Sequencing Of Hypervariable Region Research Articles

Short-read full-length 16S rRNA amplicon sequencing for characterisation of the respiratory bacteriome of captive and free-ranging African elephants (Loxodonta africana)

Hypervariable region sequencing of the 16S ribosomal RNA (rRNA) gene plays a critical role in microbial ecology by offering insights into bacterial communities within specific niches. While providing valuable genus-level information, its reliance on data from targeted genetic regions limits its overall utility. Recent advances in sequencing technologies have enabled characterisation of the full-length 16S rRNA gene, enhancing species-level classification. Although current short-read platforms are cost-effective and precise, they lack full-length 16S rRNA amplicon sequencing capability. This study aimed to evaluate the feasibility of a modified 150 bp paired-end full-length 16S rRNA amplicon short-read sequencing technique on the Illumina iSeq 100 and 16S rRNA amplicon assembly workflow by utilising a standard mock microbial community and subsequently performing exploratory characterisation of captive (zoo) and free-ranging African elephant (Loxodonta africana) respiratory microbiota. Our findings demonstrate that, despite generating assembled amplicons averaging 869 bp in length, this sequencing technique provides taxonomic assignments consistent with the theoretical composition of the mock community and respiratory microbiota of other mammals. Tentative bacterial signatures, potentially representing distinct respiratory tract compartments (trunk and lower respiratory tract) were visually identified, necessitating further investigation to gain deeper insights into their implication for elephant physiology and health.

The effect of different sweeteners on the oral microbiome: a randomized clinical exploratory pilot study

ABSTRACT Introduction The aim of the study was to evaluate the modulating effects of five commonly used sweetener (glucose, inulin, isomaltulose, tagatose, trehalose) containing mouth rinses on the oral microbiome. Methods A single-centre, double-blind, parallel randomized clinical trial was performed with healthy, 18–55-year-old volunteers (N = 65), who rinsed thrice-daily for two weeks with a 10% solution of one of the allocated sweeteners. Microbiota composition of supragingival dental plaque and the tongue dorsum coating was analysed by 16S RNA gene amplicon sequencing of the V4 hypervariable region (Illumina MiSeq). As secondary outcomes, dental plaque red fluorescence and salivary pH were measured. Results Dental plaque microbiota changed significantly for two groups: inulin (F = 2.0239, p = 0.0006 PERMANOVA, Aitchison distance) and isomaltulose (F = 0.67, p = 0.0305). For the tongue microbiota, significant changes were observed for isomaltulose (F = 0.8382, p = 0.0452) and trehalose (F = 1.0119, p = 0.0098). In plaque, 13 species changed significantly for the inulin group, while for tongue coating, three species changed for the trehalose group (ALDEx2, p < 0.1). No significant changes were observed for the secondary outcomes. Conclusion The effects on the oral microbiota were sweetener dependant with the most pronounced effect on plaque microbiota. Inulin exhibited the strongest microbial modulating potential of the sweeteners tested. Further full-scale clinical studies are required.

Effects of Scallop Mantle Toxin on Intestinal Microflora and Intestinal Barrier Function in Mice.

Previous studies have shown that feeding mice with food containing mantle tissue from Japanese scallops results in aggravated liver and kidney damage, ultimately resulting in mortality within weeks. The aim of this study is to evaluate the toxicity of scallop mantle in China's coastal areas and explore the impact of scallop mantle toxins (SMT) on intestinal barrier integrity and gut microbiota in mice. The Illumina MiSeq sequencing of V3-V4 hypervariable regions of 16S ribosomal RNA was employed to study the alterations in gut microbiota in the feces of SMT mice. The results showed that intestinal flora abundance and diversity in the SMT group were decreased. Compared with the control group, significant increases were observed in serum indexes related to liver, intestine, inflammation, and kidney functions among SMT-exposed mice. Accompanied by varying degrees of tissue damage observed within these organs, the beneficial bacteria of Muribaculaceae and Marinifilaceae significantly reduced, while the harmful bacteria of Enterobacteriaceae and Helicobacter were significantly increased. Taken together, this article elucidates the inflammation and glucose metabolism disorder caused by scallop mantle toxin in mice from the angle of gut microbiota and metabolism. SMT can destroy the equilibrium of intestinal flora and damage the intestinal mucosal barrier, which leads to glucose metabolism disorder and intestinal dysfunction and may ultimately bring about systemic toxicity.

Longitudinal evaluation of the cutaneous and rectal microbiota of German shepherd dogs with perianal fistulas undergoing therapy with ciclosporin and ketoconazole.

Perianal fistulas are painful ulcers or sinus tracts that disproportionately affect German shepherd dogs and are proposed as a spontaneous animal model of fistulising Crohn's disease. To characterise the rectal and cutaneous microbiota in German shepherd dogs with perianal fistulas and to investigate longitudinal shifts with lesion resolution during immunomodulatory therapy. Eleven German shepherd dogs with perianal fistulas and 15 healthy German shepherd dogs. Affected dogs were evaluated and swabbed at three visits, 30 days apart, while undergoing treatment with ciclosporin and ketoconazole. Healthy German shepherd dogs were contemporaneously sampled. Sites included the rectum, perianal skin and axilla. The microbiome was evaluated following sequencing of the V4 hypervariable region of the 16S ribosomal RNA (rRNA) gene. Alpha diversity was not significantly different between healthy and affected dogs at each of the three body sites (p > 0.5), yet rectal and perianal beta diversities from affected dogs differed significantly from those of healthy dogs at Day 0 (p = 0.004). Rectal and perianal relative abundance of Prevotella spp. increased and perianal Staphylococcus spp. relative abundance decreased in affected dogs over time, coincident with lesion resolution. Changes in lesional cutaneous and rectal microbiota occur in German shepherd dogs with perianal fistulas and shift over time with lesion resolution during immunomodulatory therapy. Further investigations of the role of cutaneous and enteric microbiota in the pathogenesis of perianal fistulas, and whether manipulation of microbial populations may ameliorate disease, are needed.

Bacterial communities associated with an island radiation of lichen-forming fungi.

Evolutionary radiations are one of the most striking processes biologists have studied in islands. A radiation is often sparked by the appearance of ecological opportunity, which can originate in processes like trophic niche segregation or the evolution of key innovations. Another recently proposed mechanism is facilitation mediated by the bacterial communities associated with the radiating species. Here we explore the role of the bacterial communities in a radiation of lichen-forming fungi endemic to Macaronesia. Bacterial diversity was quantified by high throughput sequencing of the V1-V2 hyper-variable region of 172 specimens. We characterized the taxonomic and phylogenetic diversity of the bacterial communities associated with the different species, tested for compositional differences between these communities, carried out a functional prediction, explored the relative importance of different factors in bacterial community structure, searched for phylosymbiosis and tried to identify the origin of this pattern. The species of the radiation differed in the composition of their bacterial communities, which were mostly comprised of Alphaproteobacteria and Acidobacteriia, but not in the functionality of those communities. A phylosimbiotic pattern was detected, but it was probably caused by environmental filtering. These findings are congruent with the combined effect of secondary chemistry and mycobiont identity being the main driver of bacterial community structure. Altogether, our results suggest that the associated bacterial communities are not the radiation's main driver. There is one possible exception, however, a species that has an abnormally diverse core microbiome and whose bacterial communities could be subject to a specific environmental filter at the functional level.

Revisiting the genotypes of Theileria equi based on the V4 hypervariable region of the 18S rRNA gene.

Equine theileriosis, an economically important disease that affects horses and other equids worldwide, is caused by a tick-borne intracellular apicomplexan protozoa Theileria equi. Genotyping of T. equi based on the 18S rRNA gene revealed the presence of two, three, four or five genotypes. In previous published reports, these genotypes have been labelled either alphabetically or numerically, and there is no uniformity in naming of these genotypes. The present study was aimed to revisit the phylogeny, genetic diversity and geographical distribution of T. equi based on the nucleotide sequences of the V4 hypervariable region of the 18S rRNA gene available in the nucleotide databases. Out of 14792 nucleotide sequences of T. equi available in the GenBank™, only 736 sequences of T. equi containing the complete V4 hypervariable region of the 18S rRNA gene (>207 bp) were used in multiple sequence alignment. Subsequently, a maximum likelihood phylogenetic tree was constructed based on the Kimura 2-parameter model (K2+I). The phylogenetic tree placed all the sequences into four distinct clades with high bootstrap values which were designated as T. equi clades/ genotypes A, B, C and D. Our results indicated that the genotype B of Nagore et al. and genotype E of Qablan et al. together formed the clade B with a high bootstrap value (95%). Furthermore, all the genotypes probably originated from clade B, which was the most dominant genotype (52.85%) followed by clades A (27.58%), and C (9.78%) and D (9.78%). Genotype C manifested a comparatively higher genetic diversity (91.0-100% identity) followed by genotypes A (93.2-99.5%), and B and D (95.7-100%). The alignment report of the consensus nucleotide sequences of the V4 hypervariable region of the 18S rRNA gene of four T. equi genotypes (A-D) revealed significant variations in one region, between nucleotide positions 113-183, and 41 molecular signatures were recognized. As far as geographical distribution is concerned, genotypes A and C exhibited far-extending geographical distribution involving 31 and 13 countries of the Asian, African, European, North American and South American continents, respectively. On the contrary, the genotypes B and D exemplified limited distribution with confinement to 21 and 12 countries of Asian, African and European continents, respectively. Interestingly, genotypes A and C have been reported from only two continents, viz., North and South America. It was observed that genotypes A and C, and B and D exhibit similar geographical distribution. The present study indicated the presence of only four previously described T. equi genotypes (A, B, C and D) after performing the molecular analyses of all available sequences of the complete V4 hypervariable region of the 18S rRNA gene of T. equi isolates in the GenBank™.

Seasonal changes of prokaryotic microbial community structure in Zhangjiayan Reservoir and its response to environmental factors

As a typical sub-deep reservoir in the upper reaches of the Yangtze River in the southwest region, Zhangjiayan Reservoir is also an important source of drinking water. Exploring the role of microorganisms in the material cycle of water bodies is of great significance for preventing the exacerbation of eutrophication in the reservoir. In this study, water samples from the overlying water of five points in the reservoir were collected four times in spring (April), summer (July), autumn (November), and winter (January) of 2022–2023 using a gas-tight water sampler. Physicochemical factors were measured, and the microbial community structure was analyzed by high-throughput MiSeq sequencing of the V3–V4 hypervariable region of 16S rRNA gene in order to explore the relationship between physicochemical factors and microbial community structure and the dominant microbial populations that affect eutrophication of the reservoir. The following results were obtained through analysis. Among the 20 overlying water samples from Zhangjiayan Reservoir, a total of 66 phyla, 202 classes, 499 orders, 835 families, 1716 genera, and 27,904 ASVs of the bacterial domain were detected. The phyla Proteobacteria and Actinobacteria were dominant in the microbial community of the overlying water in Zhangjiayan Reservoir. At the genus level, hgcI_clade and Actinobacteria had the highest abundance and was the dominant population. The microbial community in the water of Zhangjiayan Reservoir has a high level of diversity. The diversity index ranked by numerical order was winter > autumn > summer > spring. Significant differences were found in the composition and structure of the microbial community between the spring/summer and autumn/winter seasons (p < 0.05). Total phosphorus, dissolved total phosphorus, soluble reactive phosphorus, and dissolved oxygen have a significant impact on the composition and structure of the microbial community (p < 0.01). The bacterial community in the overlying water of Zhangjiayan Reservoir showed a mainly positive correlation. Sphingomonas, Brevundimonas, and Blastomonas were the central populations of the bacterial community in the overlying water of Zhangjiayan Reservoir. This study indicates that environmental factors, such as phosphorus and other nutrients, have a significant impact on the formation of the microbial community structure in different seasons. Sphingomonas, Brevundimonas, and Blastomonas are key populations that may have a significant impact on eutrophication in Zhangjiayan Reservoir.

Comparison of ruminal microbiota, IL-1β gene variation, and tick incidence between Holstein × Gyr and Holstein heifers in grazing system.

The variation in bacterial communities among breeds has been previously reported and may be one of the reasons why Holstein × Gyr dairy heifers have better development in grazing systems in tropical conditions. This study aimed to explore the ruminal microbiota composition, the IL-1β gene variation, tick incidence, and blood parameters of Holstein × Gyr (½ Holstein × ½ Gyr) and Holstein heifers grazing intensely managed Guinea grass (Panicum maximum Jacq. cv. Mombaça). Sixteen heifers were divided into two groups consisting of 8 Holstein × Gyr and 8 Holstein heifers. The experimental period was comprised of 3 periods of 21 days. Ruminal samples were taken via the stomach tube technique. The sequencing of the V4 hypervariable region of the 16S rRNA gene was performed using the Illumina MiSeq platform. Counting and collection of ticks were conducted each 21 days. Blood and skeletal muscle tissue biopsies were performed at the end of the experiment. Firmicutes were the most abundant phyla present in both breed rumen samples and Bacteroidota showed differences in relative abundance between breed groups, with greater values for Holstein heifers (p < 0.05 with FDR correction). The 10 most abundant unique OTUs identified in each breed included several OTUs of the genus Prevotella. Holstein heifers had a greater tick count and weight (9.8 ticks/animal and 1.6 g/animal, respectively) than Holstein × Gyr (2.56 ticks/animal and 0.4 g/animal, respectively). We found nucleotide substitutions in the IL-1β gene that might be related to adaptation and resistance phenotypes to tick infestation in Holstein × Gyr heifers. Blood concentrations of urea, albumin, insulin-like growth factor 1, triiodothyronine, and thyroxine were greater in Holstein × Gyr than in Holstein heifers. Adaptations in Holstein × Gyr heifers such as ruminal microbiota, tick resistance, nucleotide substitutions in IL-1β gene, and hormone concentration suggest a better energy metabolism and thermoregulation resulting in better performance in tropical grazing systems.

Partial Correspondence between Host Plant-Related Differentiation and Symbiotic Bacterial Community in a Polyphagous Insect.

Host plants play a vital role in insect population differentiation, while symbiotic associations between bacteria and insects are ubiquitous in nature. However, existing studies have given limited attention to the connection between host-related differentiation and symbiotic bacterial communities in phytophagous insects. In this study, we collected 58 samples of Aphis odinae from different host plants in southern China and constructed phylogenetic trees to investigate their differentiation in relation to host plants. We also selected aphid samples from the five most preferred host plants and analyzed their symbiotic bacterial composition using Illumina sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. The phylogeny and symbiotic bacterial community structure of A. odinae populations on different host plants showed that samples from Triadica sebifera (Euphorbiaceae) had a consistent presence of Wolbachia as the predominant secondary symbiont and suggested the possibility of undergoing differentiation. Conversely, although differentiation was observed in samples from Rhus chinensis (Anacardiaceae), no consistent presence of predominant secondary symbionts was found. Additionally, the samples from Heptapleurum heptaphyllum (Araliaceae) consistently carried Serratia, but no host differentiation was evident. In summary, this study reveals a partial correspondence between symbiotic bacterial communities and host-related differentiation in A. odinae. The findings contribute to our understanding of the microevolutionary influencing the macroevolutionary relationships between bacterial symbionts and phytophagous insects. The identification of specific symbionts associated with host-related differentiation provides valuable insights into the intricate dynamics of insect-bacteria interactions.

Inconsistencies in the Classification of the Family Cydnidae (Hemiptera: Heteroptera: Pentatomoidea) Revealed by Molecular Apomorphies in the Secondary and Tertiary Structures of 18S rRNA Length-Variable Region L (LVR L).

The SSU nuclear rDNA (encoding 18S ribosomal RNA) is one of the most frequently sequenced genes in the molecular analysis of insects. Molecular apomorphies in the secondary and tertiary structures of several 18S rRNA length-variable regions (LVRs) located within the V2, V4, and V7 hypervariable regions can be good indicators for recovering monophyletic groups within some heteropteran families. Among the LVRs that have been analysed, the LVR L in the V4 hypervariable region is the longest and most crucial for such assessments. We analysed the 18S rRNA V4 hypervariable region sequences of 45 species from the family Cydnidae, including all 6 subfamilies (Amaurocorinae, Amnestinae, Cephalocteinae, Cydninae, Garsauriinae, and Sehirinae) and three pentatomoid families (Parastrachiidae, Thaumastellidae, and Thyreocoridae), which have often been included in the broadly defined Cydnidae family. This is the first time that representatives of all Cydnidae subfamilies have been included in a molecular analysis. Only taxa from two subfamilies, Sehirinae and Cydninae, have been used in previous molecular studies. The secondary and tertiary structures of the LVR L were predicted for each species using the two-step procedure already accepted for such analyses to recover any molecular apomorphy essential for determining monophyly. The results of our comparative studies contradict the current understanding of the relationships among burrowing bugs and the current family classification.

Molecular epidemiology and characteristics of respiratory syncytial virus among hospitalized children in Guangzhou, China

BackgroundHuman respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection and hospitalization, especially in children. Highly mutagenic nature and antigenic diversity enable the RSV to successfully survive in human population. We conducted a molecular epidemiological study during 2017–2021 to investigate the prevalence and genetic characteristics of RSV.MethodsA total of 6499 nasopharyngeal (NP) swabs were collected from hospitalized children at Department of Pediatrics, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, China. All NP swab specimens were preliminary screened for common respiratory viruses and then tested for RSV using specific PCR assays. Partial G genes of RSV were amplified for phylogenetic analysis and genetic characterization.ResultsThe overall detection rate for common respiratory viruses was 16.12% (1048/6499). Among those, 405 specimens (6.20%, 405/6499) were found positive for RSV. The monthly distribution of RSV and other respiratory viruses was variable, and the highest incidence was recorded in Autumn and Winter. Based on the sequencing of hypervariable region of G gene, 93 RSV sequences were sub-grouped into RSV-A (56, 60.2%) and RSV-B (37, 39.8%). There was no coinfection of RSV-A and RSV-B in the tested samples. Phylogenetic analysis revealed that RSV-A and RSV-B strains belonged to ON1 and BA9 genotypes respectively, indicating predominance of these genotypes in Guangzhou. Several substitutions were observed which may likely change the antigenicity and pathogenicity of RSV. Multiple glycosylation sites were noticed, demonstrating high selection pressure on these genotypes.ConclusionThis study illustrated useful information about epidemiology, genetic characteristics, and circulating genotypes of RSV in Guangzhou China. Regular monitoring of the circulating strains of RSV in different parts of China could assist in the development of more effective vaccines and preventive measures.

Wide reference databases for typing Trypanosoma cruzi based on amplicon sequencing of the minicircle hypervariable region.

Trypanosoma cruzi, the etiological agent of Chagas Disease, exhibits remarkable genetic diversity and is classified into different Discrete Typing Units (DTUs). Strain typing techniques are crucial for studying T. cruzi, because their DTUs have significant biological differences from one another. However, there is currently no methodological strategy for the direct typing of biological materials that has sufficient sensitivity, specificity, and reproducibility. The high diversity and copy number of the minicircle hypervariable regions (mHVRs) makes it a viable target for typing. Approximately 24 million reads obtained by amplicon sequencing of the mHVR were analyzed for 62 strains belonging to the six main T. cruzi DTUs. To build reference databases of mHVR diversity for each DTU and to evaluate this target as a typing tool. Strains of the same DTU shared more mHVR clusters than strains of different DTUs, and clustered together. Different identity thresholds were used to build the reference sets of the mHVR sequences (85% and 95%, respectively). The 95% set had a higher specificity and was more suited for detecting co-infections, whereas the 85% set was excellent for identifying the primary DTU of a sample. The workflow's capacity for typing samples obtained from cultures, a set of whole-genome data, under various simulated PCR settings, in the presence of co-infecting lineages and for blood samples was also assessed. We present reference databases of mHVR sequences and an optimized typing workflow for T. cruzi including a simple online tool for deep amplicon sequencing analysis (https://ntomasini.github.io/cruzityping/). The results show that the workflow displays an equivalent resolution to that of the other typing methods. Owing to its specificity, sensitivity, relatively low cost, and simplicity, the proposed workflow could be an alternative for screening different types of samples.

Improving Species Level-taxonomic Assignment from 16S rRNA Sequencing Technologies.

Analysis of the bacterial community from a 16S rRNA gene sequencing technologies requires comparing the reads to a reference database. The challenging task involved in annotation relies on the currently available tools and 16S rRNA databases: SILVA, Greengenes and RDP. A successful annotation depends on the quality of the database. For instance, Greengenes and RDP have not been updated since 2013 and 2016, respectively. In addition, the nature of 16S sequencing technologies (short reads) focuses mainly on the V3-V4 hypervariable region sequencing and hinders the species assignment, in contrast to whole shotgun metagenome sequencing. Here, we combine the results of three standard protocols for 16S rRNA amplicon annotation that utilize homology-based methods, and we propose a new re-annotation strategy to enlarge the percentage of amplicon sequence variants (ASV) classified up to the species level. Following the pattern (reference) method: DADA2 pipeline and SILVA v.138.1 reference database classification (Basic Protocol 1), our method maps the ASV sequences to custom nucleotide BLAST with the SILVA v.138.1 (Basic Protocol 2), and to the 16S database of Bacteria and Archaea of NCBI RefSeq Targeted Loci Project databases (Basic Protocol 3). This new re-annotation workflow was tested in 16S rRNA amplicon data from 156 human fecal samples. The proposed new strategy achieved an increase of nearly eight times the proportion of ASV classified at the species level in contrast to the reference method for the database used in the present research. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample inference and taxonomic profiling through DADA2 algorithm. Basic Protocol 2: Custom BLASTN database creation and ASV taxonomical assignment. Basic Protocol 3: ASV taxonomical assignment using NCBI RefSeq Targeted Loci Project database. Basic Protocol 4: Definitive selection of lineages among the three methods.

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.

Bacterial communities structure in constructed wetlands for municipal and industrial wastewater treatment in Tanzania

Abstract Bacteria are the largest and most essential microorganisms in wastewater treatment systems. Despite the vital role bacteria play in enhancing wastewater treatment, its community structure and diversity remain poorly known. This study elucidated bacterial diversity and community structure in constructed wetlands (CWs) for municipal and industrial wastewater treatment using a culture-independent approach. Analysis of physicochemical parameters and Illumina high-throughput sequencing of V3 and V4 hypervariable regions of 16S rRNA gene bacterial community was performed. The results showed that the Proteobacteria were dominant (48.66%) phyla across all CWs. The Gammaproteobacteria class (27.67%), family Comamonadaceae (35.79) and Genus Flavobacterium (4.35%) were dominant across studied CWs. Bacterial abundance increased from the inlet to the outlet, with CWs having a higher abundance around the outlet, showing a good performance. The Shannon–Wiener index showed the highest species diversity (H = 1.45) in Banana Investment Limited CWs treating industrial wastewater. This study found significant bacterial diversity across studied CWs, with higher abundance and diversity at the outlet indicating better performance than those with low abundance and diversity at the outlet. We recommend a longitudinal investigation at each CWs section to confirm and establish the role of bacterial diversity on the performance pattern of CWs.

High Mitochondrial Haplotype Diversity Found in Three Pre-Hispanic Groups from Colombia.

The analysis of mitochondrial DNA (mtDNA) hypervariable region (HVR) sequence data from ancient human remains provides valuable insights into the genetic structure and population dynamics of ancient populations. mtDNA is particularly useful in studying ancient populations, because it is maternally inherited and has a higher mutation rate compared to nuclear DNA. To determine the genetic structure of three Colombian pre-Hispanic populations and compare them with current populations, we determined the haplotypes from human bone remains by sequencing several mitochondrial DNA segments. A wide variety of mitochondrial polymorphisms were obtained from 33 samples. Our results support a high population heterogeneity among pre-Hispanic populations in Colombia.

A two-cohort study on the association between the gut microbiota and bone density, microarchitecture, and strength.

The gut microbiome affects the inflammatory environment through effects on T-cells, which influence the production of immune mediators and inflammatory cytokines that stimulate osteoclastogenesis and bone loss in mice. However, there are few large human studies of the gut microbiome and skeletal health. We investigated the association between the human gut microbiome and high resolution peripheral quantitative computed tomography (HR-pQCT) scans of the radius and tibia in two large cohorts; Framingham Heart Study (FHS [n=1227, age range: 32 - 89]), and the Osteoporosis in Men Study (MrOS [n=836, age range: 78 - 98]). Stool samples from study participants underwent amplification and sequencing of the V4 hypervariable region of the 16S rRNA gene. The resulting 16S rRNA sequencing data were processed separately for each cohort, with the DADA2 pipeline incorporated in the16S bioBakery workflow. Resulting amplicon sequence variants were assigned taxonomies using the SILVA reference database. Controlling for multiple covariates, we tested for associations between microbial taxa abundances and HR-pQCT measures using general linear models as implemented in microbiome multivariable association with linear model (MaAslin2). Abundance of 37 microbial genera in FHS, and 4 genera in MrOS, were associated with various skeletal measures (false discovery rate [FDR] ≤ 0.1) including the association of DTU089 with bone measures, which was independently replicated in the two cohorts. A meta-analysis of the taxa-bone associations further revealed (FDR ≤ 0.25) that greater abundances of the genera; Akkermansia and DTU089, were associated with lower radius total vBMD, and tibia cortical vBMD respectively. Conversely, higher abundances of the genera; Lachnospiraceae NK4A136 group, and Faecalibacterium were associated with greater tibia cortical vBMD. We also investigated functional capabilities of microbial taxa by testing for associations between predicted (based on 16S rRNA amplicon sequence data) metabolic pathways abundance and bone phenotypes in each cohort. While there were no concordant functional associations observed in both cohorts, a meta-analysis revealed 8 pathways including the super-pathway of histidine, purine, and pyrimidine biosynthesis, associated with bone measures of the tibia cortical compartment. In conclusion, our findings suggest that there is a link between the gut microbiome and skeletal metabolism.

A Distinct Nasal Microbiota Signature in Peritoneal Dialysis Patients.

The nasal passages harbor both commensal and pathogenic bacteria and can be associated with infectious complications. The nasal microbiome in peritoneal dialysis (PD) patients, however, has not been well characterized. In this study, we sought to characterize the anterior nasal microbiota in peritoneal dialysis (PD) patients and assess its association with PD peritonitis. In this study, we recruited 32 PD patients, 37 kidney transplant (KTx) recipients, and 22 living donor/healthy control (HC) participants and collected their anterior nasal swabs at a single point in time. We followed the PD patients for future development of peritonitis. We performed 16S rRNA gene sequencing of the V4-V5 hypervariable region to determine the nasal microbiota. We compared nasal abundance of common genera among the 3 groups using Wilcoxon rank sum testing with Benjamini-Hochberg adjustment. DESeq2 was also utilized to compare the groups at the amplicon sequence variant levels. In the entire cohort, the most abundant genera in the nasal microbiota included: Staphylococcus, Corynebacterium, Streptococcus, and Anaerococcus. Correlational analyses revealed a significant inverse relationship between the nasal abundance of Staphylococcus and that of Corynebacterium. PD patients have a higher nasal abundance of Streptococcus than KTx recipients and HC participants. PD patients have a more diverse representation of Staphylococcus and Streptococcus than KTx recipients and HC participants. PD patients who concurrently have or who developed future Staphylococcus peritonitis had a numerically higher nasal abundance of Staphylococcus than PD patients who did not develop Staphylococcus peritonitis. We find a distinct nasal microbiota signature in PD patients compared to KTx recipients and HC participants. Given the potential relationship between the nasal pathogenic bacteria and infectious complications, further studies are needed to define the nasal microbiota associated with these infectious complications and to conduct studies on the manipulation of the nasal microbiota to prevent such complications.

Molecular Diversity of Human Respiratory Syncytial Virus before and during the COVID-19 Pandemic in Two Neighboring Japanese Cities.

Human respiratory syncytial viruses (HRSVs) are divided into subgroups A and B, which are further divided based on the nucleotide sequence of the second hypervariable region (HVR) of the attachment glycoprotein (G) gene. Understanding the molecular diversity of HRSV before and during the coronavirus disease 2019 (COVID-19) pandemic can provide insights into the effects of the pandemic on HRSV dissemination and guide vaccine development. Here, we analyzed HRSVs isolated in Fukushima Prefecture from September 2017 to December 2021. Specimens from pediatric patients were collected at two medical institutions in neighboring cities. A phylogenetic tree based on the second HVR nucleotide sequences was constructed using the Bayesian Markov chain Monte Carlo method. HRSV-A (ON1 genotype) and HRSV-B (BA9 genotype) were detected in 183 and 108 specimens, respectively. There were differences in the number of HRSV strains within clusters prevalent at the same time between the two hospitals. The genetic characteristics of HRSVs in 2021 after the COVID-19 outbreak were similar to those in 2019. HRSVs within a cluster may circulate within a region for several years, causing an epidemic cycle. Our findings add to the existing knowledge of the molecular epidemiology of HRSV in Japan. IMPORTANCE Understanding the molecular diversity of human respiratory syncytial viruses during pandemics caused by different viruses can provide insights that can guide public health decisions and vaccine development.

The urinary, vaginal and gut microbiota in women with genital lichen sclerosus – A case-control study

BackgroundLichen sclerosus (LS) is a chronic, autoimmune skin disease predominantly located in the anogenital region in women. In recent years, the role of the human microbiota in the pathogenesis of autoimmune diseases, including LS, has received interest. ObjectivesThe study aimed to evaluate and compare the composition of the urinary, vaginal and gut microbiota in women with LS versus non-affected controls. Study designWomen diagnosed with LS (n = 16) and matched controls (n = 14) were enrolled in the study. From each participant, midstream urine, upper and lower vaginal swabs, as well as faecal samples, were collected. The microbiota composition was assessed using 16S ribosomal RNA (rRNA) gene sequencing of the V4 hypervariable region. ResultsWe observed no LS-specific clustering in either of the four anatomic niches, using either hierarchical cluster analysis or weighted beta diversity metrics. However, for unweighted UniFrac, significant differences in the urinary and lower vaginal microbiota were observed when comparing women with LS to controls. These findings indicate that while the two groups have microbiota dominated by the same bacteria, variations do occur amongst less abundant bacteria. The LEfSe analysis revealed a higher relative abundance of the genus Streptococcus in the urinary and lower vaginal microbiota in women with LS compared to controls. Additionally, a higher relative abundance of phylum Euryarchaeota was observed in the gut microbiota in women with LS compared to controls. ConclusionIn this study, we demonstrated several differences amongst less abundant bacteria in the urinary, lower vaginal and faecal microbiota when comparing women with LS to controls. However, further research is required to assess whether these microbiota differences are causative or merely a result of the underlying LS disease.