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Community-wide high-throughput sequencing has transformed the study of the vaginal microbiome, and clinical applications are on the horizon. Here we outline the three main community sequencing methods: (1) amplicon sequencing, (2) shotgun metagenomic sequencing, and (3) metatranscriptomic sequencing. We discuss the advantages and limitations of community sequencing generally, and the unique strengths and weaknesses of each method. We briefly review the contributions of community sequencing to vaginal microbiome research and practice. We develop suggestions for critically interpreting research results and potential clinical applications based on community sequencing of the vaginal microbiome. TWEETABLE ABSTRACT: We review the advantages and limitations of amplicon sequencing, metagenomics, and metatranscriptomics methods for the study of the vaginal microbiome. [ABSTRACT FROM AUTHOR]

The vaginal microbiome has been linked to negative health outcomes including preterm birth. Specific taxa, including Gardnerella spp., have been identified as risk factors for these conditions. Historically, microbiome analysis methods have treated all Gardnerella spp. as one species, but the broad diversity of Gardnerella has become more apparent. We explore the diversity of Gardnerella clades and genomic species in the vaginal microbiome of pregnant women and their associations with microbiome composition and preterm birth. Relative abundance of Gardnerella clades and genomic species and other taxa was quantified in shotgun metagenomic sequencing data from three distinct cohorts of pregnant women. We also assessed the diversity and abundance of Gardnerella variants in 16S rRNA gene amplicon sequencing data from seven previously conducted studies in differing populations. Individual microbiomes often contained multiple Gardnerella variants, and the number of clades was associated with increased microbial load, or the ratio of non-human reads to human reads. Taxon co-occurrence patterns were largely consistent across Gardnerella clades and among cohorts. Some variants previously described as rare were prevalent in other cohorts, highlighting the importance of surveying a diverse set of populations to fully capture the diversity of Gardnerella . The diversity of Gardnerella both across populations and within individual vaginal microbiomes has long been unappreciated, as has been the intra-species diversity of many other members of the vaginal microbiome. The broad genomic diversity of Gardnerella has led to its reclassification as multiple species; here we demonstrate the diversity of Gardnerella found within and between vaginal microbiomes.IMPORTANCEThe present study shows that single microbiomes can contain all currently known species of Gardnerella and that multiple similar species can exist within the same environment. Furthermore, surveys of demographically distinct populations suggest that some species appear more commonly in certain populations. Further studies in broad and diverse populations will be necessary to fully understand the ecological roles of each Gardnerella sp., how they can co-exist, and their distinct impacts on microbial communities, preterm birth, and other health outcomes., Competing Interests: The authors declare no conflict of interest.

Fecal microbiota transplantation (FMT) has been demonstrated to be efficacious in preventing recurrent Clostridioides difficile (C. difficile) infections, and is being investigated for treatment of several other diseases including inflammatory bowel disease, cancer, obesity, liver disease, and diabetes. To speed up the translation of FMT into clinical practice as a safe and standardized therapeutic intervention, additional evidence-based technical and regulatory guidance is needed. To this end in May of 2022, the International Alliance for Biological Standardization (IABS) and the BIOASTER Microbiology Technology Institute hosted a second webinar to discuss key issues still impeding the advancement and standardization of FMT. The goal of this two-day webinar was to provide a forum for scientific experts to share and discuss data and key challenges with one another. Discussion included a focus on the evaluation of safety, efficacy, clinical trial design, reproducibility and accuracy in obtained microbiome measurements and data reporting, and the potential for standardization across these areas. It also focused on increasing the application potential and visibility of FMT beyond treating C. difficile infections., (Copyright © 2024. Published by Elsevier Ltd.)

Parchment, the skins of animals prepared for use as writing surfaces, offers a valuable source of genetic information. Many have clearly defined provenance, allowing for the genetic findings to be evaluated in temporal and spatial context. While these documents can yield evidence of the animal sources, the DNA contained within these aged skins is often damaged and fragmented. Previously, genetic studies targeting parchment have used destructive sampling techniques and so the development and validation of non-destructive sampling methods would expand opportunities and facilitate testing of more precious documents, especially those with historical significance. Here we present genetic data obtained by non-destructive sampling of eight parchments spanning the 15th century to the modern day. We define a workflow for enriching the mitochondrial genome (mtGenome), generating next-generation sequencing reads to permit species identification, and providing interpretation guidance. Using sample replication, comparisons to destructively sampled controls, and by establishing authentication criteria, we were able to confidently assign full/near full mtGenome sequences to 56.3% of non-destructively sampled parchments, each with greater than 90% of the mtGenome reference covered. Six of eight parchments passed all four established thresholds with at least one non-destructive sample, highlighting promise for future studies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Scheible et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aged companion dogs have a high prevalence of periodontal disease and canine cognitive dysfunction syndrome (CCDS) and the two disorders are correlated. Similarly, periodontal disease and Alzheimer's Disease are correlated in people. However, little is known about the oral microbiota of aging dogs. The goal of this project was to characterize the longitudinal changes in oral microbiota in aged dogs. Oral swabs were taken from ten senior client-owned dogs on 2-3 occasions spanning 24 months and they underwent whole genome shotgun (WGS) sequencing. Cognitive status was established at each sampling time. A statistically significant increase in alpha diversity for bacterial and fungal species was observed between the first and last study visits. Bacteroidetes and proteobacteria were the most abundant bacterial phyla. Porphyromonas gulae was the most abundant bacterial species (11.6% of total reads). The species Lactobacillus gasseri had a statistically significant increase in relative abundance with age whereas Leptotrichia sp. oral taxon 212 had a statistically significant positive longitudinal association with cognition score. There is an increased fungal and bacterial alpha diversity in aging dogs over time and nearly universal oral dysbiosis. The role of the oral microbiota, particularly Leptotrichia and P. gulae and P. gingivalis , in aging and CCDS warrants further investigation.

Background: Bacterial infection of bile is a common cause of hepatobiliary disease in cats. Whether bile harbors a core microbiota in health or in cases of suspected hepatobiliary disease in cats is unknown., Objectives: Establish if gallbladder bile in apparently healthy cats harbors a core microbiota composed of bacterial taxa common to many individuals. Compare results of bile cytology, bile culture, and 16S rRNA gene amplicon sequencing in apparently healthy cats and cats with suspected hepatobiliary disease., Animals: Forty-three client-owned cats with suspected hepatobiliary disease and 17 control cats., Methods: Bile was collected by ultrasound guided cholecystocentesis (cats with suspected hepatobiliary disease) or laparotomy after euthanasia (controls). Bile samples underwent cytologic examination, aerobic and anaerobic culture, and DNA was extracted for 16S rRNA gene amplification and sequencing., Results: Microbiome sequencing did not identify a core microbiota in control cats or cats having bile sampled because of clinical suspicion for hepatobiliary disease. Microbiome profiles from control cats were indistinguishable from profiles obtained from sampling instruments and reagents that were not exposed to bile (technical controls). Bacterial taxa that could not be explained by contamination or off-target amplification were identified only in samples from cats with bactibilia and positive bile culture results for Escherichia coli. In several E. coli positive samples, microbiome sequencing also identified a small number of potentially co-infecting bacterial genera not identified by culture., Conclusions and Clinical Importance: Cat bile does not harbor a core microbiota. Uncultured bacteria may contribute to pathogenesis of hepatobiliary disease in cats with bile E. coli infection., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Microbial community assembly remains largely unexplored in marine mammals, despite its potential importance for conservation and management. Here, neonatal microbiota assembly was studied in harbour seals (Phoca vitulina richardii) at a rehabilitation facility soon after maternal separation, through weaning, to the time of release back to their native environment. We found that the gingival and rectal communities of rehabilitated harbour seals were distinct from the microbiotas of formula and pool water, and became increasingly diverse and dissimilar over time, ultimately resembling the gingival and rectal communities of local wild harbour seals. Harbour seal microbiota assembly was compared to that of human infants, revealing the rapid emergence of host specificity and evidence of phylosymbiosis even though these harbour seals had been raised by humans. Early life prophylactic antibiotics were associated with changes in the composition of the harbour seal gingival and rectal communities and surprisingly, with transient increases in alpha diversity, perhaps because of microbiota sharing during close cohabitation with other harbour seals. Antibiotic-associated effects dissipated over time. These results suggest that while early life maternal contact may provide seeding for microbial assembly, co-housing of conspecifics during rehabilitation may help neonatal mammals achieve a healthy host-specific microbiota with features of resilience., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

We collected oral and/or rectal swabs and serum from dogs and cats living in homes with SARS-CoV-2-PCR-positive persons for SARS-CoV-2 PCR and serology testing. Pre-COVID-19 serum samples from dogs and cats were used as negative controls, and samples were tested in duplicate at different timepoints. Raw ELISA results scrutinized relative to known negative samples suggested that cut-offs for IgG seropositivity may require adjustment relative to previously proposed values, while proposed cut-offs for IgM require more extensive validation. A small number of pet dogs (2/43, 4.7%) and one cat (1/21, 4.8%) were positive for SARS-CoV-2 RNA, and 28.6 and 37.5% of cats and dogs were positive for anti-SARS-CoV-2 IgG, respectively., Competing Interests: Dorothee Bienzle is an Academic Editor for PeerJ., (©2023 Gin et al.)

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gookin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The relationship between host conditions and microbiome profiles, typically characterized by operational taxonomic units (OTUs), contains important information about the microbial role in human health. Traditional association testing frameworks are challenged by the high dimensionality and sparsity of typical microbiome profiles. Phylogenetic information is often incorporated to address these challenges with the assumption that evolutionarily similar taxa tend to behave similarly. However, this assumption may not always be valid due to the complex effects of microbes, and phylogenetic information should be incorporated in a data-supervised fashion., Results: In this work, we propose a local collapsing test called phylogeny-guided microbiome OTU-specific association test (POST). In POST, whether or not to borrow information and how much information to borrow from the neighboring OTUs in the phylogenetic tree are supervised by phylogenetic distance and the outcome-OTU association. POST is constructed under the kernel machine framework to accommodate complex OTU effects and extends kernel machine microbiome tests from community level to OTU level. Using simulation studies, we show that when the phylogenetic tree is informative, POST has better performance than existing OTU-level association tests. When the phylogenetic tree is not informative, POST achieves similar performance as existing methods. Finally, in real data applications on bacterial vaginosis and on preterm birth, we find that POST can identify similar or more outcome-associated OTUs that are of biological relevance compared to existing methods., Conclusions: Using POST, we show that adaptively leveraging the phylogenetic information can enhance the selection performance of associated microbiome features by improving the overall true-positive and false-positive detection. We developed a user friendly R package POSTm which is freely available on CRAN ( https://CRAN.R-project.org/package=POSTm ). Video Abstract., (© 2022. The Author(s).)

Background: Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge., Methods: Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads., Results: LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens., Conclusions: The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics. Video abstract.

Fluoroquinolones are a class of antimicrobial commonly used in human medicine, and deemed critical by the World Health Organization. Nonetheless, two formulations are approved for the treatment of respiratory disease in beef cattle. The objective of this study was to determine the gastrointestinal pharmacokinetics and impact on enteric bacteria of cattle when receiving one of the two dosing regimens (high: 40 mg/kg SC once or low: 20 mg/kg IM q48hr) of danofloxacin, a commonly utilized synthetic fluoroquinolone in veterinary medicine. Danofloxacin was administered to 12 steers (age 7 months) fitted with intestinal ultrafiltration devices at two different dosing regimens to assess the gastrointestinal pharmacokinetics, the shifts in the gastrointestinal microbiome and the development of resistant bacterial isolates. Our results demonstrated high intestinal penetration of danofloxacin for both dosing groups, as well as, significant differences in MIC values for E. coli and Enterococcus between dosing groups at selected time points over a 38 day period. Danofloxacin treatment consistently resulted in the Euryarchaeota phyla decreasing over time, specifically due to a decrease in Methanobrevibacter. Although microbiome differences were minor between dosing groups, the low dose group had a higher number of isolates with MIC values high enough to cause clinically relevant resistance. This information would help guide veterinarians as to appropriate dosing schemes to minimize the spread of antimicrobial resistance.

The relationship between the gut microbiota and Clostridioides difficile, and its role in the severity of C. difficile infection in humans is an area of active research. Intestinal carriage of toxigenic and non-toxigenic C. difficile strains, with and without clinical signs, is reported in animals, however few studies have looked at the risk factors associated with C. difficile carriage and the role of the host gut microbiota. Here, we isolated and characterized C. difficile strains from different animal species (predominantly canines (dogs), felines (cats), and equines (horses)) that were brought in for tertiary care at North Carolina State University Veterinary Hospital. C. difficile strains were characterized by toxin gene profiling, fluorescent PCR ribotyping, and antimicrobial susceptibility testing. 16S rRNA gene sequencing was done on animal feces to investigate the relationship between the presence of C. difficile and the gut microbiota in different hosts. Here, we show that C. difficile was recovered from 20.9% of samples (42/201), which included 33 canines, 2 felines, and 7 equines. Over 69% (29/42) of the isolates were toxigenic and belonged to 14 different ribotypes including ones known to cause CDI in humans. The presence of C. difficile results in a shift in the fecal microbial community structure in both canines and equines. Commensal Clostridium hiranonis was negatively associated with C. difficile in canines. Further experimentation showed a clear antagonistic relationship between the two strains in vitro, suggesting that commensal Clostridia might play a role in colonization resistance against C. difficile in different hosts., Competing Interests: Declaration of competing interest CMT is a scientific advisor to Locus Biosciences, a company engaged in the development of antimicrobial technologies. CMT is a consultant for Vedanta Biosciences., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In the last decade, we have witnessed a major paradigm shift in the life sciences: the recognition that the microbiome, i.e. the set of microorganisms associated with healthy animals (including humans) and plants, plays a crucial role in the sustained health and fitness of its host. Enabled by rapid advances in sequencing technologies and analytical methods, substantial advances have been achieved in both identifying the microbial taxa and understanding the relationship between microbiome composition and host phenotype. These breakthroughs are leading to novel strategies for improved human and animal health, enhanced crop yield and nutritional quality, and the control of various pests and disease agents. This article is part of the theme issue 'The role of the microbiome in host evolution'.

To survive, plants and animals must continually defend against pathogenic microbes that would invade and disrupt their tissues. Yet they do not attempt to extirpate all microbes. Instead, they tolerate and even encourage the growth of commensal microbes, which compete with pathogens for resources and via direct inhibition. We argue that hosts have evolved to cooperate with commensals in order to enhance the pathogen resistance this competition provides. We briefly describe competition between commensals and pathogens within the host, consider how natural selection might favour hosts that tilt this competition in favour of commensals, and describe examples of extant host traits that may serve this purpose. Finally, we consider ways that this cooperative immunity may have facilitated the adaptive evolution of non-pathogen-related host traits. On the basis of these observations, we argue that pathogen resistance vies with other commensal-provided benefits for being the principal evolutionary advantage provided by the microbiome to host lineages across the tree of life. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Background: Idiopathic epilepsy is a common neurological disorder of dogs characterized by recurrent seizures for which no underlying basis is identified other than a presumed genetic predisposition. The pathogenesis of the disorder remains poorly understood, but environmental factors are presumed to influence the course of disease. Within the growing body of research into the microbiota-gut-brain axis, considerable attention has focused on the protective role of Lactobacilli in the development and progression of neurological disease. Investigations into the association between the gut microbiome and epilepsy are in their infancy, but some preliminary findings support a role for Lactobacilli in drug resistant epilepsy. To date, there are no published studies evaluating the gut microbiome in dogs with epilepsy. This pilot study was undertaken to evaluate fecal Lactobacillus populations in dogs with idiopathic epilepsy compared to healthy dogs., Results: Fecal samples were obtained from 13 pairs of dogs, consisting of a drug-naïve epileptic dog and a healthy dog from the same household and maintained on the same diet. Evaluation of large-scale microbial patterns based on 16S rRNA gene amplicon sequencing identified a household effect in the study population. Differential prevalence testing at the 16S rRNA gene sequence variant and genus levels did not identify any statistically significant differences between epileptic and control dogs. Quantitative PCR of Lactobacillus species isolated through culture revealed no statistically significant difference between the epileptic and control dogs (median concentration, 3.8 log 10 CFU/g feces and 4.6 log 10 CFU/g feces, respectively). Lactobacillus in culture was not killed by exposure to phenobarbital, potassium bromide, zonisamide, or levetiracetam., Conclusions: This pilot study did not identify any difference in large-scale microbial patterns or relative or absolute abundance of Lactobacillus species in drug-naïve epileptic dogs compared to healthy dogs. Further studies are warranted to evaluate the role of the gut microbiome in disease progression and treatment response in dogs with epilepsy. Lactobacilli in culture were not killed or inhibited from growing when exposed to phenobarbital, potassium bromide, zonisamide or levetiracetam, suggesting that antiepileptic drug administration is less likely to be a confounding factor in future studies evaluating the role of Lactobacillus in epilepsy., Competing Interests: Competing interests The authors declare that they have no competing interests.

Community-wide high-throughput sequencing has transformed the study of the vaginal microbiome, and clinical applications are on the horizon. Here we outline the three main community sequencing methods: (1) amplicon sequencing, (2) shotgun metagenomic sequencing, and (3) metatranscriptomic sequencing. We discuss the advantages and limitations of community sequencing generally, and the unique strengths and weaknesses of each method. We briefly review the contributions of community sequencing to vaginal microbiome research and practice. We develop suggestions for critically interpreting research results and potential clinical applications based on community sequencing of the vaginal microbiome. TWEETABLE ABSTRACT: We review the advantages and limitations of amplicon sequencing, metagenomics, and metatranscriptomics methods for the study of the vaginal microbiome., (© 2019 Royal College of Obstetricians and Gynaecologists.)

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Antimicrobial drug concentrations in the gastrointestinal tract likely drive antimicrobial resistance in enteric bacteria. Our objective was to determine the concentration of ceftiofur and its metabolites in the gastrointestinal tract of steers treated with ceftiofur crystalline-free acid (CCFA) or ceftiofur hydrochloride (CHCL), determine the effect of these drugs on the minimum inhibitory concentration (MIC) of fecal Escherichia coli, and evaluate shifts in the microbiome. Steers were administered either a single dose (6.6 mg/kg) of CCFA or 2.2 mg/kg of CHCL every 24 hours for 3 days. Ceftiofur and its metabolites were measured in the plasma, interstitium, ileum and colon. The concentration and MIC of fecal E. coli and the fecal microbiota composition were assessed after treatment. The maximum concentration of ceftiofur was higher in all sampled locations of steers treated with CHCL. Measurable drug persisted longer in the intestine of CCFA-treated steers. There was a significant decrease in E. coli concentration (P = 0.002) within 24 hours that persisted for 2 weeks after CCFA treatment. In CHCL-treated steers, the mean MIC of ceftiofur in E. coli peaked at 48 hours (mean MIC = 20.45 ug/ml, 95% CI = 10.29-40.63 ug/ml), and in CCFA-treated steers, mean MIC peaked at 96 hours (mean MIC = 10.68 ug/ml, 95% CI = 5.47-20.85 ug/ml). Shifts in the microbiome of steers in both groups were due to reductions in Firmicutes and increases in Bacteroidetes. CCFA leads to prolonged, low intestinal drug concentrations, and is associated with decreased E. coli concentration, an increased MIC of ceftiofur in E. coli at specific time points, and shifts in the fecal microbiota. CHCL led to higher intestinal drug concentrations over a shorter duration. Effects on E. coli concentration and the microbiome were smaller in this group, but the increase in the MIC of ceftiofur in fecal E. coli was similar., Competing Interests: DMF has received research support from Zoetis. MGP has received gifts, honoraria, consulting fees, and research support from Zoetis, the manufacturer of ceftiofur. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Marker-gene and metagenomic sequencing have profoundly expanded our ability to measure biological communities. But the measurements they provide differ from the truth, often dramatically, because these experiments are biased toward detecting some taxa over others. This experimental bias makes the taxon or gene abundances measured by different protocols quantitatively incomparable and can lead to spurious biological conclusions. We propose a mathematical model for how bias distorts community measurements based on the properties of real experiments. We validate this model with 16S rRNA gene and shotgun metagenomics data from defined bacterial communities. Our model better fits the experimental data despite being simpler than previous models. We illustrate how our model can be used to evaluate protocols, to understand the effect of bias on downstream statistical analyses, and to measure and correct bias given suitable calibration controls. These results illuminate new avenues toward truly quantitative and reproducible metagenomics measurements., Competing Interests: MM, AW, BC No competing interests declared, (© 2019, McLaren et al.)

Background: The accuracy of microbial community surveys based on marker-gene and metagenomic sequencing (MGS) suffers from the presence of contaminants-DNA sequences not truly present in the sample. Contaminants come from various sources, including reagents. Appropriate laboratory practices can reduce contamination, but do not eliminate it. Here we introduce decontam ( https://github.com/benjjneb/decontam ), an open-source R package that implements a statistical classification procedure that identifies contaminants in MGS data based on two widely reproduced patterns: contaminants appear at higher frequencies in low-concentration samples and are often found in negative controls., Results: Decontam classified amplicon sequence variants (ASVs) in a human oral dataset consistently with prior microscopic observations of the microbial taxa inhabiting that environment and previous reports of contaminant taxa. In metagenomics and marker-gene measurements of a dilution series, decontam substantially reduced technical variation arising from different sequencing protocols. The application of decontam to two recently published datasets corroborated and extended their conclusions that little evidence existed for an indigenous placenta microbiome and that some low-frequency taxa seemingly associated with preterm birth were contaminants., Conclusions: Decontam improves the quality of metagenomic and marker-gene sequencing by identifying and removing contaminant DNA sequences. Decontam integrates easily with existing MGS workflows and allows researchers to generate more accurate profiles of microbial communities at little to no additional cost.

Objective: The intestinal concentrations of antimicrobial drugs that select for resistance in fecal bacteria of cattle are poorly understood. Our objective was to associate active drug concentrations in the intestine of steers with changes in the resistance profile and composition of the fecal microbiome. Methods: Steers were administered either a single dose (12.5 mg/kg) or 3 multiple doses (5 mg/kg) of enrofloxacin subcutaneously every 24 h. Enrofloxacin and ciprofloxacin concentrations in intestinal fluid were measured over 96 h, and the abundance and MIC of E. coli in culture and the composition of the fecal microbiota by 16S rRNA gene sequencing were assessed over 192 h after initial treatment. Results: Active drug concentrations in the ileum and colon exceeded plasma and interstitial fluid concentrations, but were largely eliminated by 48 h after the last dose. The concentration of E. coli in the feces significantly decreased during peak drug concentrations, but returned to baseline by 96 h in both groups. The median MIC of E. coli isolates increased for 24 h in the single dose group, and for 48 h in the multiple dose group. The median MIC was higher in the multiple dose group when compared to the single dose group starting 12 h after the initial dose. The diversity of the fecal microbiota did not change in either treatment group, and taxa-specific changes were primarily seen in phyla commonly associated with the rumen. Conclusions: Both dosing regimens of enrofloxacin achieve high concentrations in the intestinal lumen, and the rapid elimination mitigates long-term impacts on fecal E. coli resistance and the microbiota.

Microbial ecology has been transformed by the advent of high-throughput marker gene and metagenomic sequencing methods. These tools provide expansive descriptions of microbial communities, but the descriptions are framed in terms of molecular objects, such as 97% ribosomal operational taxonomic units (OTUs), rather than biological objects, such as species. A recent study by A. B. Chase and colleagues (mBio 8:e01809-17, 2017, https://doi.org/10.1128/mBio.01809-17) explores the so-called microdiversity within the Curtobacterium OTU, the most abundant OTU in a leaf litter community. Perhaps unsurprisingly, they find that some important ecologic traits, such as drought response, are coherent within the OTU, but that others vary significantly. Here we discuss their findings in relation to the more general issue of how molecular tools can be effectively used to study microbial ecology. We specifically note the need for investigators to choose the right molecular methods for their biological problem, as nature does not respect the limitations and conventions associated with our methods., (Copyright © 2018 McLaren and Callahan.)

Recent advances have made it possible to analyze high-throughput marker-gene sequencing data without resorting to the customary construction of molecular operational taxonomic units (OTUs): clusters of sequencing reads that differ by less than a fixed dissimilarity threshold. New methods control errors sufficiently such that amplicon sequence variants (ASVs) can be resolved exactly, down to the level of single-nucleotide differences over the sequenced gene region. The benefits of finer resolution are immediately apparent, and arguments for ASV methods have focused on their improved resolution. Less obvious, but we believe more important, are the broad benefits that derive from the status of ASVs as consistent labels with intrinsic biological meaning identified independently from a reference database. Here we discuss how these features grant ASVs the combined advantages of closed-reference OTUs-including computational costs that scale linearly with study size, simple merging between independently processed data sets, and forward prediction-and of de novo OTUs-including accurate measurement of diversity and applicability to communities lacking deep coverage in reference databases. We argue that the improvements in reusability, reproducibility and comprehensiveness are sufficiently great that ASVs should replace OTUs as the standard unit of marker-gene analysis and reporting.

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. Previous studies have suggested that the maternal vaginal microbiota contributes to the pathophysiology of PTB, but conflicting results in recent years have raised doubts. We conducted a study of PTB compared with term birth in two cohorts of pregnant women: one predominantly Caucasian ( n = 39) at low risk for PTB, the second predominantly African American and at high-risk ( n = 96). We profiled the taxonomic composition of 2,179 vaginal swabs collected prospectively and weekly during gestation using 16S rRNA gene sequencing. Previously proposed associations between PTB and lower Lactobacillus and higher Gardnerella abundances replicated in the low-risk cohort, but not in the high-risk cohort. High-resolution bioinformatics enabled taxonomic assignment to the species and subspecies levels, revealing that Lactobacillus crispatus was associated with low risk of PTB in both cohorts, while Lactobacillus iners was not, and that a subspecies clade of Gardnerella vaginalis explained the genus association with PTB. Patterns of cooccurrence between L. crispatus and Gardnerella were highly exclusive, while Gardnerella and L. iners often coexisted at high frequencies. We argue that the vaginal microbiota is better represented by the quantitative frequencies of these key taxa than by classifying communities into five community state types. Our findings extend and corroborate the association between the vaginal microbiota and PTB, demonstrate the benefits of high-resolution statistical bioinformatics in clinical microbiome studies, and suggest that previous conflicting results may reflect the different risk profile of women of black race., Competing Interests: The authors declare no conflict of interest.

We present the open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors (https://github.com/benjjneb/dada2). DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide. In several mock communities, DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants.

High-throughput sequencing of PCR-amplified taxonomic markers (like the 16S rRNA gene) has enabled a new level of analysis of complex bacterial communities known as microbiomes. Many tools exist to quantify and compare abundance levels or microbial composition of communities in different conditions. The sequencing reads have to be denoised and assigned to the closest taxa from a reference database. Common approaches use a notion of 97% similarity and normalize the data by subsampling to equalize library sizes. In this paper, we show that statistical models allow more accurate abundance estimates. By providing a complete workflow in R, we enable the user to do sophisticated downstream statistical analyses, including both parameteric and nonparametric methods. We provide examples of using the R packages dada2, phyloseq, DESeq2, ggplot2 and vegan to filter, visualize and test microbiome data. We also provide examples of supervised analyses using random forests, partial least squares and linear models as well as nonparametric testing using community networks and the ggnetwork package., Competing Interests: No competing interests were disclosed.

Marine mammals play crucial ecological roles in the oceans, but little is known about their microbiotas. Here we study the bacterial communities in 337 samples from 5 body sites in 48 healthy dolphins and 18 healthy sea lions, as well as those of adjacent seawater and other hosts. The bacterial taxonomic compositions are distinct from those of other mammals, dietary fish and seawater, are highly diverse and vary according to body site and host species. Dolphins harbour 30 bacterial phyla, with 25 of them in the mouth, several abundant but poorly characterized Tenericutes species in gastric fluid and a surprisingly paucity of Bacteroidetes in distal gut. About 70% of near-full length bacterial 16S ribosomal RNA sequences from dolphins are unique. Host habitat, diet and phylogeny all contribute to variation in marine mammal distal gut microbiota composition. Our findings help elucidate the factors structuring marine mammal microbiotas and may enhance monitoring of marine mammal health.

Despite the critical role of the human microbiota in health, our understanding of microbiota compositional dynamics during and after pregnancy is incomplete. We conducted a case-control study of 49 pregnant women, 15 of whom delivered preterm. From 40 of these women, we analyzed bacterial taxonomic composition of 3,767 specimens collected prospectively and weekly during gestation and monthly after delivery from the vagina, distal gut, saliva, and tooth/gum. Linear mixed-effects modeling, medoid-based clustering, and Markov chain modeling were used to analyze community temporal trends, community structure, and vaginal community state transitions. Microbiota community taxonomic composition and diversity remained remarkably stable at all four body sites during pregnancy (P > 0.05 for trends over time). Prevalence of a Lactobacillus-poor vaginal community state type (CST 4) was inversely correlated with gestational age at delivery (P = 0.0039). Risk for preterm birth was more pronounced for subjects with CST 4 accompanied by elevated Gardnerella or Ureaplasma abundances. This finding was validated with a set of 246 vaginal specimens from nine women (four of whom delivered preterm). Most women experienced a postdelivery disturbance in the vaginal community characterized by a decrease in Lactobacillus species and an increase in diverse anaerobes such as Peptoniphilus, Prevotella, and Anaerococcus species. This disturbance was unrelated to gestational age at delivery and persisted for up to 1 y. These findings have important implications for predicting premature labor, a major global health problem, and for understanding the potential impact of a persistent, altered postpartum microbiota on maternal health, including outcomes of pregnancies following short interpregnancy intervals.

Many species engage in adaptive niche construction: modification of the local environment that increases the modifying organism's competitive fitness. Adaptive niche construction provides an alternative pathway to higher fitness, shaping the environment rather than conforming to it. Yet, experimental evidence for the evolutionary emergence of adaptive niche construction is lacking, leaving its role in evolution uncertain. Here we report a direct observation of the de novo evolution of adaptive niche construction in populations of the bacteria Pseudomonas fluorescens. In a laboratory experiment, we allowed several bacterial populations to adapt to a novel environment and assessed whether niche construction evolved over time. We found that adaptive niche construction emerged rapidly, within approximately 100 generations, and became ubiquitous after approximately 400 generations. The large fitness effect of this niche construction was dominated by the low fitness of evolved strains in the ancestrally modified environment: evolved niche constructors were highly dependent on their specific environmental modifications. Populations were subjected to frequent resetting of environmental conditions and severe reduction of spatial habitat structure, both of which are thought to make adaptive niche construction difficult to evolve. Our finding that adaptive niche construction nevertheless evolved repeatably suggests that it may play a more important role in evolution than generally thought., (© 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.)

The study of evolution has entered a revolutionary new era, where quantitative and predictive methods are transforming the traditionally qualitative and retrospective approaches of the past. Genomic sequencing and modern computational techniques are permitting quantitative comparisons between variation in the natural world and predictions rooted in neo-Darwinian theory, revealing the shortcomings of current evolutionary theory, particularly with regard to large-scale phenomena like macroevolution. Current research spanning and uniting diverse fields and exploring the physical and chemical nature of organisms across temporal, spatial, and organizational scales is replacing the model of evolution as a passive filter selecting for random changes at the nucleotide level with a paradigm in which evolution is a dynamic process both constrained and driven by the informational architecture of organisms across scales, from DNA and chromatin regulation to interactions within and between species and the environment., (© 2013 New York Academy of Sciences.)

Background: PCR amplification and high-throughput sequencing theoretically enable the characterization of the finest-scale diversity in natural microbial and viral populations, but each of these methods introduces random errors that are difficult to distinguish from genuine biological diversity. Several approaches have been proposed to denoise these data but lack either speed or accuracy., Results: We introduce a new denoising algorithm that we call DADA (Divisive Amplicon Denoising Algorithm). Without training data, DADA infers both the sample genotypes and error parameters that produced a metagenome data set. We demonstrate performance on control data sequenced on Roche's 454 platform, and compare the results to the most accurate denoising software currently available, AmpliconNoise., Conclusions: DADA is more accurate and over an order of magnitude faster than AmpliconNoise. It eliminates the need for training data to establish error parameters, fully utilizes sequence-abundance information, and enables inclusion of context-dependent PCR error rates. It should be readily extensible to other sequencing platforms such as Illumina.

Central to the study of molecular evolution, and an area of long-standing debate, is the appropriate model for the fitness landscape of proteins. Much of this debate has focused on the strength and frequency of positive and purifying selection, but the form and frequency of selective correlations is also a vital element. The constituent amino acids within a protein generically interact and share selective pressures in predictable ways, which conflicts with the selective independence assumed by common caricatures of the fitness landscape. Here, I discuss a recent study by myself and coauthors that used whole-genome comparisons of orthologous molecular sequences from closely related Drosophilids to explore the form of the selective correlations and selective interactions (epistasis) between the amino acids within a protein. I outline our results and highlight our finding of a selective length scale of ten amino acids within which individual amino acids are substantially and generically more likely to share selective pressures and interact epistatically. I then focus on the evidence presented in our study supporting a substantial role for epistasis in the process of molecular evolution, and discuss further the implications of this widespread epistasis on the overdispersion of the molecular clock and the efficacy of common tests for positive selection.

Molecular adaptation is typically assumed to proceed by sequential fixation of beneficial mutations. In diploids, this picture presupposes that for most adaptive mutations, the homozygotes have a higher fitness than the heterozygotes. Here, we show that contrary to this expectation, a substantial proportion of adaptive mutations should display heterozygote advantage. This feature of adaptation in diploids emerges naturally from the primary importance of the fitness of heterozygotes for the invasion of new adaptive mutations. We formalize this result in the framework of Fisher's influential geometric model of adaptation. We find that in diploids, adaptation should often proceed through a succession of short-lived balanced states that maintain substantially higher levels of phenotypic and fitness variation in the population compared with classic adaptive walks. In fast-changing environments, this variation produces a diversity advantage that allows diploids to remain better adapted compared with haploids despite the disadvantage associated with the presence of unfit homozygotes. The short-lived balanced states arising during adaptive walks should be mostly invisible to current scans for long-term balancing selection. Instead, they should leave signatures of incomplete selective sweeps, which do appear to be common in many species. Our results also raise the possibility that balancing selection, as a natural consequence of frequent adaptation, might play a more prominent role among the forces maintaining genetic variation than is commonly recognized.

Changes in steady state minute ventilation, tidal volume and frequency were determined in unanesthetized lambs before and after bilateral cervical vagotomy while breathing 3, 6, and 9% CO2 in 20% O2 balance N2. The slope of delta VE/delta PaCO2 fell from 0.068 to 0.033 while frequency response depended on intact vagi. Electrical stimulation of the cut proximal vagus (after bilateral vagotomy) restored CO2 responsiveness to control values. From these observations, it may be concluded that the afferent vagus is involved in the control of ventilation during hypercapnia and helps to modulate ventilatory frequency by inhibiting tidal volume.