus sanguineus ticks in Bitung City. A hypervariable region of 16S rRNA called V3-V4 was selected for the DNA barcode area. Dogs' ticks have been collected from several areas in Bitung. The microbes inside the digestive tract were analyzed using genomic DNA with a gSYNC DNA extraction kit, and PCR amplification using KOD FX Neo and MyTaq HS red mix. At First Base Singapore, gel electrophoresis and sequencing were carried out. The collected sequencing data were analyzed using MEGA X and the BLAST, connected online with the NCBI's GenBank. The nine distinct bacteria were identified inside the digestive tract of ticks. Pseudomonas stutzeri (35%), Brevundimonas sp. (21%), and Aquamicrobium lusatiense (17%) were the pathogens that were most frequently found. These results enhance knowledge of the pathogen microbes that may impact either humans or animals.

Bacterial Compositional Shifts of Gut Microbiomes in Patients with Rheumatoid Arthritis in Association with Disease Activity

Background: Rheumatoid arthritis (RA) is a chronic inflammatory disabling autoimmune disorder. Little is known regarding the association between the gut microbiome and etiopathogenesis of RA. We aimed to dissect the differences in gut microbiomes associated with RA in comparison to healthy individuals and, in addition, to identify the shifts in the bacterial community in association with disease activity; Methods: In order to identify compositional shifts in gut microbiomes of RA patients, V3-V4 hypervariable regions of 16S rRNA were sequenced using Illumina MiSeq. In total, sixty stool samples were collected from 45 patients with RA besides 15 matched healthy subjects; Results: Notably, RA microbiomes were significantly associated with diverse bacterial communities compared with healthy individuals. Likewise, a direct association between bacterial diversity and disease activity was detected in RA patients (Kruskal Wallis; p = 0.00047). In general, genus-level analysis revealed a positive coexistence between RA and Megasphaera, Adlercreutzia, Ruminococcus, Bacteroides, Collinsella, and Acidaminococcus. Furthermore, Spearman correlation analysis significantly stratified the most dominant genera into distinct clusters that were mainly based on disease activity (r ≥ 0.6; p ≤ 0.05). The predictive metabolic profile of bacterial communities associated with RA could support the potential impact of gut microbiomes in either the development or recovery of RA; Conclusions: The overall shifts in bacterial composition at different disease statuses could confirm the cross-linking of certain genera either to causation or progression of RA.

Detection of human pathogenic bacteria in rectal DNA samples from Zalophus californianus in the Gulf of California, Mexico

Human intrusions into undisturbed wildlife areas greatly contribute to the emergence of infectious diseases. To minimize the impacts of novel emerging infectious diseases (EIDs) on human health, a comprehensive understanding of the microbial species that reside within wildlife species is required. The Gulf of California (GoC) is an example of an undisturbed ecosystem. However, in recent decades, anthropogenic activities within the GoC have increased. Zalophus californianus has been proposed as the main sentinel species in the GoC; hence, an assessment of sea lion bacterial microbiota may reveal hidden risks for human health. We evaluated the presence of potential human pathogenic bacterial species from the gastrointestinal (GI) tracts of wild sea lions through a metabarcoding approach. To comprehensively evaluate this bacterial consortium, we considered the genetic information of six hypervariable regions of 16S rRNA. Potential human pathogenic bacteria were identified down to the species level by integrating the RDP and Pplacer classifier outputs. The combined genetic information from all analyzed regions suggests the presence of at least 44 human pathogenic bacterial species, including Shigella dysenteriae and Bacillus anthracis. Therefore, the risks of EIDs from this area should be not underestimated.

Microbiota profiling on itchy scalp with undetermined origin.

Scalp pruritus is a common skin problem that remains therapeutic challenge. The relationships between the dysbiosis of microbiota and skin diseases have caught attention recently. However, there are few reports about microbiota on itchy scalp. Thisstudy investigated scalp microbial characteristics of subjects with mild scalp pruritus of undetermined origin and preliminarily screened physiological factors and bacteria potentially related to pruritus. The pruritus severity of 17 qualified females was evaluated by Visual Analogue Scale (VAS). Microbiota collection was done at both itchy (n = 20) and non-itchy sites (n = 27) at occiput and crown of the same subject and Illumina sequencing was performed at the V3-V4 hypervariable regions of 16S rRNA. The corresponding sebum content, hydration, pH, trans-epidermal water loss, erythema index and porphyrin numbers were also measured by skin tester. We identified 3044 ampliconsequence variants from 821 genera. The itchy and non-itchy sites had different microbiota structures (p = 0.045, by multivariate analysis of variance), while there were large inter- and intra-individual variations. Both sites had Staphylococcus, Cutibacterium and Lawsonella as predominant genera, which were not significantly related to pruritus. The use of three genera Lactobacillus, Morganella and Pseudomonas, could well distinguish non-itchy from itchy groups, whereas different composition patterns existed inside each group. Our investigation indicated that though the bacterial community structure on itchy scalp was individual specific, there was difference between itchy and non-itchy sites. The study provides new insights into microbiota profiling on itchy scalp, which will help microbiota-targeted therapeutic experiment or products design for scalp pruritus.

Comparison of Gut Microbiota Diversity Between Captive and Wild Tokay Gecko (Gekko gecko).

Captive animals and wild animals may exhibit different characteristics due to the heterogeneity of their living environments. The gut microbiota play an important role in the digestion and absorption, energy metabolism, immune regulation, and physiological health of the host. However, information about the gut microbiota of captive and wild Gekko gecko is currently limited. To determine the difference in gut microbiota community composition, diversity, and structure between captive and wild geckos, we used the Illumina miseq platform to conduct high-throughput sequencing and bioinformatics analysis of the v3–v4 hypervariable region of 16S rRNA in 54 gecko samples. Our results showed that Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the dominant gut microbiota phyla of the gecko. The dominant genera comprised mainly Pseudomonas, Burkholderia-caballeronia-paraburkholderia, Ralstonia, Romboutsia, and Bacteroides. Captive geckos had significantly higher alpha diversity and potential pathogenic bacteria than wild populations. Moreover, significant differences in beta diversity of gut microbiota were observed between two populations. Functional prediction analysis showed that the relative abundance of functional pathways of wild geckos was more higher in metabolism, genetic information processing and organismal system function than those in captive geckos. Total length significantly affected gut microbial community (R2 = 0.4527, p = 0.001) and explained 10.45% of the total variation for gut microbial community variance between two groups. These results may be related to differences in diet and living environment between two populations, suggesting that the management of captive populations should mimic wild environments to the greatest extent possible to reduce the impact on their gut microbiota.

Microbiota of the Tongue and Systemic Connections: The Examination of the Tongue as an Integrated Approach in Oral Medicine

The tongue is able to quickly reflect the state of health or disease of the human body. Tongue inspection is an important diagnostic approach. It is a unique method that allows to explore the pathogenesis of diseases based on the guiding principles of the holistic concept that involves the observation of changes in the lining of the tongue in order to understand the physiological functions and pathological changes of the body. It is a potential method of screening and early detection of cancer. However, the subjective inspection of the tongue has a low reliability index, and therefore computerized systems of acquisition of diagnostic bioinformation have been developed to analyze the lining of the tongue. Next-generation sequencing technology is used to determine the V2–V4 hypervariable regions of 16S rRNA to study the microbiota. A lot of neoplasms are identified only at an advanced phase, while in the early stages, many subjects remain in an asymptomatic form. On the contrary, the early diagnosis is able to increase the prognosis of cancer and improve the survival rates of subjects. Evidently, it is necessary to develop new strategies in oral medicine for the early diagnosis of diseases, and the diagnosis of the tongue as a minimally invasive method is certainly one of them.

Gut microbiome, body weight, and mammographic breast density in healthy postmenopausal women.

We examined gut microbiome (GM) profiles in relation to mammographic breast density (BD) and body mass index (BMI) in healthy postmenopausal women. Eligible women were postmenopausal, had a BMI ≤ 35kg/m2, and had not recently taken oral/IV antibiotics. All women provided a fecal sample and information on breast cancer risk factors. Mammographic BD was classified with the American College of Radiology's BI-RADS BD classification system. Bacterial DNA was isolated from fecal samples and the V1-V2 hypervariable regions of 16S rRNA were sequenced on the Illumina MiSeq platform. We examined associations of GM with indices of within-sample (alpha) diversity and the ratio of the two main phyla (Firmicutes and Bacteroidetes; F/B ratio) with BD and BMI. Among 69 women with BD data, 39 had low BD (BI-RADS I/II) and 30 had high BD (BI-RADS III/IV). BMI was inversely associated with BD (mean BMI = 23.8 and 28.0 in women with high and low BD, respectively, p = 1.07 × 10-5). Similar levels of GM diversity were found across weight groups according to Shannon (p = 0.83); Inverse Simpson (p = 0.97); and Chao1 (p = 0.31) indices. F/B ratio and microbiota diversity were suggestively greater in women with high vs. low BD (p = 0.35, 0.14, 0.15, and 0.17 for F/B ratio, Shannon, Inverse Simpson and Chao1, respectively). Suggestive differences observed in women with high and low BD with respect to GM alpha diversity and prevalence of specific GM taxa need to be confirmed in larger studies.

Microbial community structure in a host–parasite system: the case of Prussian carp and its parasitic crustaceans

The aim of the study was to investigate the skin microbiota of Prussian carp infested by ectoparasites from the genera Argulus and Lernaea. Associated microbiota of skin of Prussian carp and ectoparasites were investigated by sequencing of the V3, V4 hypervariable regions of 16S rRNA using Illumina MiSeq sequencing platform. According to the Spearman rank correlation test, the increasing load of ulcerations of the skin of Prussian carp was weakly negatively correlated with reduction in the abundance of the following taxa: Acrobacter, bacteria C39 (Rhodocyclaceae), Rheinheimera, Comamonadaceae, Helicobacteraceae and Vogesella. In this study, the microbiota of ectoparasites from the genera Lernaea and Argulus were characterized for the first time. The microbiota associated with L. cyprinacea was significantly different from microbial communities of intact skin mucosa of both infested and uninfested fish and skin ulcers (ADONIS, P≤0·05). The microbiota associated with parasitic crustaceans L. cyprinacea were dominated by unclassified bacteria from Comamonadaceae, Aeromonadaceae families and Vogesella. The dominant microbiota of A. foliaceus were represented by Flavobacterium, Corynebacterium and unclassified Comamonadaceae. Results from these studies indicate that ectoparasites have the potential to alter skin microbiota, which can play a possible role in the transmission of secondary bacterial infections in fish, caused by pathogenic bacteria.

Effects of Increasing Stocking Density on the Performance and Ileal Microbiota of Broilers.

This study was conducted to investigate the effects of increasing stocking density under suitable environmental conditions on the performance and ileal microbiota of broilers. A total of 108 Arbor Acres male broilers (28 days old) were allocated to a normal stocking density (NSD, normal stocking density; 31 kg/m2) and a maximum allowed stocking density group (MSD, maximum stocking density; 39 kg/m2). All birds were reared at a constant temperature of 21°C. At 42 days of age, bacterial DNA was extracted from ileal content, and the V3–4 hypervariable region of 16S rRNA was amplified. Increasing stocking density had no significant effect on average daily gain, average daily feed intake, and feed conversion ratio (P>0.05). The alpha and beta diversities of the ileal microbiomes did not differ significantly between the NSD and MSD groups; however, increasing stocking density altered the composition of ileal microbiota. The relative abundance of Lactobacillales, including Lactobacillus, Enterococcus, and Streptococcus, significantly decreased in MSD broilers, compared with NSD broilers. The present results suggest that even under suitable environmental conditions, an increase in stocking density to a level of 39 kg/m2 may disturb the composition of ileal microbiota in broilers. Further studies are needed to determine the reasons and the potential consequences for animal health and physiology.

Comparison of different hypervariable regions of 16S rRNA for taxonomic profiling of vaginal microbiota using next-generation sequencing.

The exploration of vaginal microbiota by using next-generation sequencing (NGS) of 16S ribosomal RNA (rRNA) gene is widely used. Up to now, different hypervariable regions have been selected to study vaginal microbiota by NGS and there is no standard method for analysis. The study aimed to characterize vaginal microbiota from clinical samples using NGS targeting the 16S rRNA gene and to determine the performance of individual and concatenated hypervariable region sequences to generate the taxonomic profiles of the vaginal microbiota. Fifty-one vaginal DNA samples were subjected to 16S rRNA gene NGS based on the Ion Torrent PGM platform with the use of two primer sets spanning seven hypervariable regions of the 16S rRNA gene. Our analysis revealed that the predominant bacterial genera were Lactobacillus, Gardnerella and Atopobium, which accounted for 78%, 14% and 2%, respectively, of sequences from all vaginal bacterial genera. At the species level, Lactobacillus iners, Gardnerella vaginalis and Atopobium vaginae accounted for 72%, 10% and 6%, respectively, of the bacterial cells present. Analyses using the V3 region generally indicated the highest bacterial diversity followed by the V6-V7 and V4 regions, while the V9 region gave the lowest bacterial resolution. NGS based on the 16S rRNA gene can give comprehensive estimates of the diversity of vaginal bacterial communities. Selection of sequences from appropriate hypervariable regions is necessary to provide reliable information on bacterial community diversity.

Data-Driven Modeling for Species-Level Taxonomic Assignment From 16S rRNA: Application to Human Microbiomes.

With the emergence of next-generation sequencing (NGS) technology, there have been a large number of metagenomic studies that estimated the bacterial composition via 16S ribosomal RNA (16S rRNA) amplicon sequencing. In particular, subsets of the hypervariable regions in 16S rRNA, such as V1–V2 and V3–V4, are targeted using high-throughput sequencing. The sequences from different taxa are assigned to a specific taxon based on the sequence homology. Since such sequences are highly homologous or identical between species in the same genus, it is challenging to determine the exact species using 16S rRNA sequences only. Therefore, in this study, homologous species groups were defined to obtain maximum resolution related with species using 16S rRNA. For the taxonomic assignment using 16S rRNA, three major 16S rRNA databases are independently used since the lineage of certain bacteria is not consistent among these databases. On the basis of the NCBI taxonomy classification, we re-annotated inconsistent lineage information in three major 16S rRNA databases. For each species, we constructed a consensus sequence model for each hypervariable region and determined homologous species groups that consist of indistinguishable species in terms of sequence homology. Using a k-nearest neighbor method and the species consensus sequence models, the species-level taxonomy was determined. If the species determined is a member of homologous species groups, the species group is assigned instead of a specific species. Notably, the results of the evaluation on our method using simulated and mock datasets showed a high correlation with the real bacterial composition. Furthermore, in the analysis of real microbiome samples, such as salivary and gut microbiome samples, our method successfully performed species-level profiling and identified differences in the bacterial composition between different phenotypic groups.

Association between Altered Blood Parameters and Gut Microbiota after Synbiotic Intake in Healthy, Elderly Korean Women.

Synbiotics intake can alter the composition of intestinal microbes beneficially. We aimed to detect the changes in the intestinal microbiomes of 37 healthy elderly Korean women after the intake of a synbiotic drink. This was a longitudinal study controlled with a temporal series, including a control period of 3 weeks before intake, synbiotic intake for 3 weeks, and a washout period of 3 weeks. Fecal microbiota composition was analyzed by sequencing the V3-V4 hypervariable regions of 16S rRNA. Physical fecal activity increased with improvement in fecal shape. Thirty intestinal bacterial taxa were observed to change only after the intake period. In particular, Ellagibacter appeared only after ingestion. In addition, the abundance of Terrisporobacter showed a positive correlation with C-reactive protein, triglyceride. Lachnospiraceae_uc, Eubacterium_g5, and Blautia had a positive correlation with creatinine, whereas PAC001100_g had a negative correlation with creatinine. Short-term (3 weeks) intake of symbiotic organisms changes the composition of the gut microbiota in healthy elderly Korean women.

Effect of age and the individual on the gastrointestinal bacteriome of ponies fed a high-starch diet.

Bacteria residing in the gastrointestinal tract of mammals are crucial for the digestion of dietary nutrients. Bacterial community composition is modified by age and diet in other species. Although horses are adapted to consuming fibre-based diets, high-energy, often high-starch containing feeds are increasingly used. The current study assessed the impact of age on the faecal bacteriome of ponies transitioning from a hay-based diet to a high-starch diet. Over two years, 23 Welsh Section A pony mares were evaluated (Controls, 5–15 years, n = 6/year, 12 in total; Aged, ≥19 years, n = 6 Year 1; n = 5 Year 2, 11 in total). Across the same 30-week (May to November) period in each year, animals were randomly assigned to a 5-week period of study and were individually fed the same hay to maintenance (2% body mass as daily dry matter intake) for 4-weeks. During the final week, 2g starch per kg body mass (micronized steam-flaked barley) was incorporated into the diet (3-day transition and 5 days at maximum). Faecal samples were collected for 11 days (final 3 days hay and 8 days hay + barley feeding). Bacterial communities were determined using Ion Torrent Sequencing of amplified V1–V2 hypervariable regions of 16S rRNA. Age had a minimal effect on the bacteriome response to diet. The dietary transition increased Candidatus Saccharibacteria and Firmicutes phyla abundance and reduced Fibrobactres abundance. At the genera level, Streptococcus abundance was increased but not consistently across individual animals. Bacterial diversity was reduced during dietary transition in Streptococcus ‘responders’. Faecal pH and VFA concentrations were modified by diet but considerable inter-individual variation was present. The current study describes compositional changes in the faecal bacteriome associated with the transition from a fibre-based to a high-starch diet in ponies and emphasises the individual nature of dietary responses, which may reflect functional differences in the bacterial populations present in the hindgut.