Comparison of two commercial DNA extraction kits for the analysis of nasopharyngeal bacterial communities

Characterization of microbial communities via next-generation sequencing (NGS) requires an extraction ofmicrobial DNA. Methodological differences in DNA extraction protocols may bias results and complicate inter-study comparisons. Here we compare the effect of two commonly used commercial kits (Norgen and Qiagen)for the extraction of total DNA on estimatingnasopharyngeal microbiome diversity. The nasopharynxis a reservoir for pathogens associated with respiratory illnesses and a key player in understandingairway microbial dynamics. Total DNA from nasal washes corresponding to 30 asthmatic children was extracted using theQiagenQIAamp DNA and NorgenRNA/DNA Purification kits and analyzed via IlluminaMiSeq16S rRNA V4 ampliconsequencing. The Norgen samples included more sequence reads and OTUs per sample than the Qiagen samples, but OTU counts per sample varied proportionallybetween groups (r = 0.732).Microbial profiles varied slightly between sample pairs, but alpha- and beta-diversity indices (PCoAand clustering) showed highsimilarity between Norgen and Qiagenmicrobiomes. Moreover, no significant differences in community structure (PERMANOVA and adonis tests) and taxa proportions (Kruskal-Wallis test) were observed betweenkits. Finally, aProcrustes analysis also showed low dissimilarity (M2 = 0.173; P\u3c 0.001) between the PCoAs of the two DNA extraction kits. Contrary to what has been observed in previous studies comparing DNA extraction methods, our 16S NGS analysis of nasopharyngeal washes did not reveal significant differences in community composition or structure between kits. Our findingssuggest congruence between column-based chromatography kits and supportthe comparison of microbiomeprofilesacross nasopharyngeal metataxonomic studies

How Long Does Evolution of the Troglomorphic Form Take? Estimating Divergence Times in Astyanax Mexicanus

Features including colonization routes (stream capture) and the existence of both epigean and cave-adapted hypogean populations make Astyanax mexicanus an attractive system for investigating the subterranean evolutionary time necessary for acquisition of the troglomorphic form. Using published sequences, we have estimated divergence times for A. mexicanus using: 1) two different population-level mitochondrial datasets (cytochrome b and NADH dehydrogenase 2) with both strict and relaxed molecular clock methods, and 2) broad phylogenetic approaches combining fossil calibrations and with four nuclear (recombination activating gene, seven in absentia, forkhead, and α-tropomyosin) and two mitochondrial (16S rDNA and cytochrome b) genes. Using these datasets, we have estimated divergence times for three events in the evolutionary history of troglomorphic A. mexicanus populations. First, divergence among cave haplotypes occurred in the Pleistocene, possibly correlating with fluctuating water levels allowing the colonization and subsequent isolation of new subterranean habitats. Second, in one lineage, A. mexicanus cave populations experienced introgressive hybridization events with recent surface populations (0.26-2.0 Ma), possibly also correlated with Pleistocene events. Finally, using divergence times from surface populations in the lineage without evidence of introgression as an estimate, the acquisition of the troglomorphic form in A. mexicanus is younger than 2.2 (fossil calibration estimates) – 5.2 (cytb estimate) Ma (Pliocene)

Two sampling methods yield distinct microbial signatures in the nasopharynges of asthmatic children.

Background The nasopharynx is a reservoir for pathogens associated with respiratory illnesses, such as asthma. Next-generation sequencing (NGS) has been used to characterize the nasopharyngeal microbiome during health and disease. Most studies so far have surveyed the nasopharynx as a whole; however, less is known about spatial variation (biogeography) in nasal microenvironments and how sampling techniques may capture that microbial diversity. Findings We used targeted 16S rRNA MiSeq sequencing and two different sampling strategies [nasal washes (NW) and nasal brushes (NB)] to characterize the nasopharyngeal microbiota in 30 asthmatic children. Nasal brushing is more abrasive than nasal washing and targeted the inner portion of the inferior turbinate. This region is expected to be different from other nasal microenvironments. Nasal washing is not spatially specific. Our 30 × 2 nasal microbiomes generated 1,474,497 sequences, from which we identified an average of 157 and 186 OTUs per sample in the NW and NB groups, respectively. Microbiotas from NB showed significantly higher alpha-diversity than microbiotas from NW. Similarly, both nasal microbiotas were distinct from each other (PCoA) and significantly differed in their community composition and abundance in at least 9 genera (effective size ≥1 %). Conclusions Nasopharyngeal microenvironments in asthmatic children contain microbiotas with different diversity and structure. Nasal washes and brushes capture that diversity differently. Future microbial studies of the nasopharynx need to be aware of potential spatial variation (biogeography)

Diversity, structure and sources of bacterial communities in earthworm cocoons

Animals start interactions with the bacteria that will constitute their microbiomes at embryonic stage. After mating, earthworms produce cocoons externally which will be colonized with bacteria from their parents and the environment. Due to the key role bacterial symbionts play on earthworm fitness, it is important to study bacterial colonization during cocoon formation. Here we describe the cocoon microbiome of the earthworms Eisenia andrei and E. fetida, which included 275 and 176 bacterial species, respectively. They were dominated by three vertically-transmitted symbionts, Microbacteriaceae, Verminephrobacter and Ca. Nephrothrix, which accounted for 88% and 66% of the sequences respectively. Verminephrobacter and Ca. Nephrothrix showed a high rate of sequence variation, suggesting that they could be biparentally acquired during mating. The other bacterial species inhabiting the cocoons came from the bedding, where they accounted for a small fraction of the diversity (27% and 7% of bacterial species for E. andrei and E. fetida bedding). Hence, earthworm cocoon microbiome includes a large fraction of the vertically-transmitted symbionts and a minor fraction, but more diverse, horizontally and non-randomly acquired from the environment. These data suggest that horizontally-transmitted bacteria to cocoons may play an important role in the adaptation of earthworms to new environments or diets.Ministerio de Economía y Competitividad | Ref. CTM2013-42540-RMinisterio de Economía y Competitividad | Ref. AGL2017-86813-RXunta de Galicia | Ref. ED431B-2016/04

Spatial diversity of the skin bacteriome

The bacterial communities of the human skin impact its physiology and homeostasis, hence elucidating the composition and structure of the healthy skin bacteriome is paramount to understand how bacterial imbalance (i.e., dysbiosis) may lead to disease. To obtain an integrated view of the spatial diversity of the skin bacteriome, we surveyed from 2019 to 2023 five skin regions (belly button, behind ears, between toes, calves and forearms) with different physiological characteristics (dry, moist and sebaceous) in 129 healthy adults (579 samples – after data cleaning). Estimating bacterial diversity through 16S rRNA metataxonomics, we identified significant (p < 0.0001) differences in the bacterial relative abundance of the four most abundant phyla and 11 genera, alpha- and beta-diversity indices and predicted functional profiles (36 to 400 metabolic pathways) across skin regions and microenvironments. No significant differences, however, were observed across genders, ages, and ethnicities. As previously suggested, dry skin regions (forearms and calves) were more even, richer, and functionally distinct than sebaceous (behind ears) and moist (belly button and between toes) regions. Within skin regions, bacterial alpha- and beta-diversity also varied significantly for some of the years compared, suggesting that skin bacterial stability may be region and subject dependent. Our results, hence, confirm that the skin bacteriome varies systematically across skin regions and microenvironments and provides new insights into the internal and external factors driving bacterial diversity

A 28-Year History of HIV-1 Drug Resistance and Transmission in Washington, DC

Washington, DC consistently has one of the highest annual rates of new HIV-1 diagnoses in the United States over the last 10 years. To guide intervention and prevention strategies to combat DC HIV infection, it is helpful to understand HIV transmission dynamics in a historical context. Toward this aim, we conducted a retrospective study (years 1987–2015) of 3,349 HIV pol sequences (1,026 bp) from 1,995 individuals living in the DC area belonging to three different cohorts. We coupled HIV sequence data with clinical information (sex, risk factor, race/ethnicity, viral load, subtype, anti-retroviral regimen) to identify circulating drug resistant mutations (DRM) and transmission clusters and assess their persistence over time. Of the transmission clusters identified in the DC area, 78.0 and 31.7% involved MSM and heterosexuals, respectively. The longest spread of time for a single cluster was 5 years (2007–2012) using a distance-based network inference approach and 27 years (1987–2014) using a maximum likelihood phylogenetic approach. We found eight subtypes and nine recombinants. Genetic diversity increased steadily over time with a slight peak in 2009 and remained constant thereafter until 2015. Nucleotide diversity also increased over time while relative genetic diversity (BEAST) remained relatively steady over the last 28 years with slight increases since 2000 in subtypes B and C. Sequences from individuals on drug therapy contained the highest total number of DRMs (1,104–1,600) and unique DRMs (63–97) and the highest proportion (>20%) of resistant individuals. Heterosexuals (43.94%), MSM (40.13%), and unknown (44.26%) risk factors showed similar prevalence of DRMs, while injection drug users had a lower prevalence (33.33%). Finally, there was a 60% spike in the number of codons with DRMs between 2007 and 2010. Past patterns of HIV transmission and DRM accumulation over time described here will help to predict future efficacy of ART drugs based on DRMs persisting over time and identify risk groups of interest for prevention and intervention efforts within the DC population. Our results show how longitudinal data can help to understand the temporal dynamics of HIV-1 at the local level

Glucocorticoids modulate gastrointestinal microbiome in a wild bird.

It has recently been hypothesized that stress exposure (e.g. via glucocorticoid secretion) may dysregulate the bacterial gut microbiome, a crucial \u27organ\u27 in animal health. However, whether stress exposure (e.g. via glucocorticoid secretion) affects the bacterial gut microbiome of natural populations is unknown. We have experimentally altered the basal glucocorticoid level (corticosterone implants) in a wild avian species, the yellow-legged gul

Integrating microbial and host transcriptomics to characterize asthma-associated microbial communities.

BACKGROUND: The relationships between infections in early life and asthma are not completely understood. Likewise, the clinical relevance of microbial communities present in the respiratory tract is only partially known. A number of microbiome studies analyzing respiratory tract samples have found increased proportions of gamma-Proteobacteria including Haemophilus influenzae, Moraxella catarrhalis, and Firmicutes such as Streptococcus pneumoniae. The aim of this study was to present a new approach that combines RNA microbial identification with host gene expression to characterize and validate metagenomic taxonomic profiling in individuals with asthma. METHODS: Using whole metagenomic shotgun RNA sequencing, we characterized and compared the microbial communities of individuals, children and adolescents, with asthma and controls. The resulting data were analyzed by partitioning human and microbial reads. Microbial reads were then used to characterize the microbial diversity of each patient, and potential differences between asthmatic and healthy groups. Human reads were used to assess the expression of known genes involved in the host immune response to specific pathogens and detect potential differences between those with asthma and controls. RESULTS: Microbial communities in the nasal cavities of children differed significantly between asthmatics and controls. After read count normalization, some bacterial species were significantly overrepresented in asthma patients (Wald test, p-value \u3c 0.05), including Escherichia coli and Psychrobacter. Among these, Moraxella catarrhalis exhibited ~14-fold over abundance in asthmatics versus controls. Differential host gene expression analysis confirms that the presence of Moraxella catarrhalis is associated to a specific M. catarrhalis core gene signature expressed by the host. CONCLUSIONS: For the first time, we show the power of combining RNA taxonomic profiling and host gene expression signatures for microbial identification. Our approach not only identifies microbes from metagenomic data, but also adds support to these inferences by determining if the host is mounting a response against specific infectious agents. In particular, we show that M. catarrhalis is abundant in asthma patients but not in controls, and that its presence is associated with a specific host gene expression signature