Ecological and evolutionary consequences of microbial community responses to environmental changeAbstract
Global changes such as increased frequency of fire, drought, and nitrogen deposition, perturb
microorganisms and the higher trophic life forms they support. Microorganisms play key roles in
carbon and nutrient cycling, which are important to agriculture and ecosystem health. Although
microorganisms are pivotal in an ecosystem's response to environmental changes, little is known
about how abundant and diverse microbial communities adapt to such changes. The overarching
aim of my thesis is to investigate how bacterial communities respond to global change and in
particular, their ability to quickly adapt to environmental perturbations.
I first investigated how microbial responses to global changes are influenced by
interactions with plant communities using the Loma Ridge Global Change Experiment, a decadelong experiment that manipulates rainfall and nitrogen levels across two adjacent ecosystems
(Chapter 1). My findings underscore the importance of plant–microbe interactions when
considering the transferability of the results of global change experiments across ecosystems.
Next, I investigated traits found on plasmids, a type of mobile genetic element (MGE) that can
facilitate rapid evolution in bacteria. I asked what are the ecologically-relevant plasmid genes
that may serve as reservoirs of environmental-adaptive traits in bacteria (Chapter 2). The findings of this chapter suggest that plasmid traits may contribute to host adaptation in
environmental microbiomes. Lastly, I extended this work to a cosmopolitan soil taxon,
Curtobacterium, an abundant genus of bacteria in southern California ecosystems. This taxon
shows marked shifts in relative abundance in response to simulated drought and is amenable to
culturing, providing a tractable system for investigating both genotypic and phenotypic
characteristics of this organism. Previous experiments have shown Curtobacterium rapidly
evolve via de novo mutations in response to environmental changes. I asked what MGE and
associated traits are found in Curtobacterium, and determined whether MGE and traits showed
any environment- versus clade- specific genomic signatures (Chapter 3). The findings of this
chapter highlight the potential of traits found on plasmids to be mobilized within the bacterial
communities where these Curtobacterium were isolated. Overall, my thesis work highlights the
importance for considering the intersection of evolution and ecology in understanding how
microbial communities adapt to environmental changes.
Comparison of three artificial rumen systems for rumen microbiome modelingAbstract
The rumen contains a complex mixture of microbes, which are crucial for ruminant health and feed
fermentation. During the fermentation process some of the feed-derived carbon becomes carbon dioxide
and methane, which are released into the atmosphere where they act as greenhouse gases and contribute
to climate change. There is growing interest in reducing the loss of feed-derived carbon and making it
available to the animal, improving animal productivity, while also reducing the carbon footprint of the
ruminant industry. To this end, artificial rumen systems (ARS) have been used for evaluating novel feed
additives for their effect on the rumen microbiome and rumen function prior to conducting resource
intensive animal trials. Whereas ARS are capable of predicting the response of the rumen and its
microbiome, it is unclear how accurately different in vitro systems simulate the natural system and how
results compare between the artificial systems that are being employed. Here we evaluated physical,
chemical and microbiome metrics of three ARS over five days and compared them to those metrics in the
in vivo rumen.
Over a 48 hrs sampling period, the batch style platform (Ankom) was able to replicate pH, volatile fatty
acid profile, and bacterial and fungal microbiome of the in vivo rumen, but its accuracy of mimicking in
vivo metrics dropped significantly beyond 48 hrs. In contrast, the semi-continuous RUSITEC models,
RUSITEC PP and RUSITEC prime, were able to mimic the volatile fatty acid profile and microbiota of the in
vivo rumen for up to 120 hrs of rumen simulation. Comparison of gas production across vessel types
demonstrated that the semi-continuous RUSITEC platforms display less variability among vessel
replicates and time compared to the Ankom system.
In this study, we found that three widely used ARS were able to simulate the rumen ecosystem adequately
for the first 48 hrs, with predictions from the more advanced semi-continuous ARS being more accurate
when simulations extended over 48 hrs. Findings of this study will help to select the appropriate in vitro
system for evaluating the response of the complex rumen microbiome to feed additives. Further work is
necessary to improve the capabilities of these platforms and to standardize the methodology for largescale application.
Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. ×ananassa)Abstract
Phytophthora crown rot (PhCR) caused by P. cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently applied to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2- 1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product.
Microbiome of rehydrated corn and sorghum grain silages treated with microbial inoculants in different fermentation periodsAbstract
Due to the co-evolved intricate relationships and mutual influence between changes in the microbiome and silage fermentation quality, we explored the effects of Lactobacillus plantarum and Propionibacterium acidipropionici (Inoc1) or Lactobacillus buchneri (Inoc2) inoculants on the diversity and bacterial and fungal community succession of rehydrated corn (CG) and sorghum (SG) grains and their silages using Illumina Miseq sequencing after 0, 3, 7, 21, 90, and 360 days of fermentation. The effects of inoculants on bacterial and fungal succession differed among the grains. Lactobacillus and Weissella species were the main bacteria involved in the fermentation of rehydrated corn and sorghum grain silage. Aspergillus spp. mold was predominant in rehydrated CG fermentation, while the yeast Wickerhamomyces anomalus was the major fungus in rehydrated SG silages. The Inoc1 was more efficient than CTRL and Inoc2 in promoting the sharp growth of Lactobacillus spp. and maintaining the stability of the bacterial community during long periods of storage in both grain silages. However, the bacterial and fungal communities of rehydrated corn and sorghum grain silages did not remain stable after 360 days of storage.
Performance of Conventional Urine Culture Compared to 16S rRNA Gene Amplicon Sequencing in Children with Suspected Urinary Tract InfectionAbstract
Because some organisms causing urinary tract infection (UTI) may be difficult
to culture, examination of bacterial gene sequences in the urine may provide a more accurate view of bacteria present during a UTI. Our objective was to estimate how often access
to 16S rRNA gene amplicon sequencing alters diagnosis and/or clinical management. The
study was designed as a cross-sectional study of a convenience sample of children with suspected UTI. The setting was the emergency department or outpatient clinic at six pediatric
centers. Participants included children 2 months to 10 years of age suspected of UTI. We
categorized the results of urine culture as follows: “likely UTI” ($100,000 CFU/ml of a single
uropathogen), “possible UTI” (10,000 to 99,000 CFU/ml of a uropathogen or $100,000 CFU/
ml of a single uropathogen plus other growth), and “unlikely UTI” (no growth or growth of
nonuropathogens). Similarly, we categorized the results of 16S rRNA gene sequencing into
the same three categories using the following criteria: likely UTI ($90% relative abundance
of a uropathogen), possible UTI (50 to 89% relative abundance of a uropathogen), and
unlikely UTI (remainder of samples). The main study outcome was concordance between
conventional culture results and 16S rRNA gene sequencing. Concordance between the
two methods was high in children with likely and unlikely UTI by conventional culture
(95% and 87%, respectively). In children with possible UTI according to conventional culture, 71% had a single uropathogen at a relative abundance of $90% according to 16S
rRNA gene sequencing data. Concordance between conventional culture and 16S rRNA
gene amplicon sequencing appears to be high. In children with equivocal culture results,
16S rRNA gene results may provide information that may help clarify the diagnosis.
IMPORTANCE Concordance between conventional culture and 16S rRNA gene amplicon
sequencing appears to be high. In children with equivocal culture results, 16S rRNA gene
results may provide information that may help clarify the diagnosis.
Potential improvements of the cognition of piglets through a synbiotic supplementation from 1 to 28 days via the gut microbiotaAbstract
The influence of feed supplements on behavior and memory has been recently studied in livestock. The objectives of the study were to evaluate the effects of a synbiotic on: an episodic-like (SOR: Spontaneous Object Recognition), a working (BARR: Fence barrier task), a long-term (TMAZE: Spatial T-maze task) memory test and on gut microbiota composition. Eighteen female piglets were supplemented from 1 to 28 days of age with a synbiotic (SYN), while 17 served as control (CTL). Feces were collected on days 16, 33 and 41 for 16S rRNA gene composition analyses. In the SOR, SYN piglets interacted more quickly with the novel object than CTL piglets. In the BARR, SYN piglets had shorter distances to finish the test in trial 3. In the TMAZE, SYN piglets were quicker to succeed on specific days and tended to try the new rewarded arm earlier during the reversal stage. Difference of microbiota composition between treatments was nonexistent on D16, a tendency on D33 and significant on D41. The synbiotic supplement may confer memory advantages in different cognitive tasks, regardless of the nature of the reward and the memory request. Difference in memory abilities can potentially be explained by differences in microbiota composition.
Microbiomes: biogeographic patterns and the influence of dispersalAbstract
Bacteria are essential parts of ecosystems and are the most diverse organisms on the planet. Yet,
we still do not know which habitats support the highest diversity of bacteria across multiple
scales. We analyzed alpha-, beta-, and gamma-diversity of bacterial assemblages using 11,680
samples compiled by the Earth Microbiome Project. We found that soils contained the highest
bacterial richness within a single sample (alpha-diversity), but sediment assemblages displayed
the highest gamma-diversity. Sediment, biofilms/mats, and inland water exhibited the most
variation in community composition among geographic locations (beta-diversity). Within soils,
agricultural lands, hot deserts, grasslands, and shrublands contained the highest richness, while
forests, cold deserts, and tundra biomes consistently harbored fewer bacterial species.
Surprisingly, agricultural soils encompassed similar levels of beta-diversity as other soil biomes.
These patterns were robust to the alpha- and beta- diversity metrics used and the taxonomic
binning approach. Overall, the results support the idea that spatial environmental heterogeneity is
an important driver of bacterial diversity.
Constitutive and differential expression of transport protein genes inParascaris univalenslarvae and adult tissues afterin vitroexposure toanthelmintic drugsAbstract
The equine roundworm Parascaris univalens has developed resistance to the three anthelmintic substances most commonly used in horses. The mechanisms responsible for resistance are believed to be multi-genic, and transport proteins such as the P-glycoprotein (Pgp) family have been suggested to be involved in resistance in several parasites including P. univlaens. To facilitate further research into the mechanisms behind drug metabolism and resistance development in P. univalens we aimed to develop an in vitro model based on larvae. We developed a fast and easy protocol for hatching P. univalens larvae for in vitro studies, resulting in a hatching rate of 92 %. The expression of transport protein genes pgp-2, pgp-9, pgp-11.1, pgp-16.1 and major facilitator superfamily (MFS) genes PgR006_g137 and PgR015_g078 were studied in hatched larvae exposed to the anthelmintic drugs ivermecin (IVM) 10-9 M, pyrantel citrate (PYR) 10-6 M and thiabendazole (TBZ) 10-5 M for 24 h. In comparison, the expression of these transport protein genes was studied in the anterior end and intestinal tissues of adult worms in vitro exposed to IVM, TBZ and PYR, at the same concentrations as larvae, for 3 h, 10 h and 24 h. Larval exposure to sub-lethal doses of IVM for 24 h did not affect the expression levels of any of the investigated genes, however larvae exposed to PYR and TBZ for 24 h showed significantly increased expression of pgp-9. In vitro drug exposure of adult worms did not result in any significant increases in expression of transport protein genes. Comparisons of constitutive expression between larvae and adult worm tissues showed that pgp-9, pgp-11.1, pgp-16.1 and MFS gene PgR015_g078 were expressed at lower levels in larvae than in adult tissues, while pgp-2 and MFS gene PgR006_g137 had similar expression levels in larvae and adult worms. All investigated transport protein genes were expressed at higher rates in the intestine than in the anterior end of adult worms, except pgp-11.1 where the expression was similar between the two tissues. This high constitutive expression in the intestine suggests that this is an important site for xenobiotic efflux in P. univalens. Despite the fact that the results of this study show differences in expression of transport protein genes between larvae and adult tissues, we believe that the larval assay system described here will be an important tool for further research into the molecular mechanisms behind anthelmintic resistance development and for other in vitro studies.
A Comparison of PCR and ELISA Methods to Detect Different Stages of Plasmodium Vivax in Anopheles Arabiensis.Abstract
Background: In characterizing malaria epidemiology, measuring mosquito infectiousness informs the
entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have
been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider
application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite
stage. We compared a PCR method for detecting the parasite’s cytochrome oxidase I (COX-I) gene with
ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite
during development within a mosquito.
Methods: A PCR-based method targeting the Plasmodium COX-I gene was compared with the CSP ELISA
method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients
infected with Plasmodium vivax. Mosquitoes were tested at six post-infection timepoints (days 0.5, 1, 6,
9, 12, 15). Head and thoraces, and abdomens for each specimen were tested separately with both
methods. Agreement between methods at each infection stage was measured using Cohen’s Kappa
measure of test association.
Results: Infection status of mosquitoes was assessed in approximately 90 head and thoraces and 90
abdomens at each time point; in total 538 head and thoraces and 534 abdomens were tested. In
mosquitoes bisected after 0.5, 1-, and 6-days post-infection, the COX-I PCR detected Plasmodium DNA in
both the abdomens (88%, 78%, and 67%, respectively) and head and thoraces (69%, 60%, and 44%,
respectively) whilst CSP-ELISA detect sporozoites in only 1 abdomen on day 6 post-infection. PCR was
also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9-, 12-, and 15-
days post-infection in both the head and thoraces and abdomens. There was fair agreement between
both methods for time points 9 -15 days post-infection (κ = 0.312, 95% CI: 0.230 – 0.394).
Conclusion: The COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in
mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the
head and thorax from the abdomen prior to PCR. Thus, the COX-I PCR is a poor candidate for identifying
Microbiome Analyses Demonstrate Specific Communities Within Five Shark SpeciesAbstract
Profiles of symbiotic microbial communities (“microbiomes”) can provide insight into the natural history and ecology of their hosts. Using high throughput DNA sequencing of the 16S rRNA V4 region, microbiomes of five shark species in South Florida (nurse, lemon, sandbar, Caribbean reef, and tiger) have been characterized for the first time. The microbiomes show species specific microbiome composition, distinct from surrounding seawater. Shark anatomical location (gills, teeth, skin, cloaca) affected the diversity of microbiomes. An in-depth analysis of teeth communities revealed species specific microbial communities. For example, the genus Haemophilus, explained 7.0% of the differences of the teeth microbiomes of lemon and Caribbean reef sharks. Lemon shark teeth communities (n = 11) contained a high abundance of both Vibrio (10.8 ± 26.0%) and Corynebacterium (1.6 ± 5.1%), genera that can include human pathogenic taxa. The Vibrio (2.8 ± 6.34%) and Kordia (3.1 ± 6.0%) genera and Salmonella enterica (2.6 ± 6.4%) were the most abundant members of nurse shark teeth microbial communities. The Vibrio genus was highly represented in the sandbar shark (54.0 ± 46.0%) and tiger shark (5.8 ± 12.3%) teeth microbiomes. The prevalence of genera containing potential human pathogens could be informative in shark bite treatment protocols and future research to confirm or deny human pathogenicity. We conclude that South Florida sharks host species specific microbiomes that are distinct from their surrounding environment and vary due to differences in microbial community composition among shark species and diversity and composition among anatomical locations. Additionally, when considering the confounding effects of both species and location, microbial community diversity and composition varies.
Influence of pH on the balance between methanogenesis and iron reductionAbstract
Methanogenesis and iron reduction play major roles in determining global fluxes of
greenhouse gases. Despite their importance, environmental factors that influence their
interactions are poorly known. Here we present evidence that pH significantly influences the
balance between each reaction in anoxic environments that contain ferric (oxyhydr)oxide
minerals. In sediment bioreactors that contained goethite as a source of ferric iron, both iron
reduction and methanogenesis occurred but the balance between them varied significantly with
pH. Compared to bioreactors receiving acidic media (pH 6), electron donor oxidation was 85%
lower for iron reduction and 61% higher for methanogenesis in bioreactors receiving alkaline
media (pH 7.5). Thus, methanogenesis displaced iron reduction considerably at alkaline pH.
Geochemistry data collected from U.S. aquifers demonstrate that a similar pattern also exists on
a broad spatial scale in natural settings. In contrast, in bioreactors that were not augmented with
goethite, clay minerals served as the source of ferric iron and the balance between each reaction
did not vary significantly with pH. We therefore conclude that pH can regulate the relative
contributions of microbial iron reduction and methanogenesis to carbon fluxes from terrestrial
environments. We further propose that the availability of ferric (oxyhydr)oxide minerals
influences the extent to which the balance between each reaction is sensitive to pH. The results
of this study advance our understanding of environmental controls on microbial methane
generation and provide a basis for using pH and the occurrence of ferric minerals to refine
predictions of greenhouse gas fluxes.
Characterization of maize root microbiome in two different soils by minimizing plant DNA contamination in metabarcoding analysisAbstract
A micropore-filtration method was used to reduce the proportion of plant DNA in microbial DNA samples isolated from roots prior to sequencing. We tested the impact of this pre-sequencing filtration methodology and used it to characterize the root microbiome of maize grown on two soils with different fertility levels. The micropore filtration reduced plant DNA contamination and unveiled potential in the N-poor soil for N fixation in roots and phosphate uptake by roots in the phosphate-poor soil. Our methodology and findings allude to the potential capability of plants to initiate plant-microbe interactions under sub-optimal soil fertility.
Assessing the comparability of different DNA extraction and amplification methods in gut microbial community profilingAbstract
Automated, high-throughput technologies are becoming increasingly common in microbiome studies to decrease costs and increase efficiency. However, in microbiome studies, small differences in methodology – including storage conditions, wet lab methods, sequencing platforms and data analysis – can influence the reproducibility and comparability of data across studies. There has been limited testing of the effects of high-throughput methods, including microfluidic PCR technologies. In this paper, we compare two extraction methods (the QIAamp DNA Stool Mini Kit and the MoBio PowerSoil DNA Isolation kit), two taq polymerase enzymes (MyTaq HS Red Mix and Accustart II PCR ToughMix), two primer sets (V3–V4 and V4–V5) and two amplification methods (a common two-step PCR protocol and amplicon library preparation on the Fluidigm Access Array system that allows automated multiplexing of primers). Gut microbial community profiles were significantly affected by all variables. While there were no significant differences in alpha diversity measured between the two extraction methods, there was an effect of extraction method on community composition measured by unweighted UniFrac distances. Both amplification method and primers had a significant effect on both alpha diversity and community composition. The relative abundance of Actinobacteria was significantly lower when using the MoBio kit or Fluidigm amplification method, and the relative abundance of Firmicutes was lower when using the Qiagen kit. Microbial community profiles based on Fluidigm-generated amplicon libraries were not comparable to those generated with more commonly used methods. Researchers should carefully consider the limitations and biases that different extraction and amplification methods can introduce into their results. Additionally, more thorough benchmarking of automated and multiplexing methods is necessary to determine the magnitude of the potential trade-off between the quality and the quantity of data.
Predictive Metagenomic Profiling, Urine Metabolomics, and Human Marker Gene Expression as an Integrated Approach to Study Alopecia AreataAbstract
Involvement of the microbiome in many different scalp conditions has been investigated over the years. Studies on the role of the scalp microbiome in specific diseases, such as those involving hair growth alterations like non-cicatricial [androgenetic alopecia (AGA), alopecia areata (AA)] and cicatricial alopecia lichen planopilaris, are of major importance. In the present work, we highlighted the differences in microbial populations inhabiting the scalp of AA subjects and a healthy sample cohort by using an integrated approach relying on metagenomic targeted 16S sequencing analysis, urine metabolomics, and human marker gene expression. Significant differences in genera abundances (p < 0.05) were found in the hypodermis and especially the dermis layer. Based on 16S sequencing data, we explored the differences in predicted KEGG pathways and identified some significant differences in predicted pathways related to the AA pathologic condition such as flagellar, assembly, bacterial chemotaxis, mineral absorption, ABC transporters, cellular antigens, glycosaminoglycan degradation, lysosome, sphingolipid metabolism, cell division, protein digestion and absorption, and energy metabolism. All predicted pathways were significantly enhanced in AA samples compared to expression in healthy samples, with the exceptions of mineral absorption, and ABC transporters. We also determined the expression of TNF-α, FAS, KCNA3, NOD-2, and SOD-2 genes and explored the relationships between human gene expression levels and microbiome composition by Pearson's correlation analysis; here, significant correlations both positive (SOD vs. Staphylococcus, Candidatus Aquiluna) and negative (FAS and SOD2 vs. Anaerococcus, Neisseria, and Acinetobacter) were highlighted. Finally, we inspected volatile organic metabolite profiles in urinary samples and detected statistically significant differences (menthol, methanethiol, dihydrodehydro-beta-ionone, 2,5-dimethylfuran, 1,2,3,4, tetrahydro-1,5,7-trimethylnapthalene) when comparing AA and healthy subject groups. This multiple comparison approach highlighted potential traits associated with AA and their relationship with the microbiota inhabiting the scalp, opening up novel therapeutic interventions in such kind of hair growth disorders mainly by means of prebiotics, probiotics, and postbiotics.
Relating the gut metagenome and metatranscriptome to immunotherapy responses in melanoma patientsAbstract
Recent evidence suggests that immunotherapy efficacy in melanoma is modulated by gut microbiota. Few studies have examined this phenomenon in humans, and none have incorporated metatranscriptomics, important for determining expression of metagenomic functions in the microbial community.
In melanoma patients undergoing immunotherapy, gut microbiome was characterized in pre-treatment stool using 16S rRNA gene and shotgun metagenome sequencing (n = 27). Transcriptional expression of metagenomic pathways was confirmed with metatranscriptome sequencing in a subset of 17. We examined associations of taxa and metagenomic pathways with progression-free survival (PFS) using 500 × 10-fold cross-validated elastic-net penalized Cox regression.
Higher microbial community richness was associated with longer PFS in 16S and shotgun data (p < 0.05). Clustering based on overall microbiome composition divided patients into three groups with differing PFS; the low-risk group had 99% lower risk of progression than the high-risk group at any time during follow-up (p = 0.002). Among the species selected in regression, abundance of Bacteroides ovatus, Bacteroides dorei, Bacteroides massiliensis, Ruminococcus gnavus, and Blautia producta were related to shorter PFS, and Faecalibacterium prausnitzii, Coprococcus eutactus, Prevotella stercorea, Streptococcus sanguinis, Streptococcus anginosus, and Lachnospiraceae bacterium 3 1 46FAA to longer PFS. Metagenomic functions related to PFS that had correlated metatranscriptomic expression included risk-associated pathways of L-rhamnose degradation, guanosine nucleotide biosynthesis, and B vitamin biosynthesis.
This work adds to the growing evidence that gut microbiota are related to immunotherapy outcomes, and identifies, for the first time, transcriptionally expressed metagenomic pathways related to PFS. Further research is warranted on microbial therapeutic targets to improve immunotherapy outcomes.
Metagenomic Characterization of Indoor Dust Bacterial and Fungal Microbiota in Homes of Asthma and Non-asthma Patients Using Next Generation SequencingAbstract
Background: The exposure of house occupants to indoor air pollutants has increased in recent decades. Among microbiological contaminants, bacterial and fungal aerosols remain poorly studied and the debate on the impact of these aerosols on respiratory health is still open. This study aimed to assess the diversity of indoor microbial communities in relationship with the health of occupants.
Methods: Measurements were taken from dwellings of 2 cohorts in Brittany (France), one with children without any pathology and the other with children and adults with asthma. Thirty dust samples were analyzed by next generation sequencing with a 16S and 18S targeted metagenomics approach. Analysis of sequencing data was performed using qiime 2, and univariate and multivariate statistical analysis using R software and phyloseq package.
Results: A total of 2,637 prokaryotic (589 at genus level) and 2,153 eukaryotic taxa were identified (856 fungal taxa (39%) and 573 metazoa (26%)). The four main bacterial phyla were identified: Proteobacteria (53%), Firmicutes (27%), Actinobacteria (11%), Bacteroidetes (8%). Among Fungi, only 136 taxa were identified at genus level. Three main fungal phyla were identified: Ascomycota (84%), Basidiomycota (12%) and Mucoromycota (3%). No bacterial nor fungal phyla were significantly associated with asthma versus control group. A significant over representation in control group versus asthma was observed for Christensenellaceae family (p-value = 0.0015, adj. p-value = 0.033). Besides, a trend for over representation in control group was observed with Dermabacteraceae family (p-value = 0.0002, adj. p-value = 0.815).
Conclusions: Our findings provide evidence that dust samples harbor a high diversity of human-associated bacteria and fungi. Molecular methods such as next generation sequencing are reliable tools for identifying and tracking the bacterial and fungal diversity in dust samples, a less easy strategy for the detection of eukaryotes at least using18S metagenomics approach. This study showed that the detection of some bacteria might be associated to indoor air of asthmatic patients. Regarding fungi, a higher number of samples and sequencing with more depth could allow reaching significant signatures.
Phylogenetic farming: Can evolutionary history predict crop rotation via the soil microbiome?Abstract
Agriculture has long employed phylogenetic rules whereby farmers are encouraged to rotate taxonomically unrelated plants in shared soil. Although this forms a central tenet of sustainable agriculture, strangely, this on-farm ‘rule of thumb’ has never been rigorously tested in a scientific framework. To experimentally evaluate the relationship between phylogenetic distance and crop performance, we used a plant-soil feedback approach whereby 35 crops and weeds varying in their relatedness to tomato (Solanum lycopersicum) were tested in a two-year field experiment. We used community profiling of the bacteria and fungi to determine the extent to which soil microbes contribute to phenotypic differences in crop growth. Overall, tomato yield was ca. 15% lower in soil previously cultivated with tomato; yet, past the species-level there was no effect of phylogenetic distance on crop performance. Soil microbial communities, on the other hand, were compositionally more similar between close plant relatives. Random Forest regression predicted log10 phylogenetic distance to tomato with moderate accuracy (R2 = 0.52), primarily driven by bacteria in the genus Sphingobium. These data indicate that, beyond avoiding conspecifics, evolutionary history contributes little to understanding plant-soil feedbacks in agricultural fields; however, microbial legacies can be predicted by species identity and relatedness.
High diversity and pan-oceanic distribution of deep-sea polychaetes: Prionospio and Aurospio (Annelida: Spionidae) in the Atlantic and Pacific OceanAbstract
Prionospio Malmgren 1867 and Aurospio Maciolek 1981 (Annelida: Spionidae) are polychaete genera commonly found in the deep sea. Both genera belong to the Prionospio complex, whose members are known to have limited distinguishing characters. Morphological identification of specimens from the deep sea is challenging, as fragmentation and other damages are common during sampling. These issues impede investigations into the distribution patterns of these genera in the deep sea. In this study, we employ two molecular markers (16S rRNA and 18S) to study the diversity and the distribution patterns of Prionospio and Aurospio from the tropical North Atlantic, the Puerto Rico Trench and the central Pacific. Based on different molecular analyses (Automated Barcode Gap Discovery, GMYC, pairwise genetic distances, phylogenetics, haplotype networks), we were able to identify and differentiate 21 lineages (three lineages composed solely of GenBank entries) that represent putative species. Seven of these lineages exhibited pan-oceanic distributions (occurring in the Atlantic as well as the Pacific) in some cases even sharing identical 16S rRNA haplotypes in both oceans. Even the lineages found to be restricted to one of the oceans were distributed over large regional scales as for example across the Mid-Atlantic Ridge from the Caribbean to the eastern Atlantic (> 3389 km). Our results suggest that members of Prionospio and Aurospio may have the potential to disperse across large geographic distances, largely unaffected by topographic barriers and possibly even between oceans. Their high dispersal capacities are probably explained by their free-swimming long-lived planktonic larvae.
Investigation of the relationship between virulence factors and antibiotic resistance of Enterococci isolatesAbstract
The aim of this study was to determine the relationship between aggregation factor (asa1), enterococcal surface protein (esp), cytolysin (cyl), gelatinase (gelE), hyaluronidase (hyl) virulence factors and antibiotic resistance in Enterococci. VITEK 2 ID system was used to identify the isolates and determine
their antibiotic susceptibility. Virulence genes were investigated by polymerase chain reaction. Of the 93 isolates, 62 (66 %) were Enterococcus faecium, 31 (44
%) were Enterococcus faecalis (E. faecialis ). E. faecium isolates were more resistant to ampicillin, ciprofloxacin, linezolid, teicoplanin and vancomycin than E.
faecalis. High-level gentamycin rate were higher in E. faecium than E. faecalis (p <0.05). The most prevalant virulence genes were esp (60.9 %) and asa1 (25 %)
followed by gelE (22.8 %), cyl (16.3 %) and hyl (8.7 %). Asa1, cyl, gelE genes positivity were higer in E. faecalis than E. faecium. Hyl positivity was higher in E.
faecalis than E. faecium isolates. Ampicillin resistance was higher in gelE positive E. faecalis than gelE negative E. faecalis (p <0.05). Ciprofloxacin resistance
was higher in gelE negative E. faecalis than gelE positive E. faecalis (p <0.05). Asa, cyl, hyl, gelE positive E. faecium isolates were more susceptible to teicoplanin than the isolates that did not have these genes (p <0.05). Cyl, asa, gelE positive E. faecalis isolates were more susceptible to vancomycin than cyl, asa, gelE
negative E. faecalis isoates (p <0.05). Hyl positive E. faecium isolates were more susceptible to vancomycin than hyl negative E. faecium isolates (p <0.05). E.
faecalis isolates that have virulence genes were more susceptible to vancomycin (p <0.05). The resistance to antibiotics in E. faecalis should be a concern for the
treatment of infectious disease.
Diversity and distribution of Laonice species (Annelida: Spionidae) in the tropical North Atlantic and Puerto Rico TrenchAbstract
Laonice Malmgren, 1867 (Annelida: Spionidae) is a common polychaete genus in the deep-sea. Although most species are quite well studied morphologically, fragmentation and other damage that occurs during sampling often hampers morphological species identification of deep-sea specimens. In this study, we employ three molecular markers (16S, COI and 18S) to study the biodiversity and the distribution patterns of Laonice from the tropical North Atlantic and the Puerto Rico Trench. Based upon different molecular analyses (Automated Barcode Gap Discovery, pairwise genetic distances, phylogenetics, haplotype networks) we were able to identify and differentiate eight Laonice species. Up to four of these species co-occurred sympatrically at the same station. The majority of species were found at multiple stations and two species in the eastern as well as western Atlantic had ranges of up to 4,000 km. Genetic differentiation across these extensive geographic distances was very low. Surprisingly, one 16S haplotype was shared between individuals 2,776 km apart and individuals from the Caribbean and the abyssal plain in the eastern Atlantic (>3,389 km) differed in only a single mutation in 16S. Our results suggest that members of this genus successfully disperse across large geographic distances and are largely unaffected by topographic barriers.
The genus Syrrhoe (Crustacea, Amphipoda, Synopiidae) from the North AtlanticAbstract
Three species of the amphipod genus Syrrhoe are described from the North Atlantic. The differences between these species are primarily the patterns of serration of the posterior margins of pleonite 3 and urosomite 1 and 2: Syrrhoe affinis has a wide convex space on the posterior margin between the epimeron 3 and the dorsal serration. In Syrrhoe crenulata and Syrrhoe anneheleneae sp. nov. there is only a small notch on the posterior margin of pleonite 3. Syrrhoe anneheleneae sp. nov., otherwise similar to S. crenulata, has an additional serration on the posterior margin of urosomite 1. The inter- and intraspecific distances analyzed from COI confirm the morphological species concept of North Atlantic Syrrhoe.
Identification and removal of contaminating microbial DNA from PCR reagents: impact on low‐biomass microbiome analysesAbstract
Reagent‐derived contamination can compromise the integrity of microbiome data, particularly in low microbial biomass samples. This contamination has recently been attributed to the ‘kitome’ (contamination introduced by the DNA extraction kit), prior to which attention was mostly paid to potential contamination introduced by PCR reagents. In this study, we assessed the proportion to which our DNA extraction kit and PCR master mix introduce contaminating microbial DNA to bacterial microbial profiles generated by 16S rRNA gene sequencing. Utilizing a commercial dsDNase treatment protocol to decontaminate the PCR master mix, we demonstrated that the vast majority of contaminating DNA was derived from the PCR master mix. Importantly, this contamination was almost completely eliminated using the simple dsDNase treatment, resulting in a 99% reduction in contaminating bacterial reads. We suggest that dsDNase treatment of PCR reagents should be explored as a simple and effective way of reducing contamination in low‐biomass microbiome studies and producing more robust and reliable data.
Scalp bacterial shift in Alopecia areataAbstract
The role of microbial dysbiosis in scalp disease has been recently hypothesized. However,
little information is available with regards to the association between microbial population on
the scalp and hair diseases related to hair growth. Here we investigated bacterial communities in healthy and Alopecia areata (AA) subjects. The analysis of bacterial distribution at the
genus level highlighted an increase of Propionibacterium in AA subjects alongside a general
decrease of Staphylococcus. Analysis of log Relative abundance of main bacterial species
inhabiting the scalp showed a significant increase of Propionibacterium acnes in AA subjects compared to control ones. AA scalp condition is also associated with a significant
decrease of Staphylococcus epidermidis relative abundance. No significant changes were
found for Staphylococcus aureus. Therefore, data from sequencing profiling of the bacterial
population strongly support a different microbial composition of the different area surrounded hair follicle from the epidermis to hypodermis, highlighting differences between normal and AA affected the scalp. Our results highlight, for the first time, the presence of a
microbial shift on the scalp of patients suffering from AA and gives the basis for a larger and
more complete study of microbial population involvement in hair disorders.
A novel perspective on MOL-PCR optimization and MAGPIX analysis of in-house multiplex foodborne pathogens detection assayAbstract
Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.
The Microbial Communities of Leaves and Roots Associated with Turtle Grass (Thalassia testudinum) and Manatee Grass (Syringodium filliforme) are Distinct from Seawater and Sediment Communities, but Are Similar between Species and Sampling SitesAbstract
Seagrasses are vital members of coastal systems, which provide several important ecosystem services such as improvement of water quality, shoreline protection, and serving as shelter, food, and nursery to many species, including economically important fish. They also act as a major carbon sink and supply copious amounts of oxygen to the ocean. A decline in seagrasses has been observed worldwide, partly due to climate change, direct and indirect human activities, diseases, and increased sulfide concentrations in the coastal porewaters. Several studies have shown a symbiotic relationship between seagrasses and their microbiome. For instance, the sulfur, nitrogen, and carbon cycles are important biochemical pathways that seem to be linked between the plant and its microbiome. The microbiome presumably also plays a key role in the health of the plant, for example in oxidizing phyto-toxic sulfide into non-toxic sulfate, or by providing protection for seagrasses from pathogens. Two of the most abundant seagrasses in Florida include Thalassia testudinum (turtle grass) and Syringodium filliforme (manatee grass), yet there is little data on the composition of the microbiome of these two genera. In this study, the microbial composition of the phyllosphere and rhizosphere of Thalassia testudinum and Syringodium filiforme were compared to water and sediment controls using amplicon sequencing of the V4 region of the 16S rRNA gene. The microbial composition of the leaves, roots, seawater, and sediment differ from one another, but are similar between the two species of seagrasses.
High-throughput marker assays for FaRPc2-mediated resistance to Phytophthora crown rot in octoploid strawberryAbstract
Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is a destructive disease of the allo-octoploid cultivated strawberry (Fragaria ×ananassa Duch). Many major strawberry cultivars grown worldwide are susceptible to PhCR. Resistance is conferred by the recently-discovered FaRPc2 locus, but high-throughput markers are not yet available for marker-assisted breeding. In the current study, we developed DNA markers for two haplotypes at the FaRPc2 locus associated with resistance, H2 and H3. Marker validation and marker-assisted selection were performed in University of Florida (UF) breeding population. Seven single nucleotide polymorphism-based high resolution melting (HRM) markers linked to H2 and four HRM markers for H3 were developed. One HRM marker, RPCHRM3 linked to H3, was converted to a Kompetitive Allele Specific PCR (KASP) marker. To further examine the utility of the markers, they were screened in University of California Davis cultivars with known phenotypes as well as in 20 diverse accessions with phenotypes that are reported in the literature and that are preserved at the USDA-ARS National Clonal Germplasm Repository, in Corvallis, Oregon. The most informative markers for FaRPc2 resistance are being implemented in the UF strawberry breeding program to improve PhCR resistance.
Development of High-Throughput SNP Genotyping Assays for Rapid Detection of Strawberry Colletotrichum Species and the G143A MutationAbstract
Colletotrichum species cause major diseases of strawberry and disease management depends on the species present. However, species identification based on symptoms and spore morphology is difficult. Therefore, development of molecular techniques for trustworthy and high-throughput identification of Colletotrichum species is vital for the accurate diagnosis. A High-Resolution Melting (HRM) assay was developed for simultaneous identification and differentiation of Colletotrichum species from fungal colonies or from symptomatic strawberry tissue. HRM markers were designed based on the ITS region of C. acutatum and C. gloeosporioides from strawberry, and accurately identified and differentiated the two species. In addition, for the rapid detection of a single nucleotide polymorphism (SNP) in the cytochrome b (cytb) gene of C. acutatum and C. gloeosporioides associated with resistance to quinone-outside inhibitor fungicides, an endpoint SNP genotyping analysis was developed. The HRM and endpoint SNP genotyping assays are useful methods that can be implemented in plant diagnostic clinics for the rapid and accurate identification of Colletotrichum species and detection of the G143A mutation in the cytb gene of C. acutatum and C. gloeosporioides.
FaRCg1: a quantitative trait locus conferring resistance to Colletotrichum crown rot caused by Colletotrichum gloeosporioides in octoploid strawberryAbstract
Colletotrichum crown rot (CCR) is an important disease of strawberry (Fragaria ×ananassa) throughout the Southeastern US and in subtropical climates around the world, where hot and humid conditions facilitate rapid disease development. Yet no resistance loci have been described to date, as genetic studies have been historically difficult in allo-octoploid (2n = 8x = 56) strawberry. In the present study, we investigate the genetic architecture of resistance to CCR. Four population sets from the University of Florida were inoculated in four different seasons from 2013–2014 to 2016–2017. Two large, multiparental discovery population sets were used for QTL discovery, and two validation sets of cultivars and advanced selections representing the parent pool of the breeding program were also assessed. Subgenome-specific single-nucleotide polymorphism (SNP) markers were mapped, and FlexQTL™ software was utilized to perform a Bayesian, pedigree-based QTL analysis. A quantitative trait locus on linkage group 6B, which we name FaRCg1, accounts for most of the genetic variation for resistance in the discovery sets (26.8–29.8% in 2013–2014 and 17% in 2015–2016). High-throughput marker assays were developed for the most significant SNPs which correlated with the mode of the QTL region. The discovery and characterization of the FaRCg1 locus and the molecular tools developed from it will be utilized to achieve increased genetic gains for resistance.
VKORC1 sequence variants associated with resistance to anticoagulant rodenticides in Irish populations of Rattus norvegicus and Mus musculus domesticusAbstract
While resistance to anticoagulant rodenticides is known to occur in many European populations of Norway rat and house mouse, to-date no data is available on the occurrence in Ireland of such resistance. No genetic evidence for the occurrence of resistance was found in 65 Norway rat samples analysed, indicative of an absence, or low prevalence, of resistance in rats in at least the Eastern region of the island of Ireland. The presence of two of the most commonly found amino acid substitutions Leu128Ser and Tyr139Cys associated with house mouse resistance to anticoagulant rodenticides was confirmed. The occurrence of two such mutations is indicative of the occurrence of resistance to anticoagulant rodenticides in house mice in the Eastern region of the island of Ireland.
Comparison of the oral microbiome in mouthwash and whole saliva samplesAbstract
Population-based epidemiologic studies can provide important insight regarding the role of the microbiome in human health and disease. Buccal cells samples using commercial mouthwash have been obtained in large prospective cohorts for the purpose of studying human genomic DNA. We aimed to better understand if these mouthwash samples are also a valid resource for the study of the oral microbiome. We collected one saliva sample and one Scope mouthwash sample from 10 healthy subjects. Bacterial 16S rRNA genes from both types of samples were amplified, sequenced, and assigned to bacterial taxa. We comprehensively compared these paired samples for bacterial community composition and individual taxonomic abundance. We found that mouthwash samples yielded similar amount of bacterial DNA as saliva samples (p from Student’s t-test for paired samples = 0.92). Additionally, the paired samples had similar within sample diversity (p from = 0.33 for richness, and p = 0.51 for Shannon index), and clustered as pairs for diversity when analyzed by unsupervised hierarchical cluster analysis. No significant difference was found in the paired samples with respect to the taxonomic abundance of major bacterial phyla, Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria (FDR adjusted q values from Wilcoxin signed-rank test = 0.15, 0.15, 0.87, 1.00 and 0.15, respectively), and all identified genera, including genus Streptococcus (q = 0.21), Prevotella (q = 0.25), Neisseria (q = 0.37), Veillonella (q = 0.73), Fusobacterium (q = 0.19), and Porphyromonas (q = 0.60). These results show that mouthwash samples perform similarly to saliva samples for analysis of the oral microbiome. Mouthwash samples collected originally for analysis of human DNA are also a resource suitable for human microbiome research.