Performance characteristics of next-generation sequencing for the detection of antimicrobial resistance determinants in Escherichia coli genomes and metagenomes

Short-read sequencing can provide detection of multiple genomic determinants of antimicrobial resistance from single bacterial genomes and metagenomic samples. Despite its increasing application in human, animal, and environmental microbiology, including human clinical trials, the performance of short-read Illumina sequencing for antimicrobial resistance gene (ARG) detection, including resistance-conferring single nucleotide polymorphisms (SNPs), has not been systematically characterized. Using paired-end 2 x 150 bp (base pair) Illumina sequencing and an assembly-based method for ARG prediction, we determined sensitivity, positive predictive value (PPV), and sequencing depths required for ARG detection in an Escherichia coli isolate of sequence type (ST) 38 spiked into a synthetic microbial community at varying abundances. Approximately 300,000 reads or 15x genome coverage was sufficient to detect ARGs in E. coli ST38, with comparable sensitivity and PPV to ~100x genome coverage. Using metagenome assembly of mixed microbial communities, ARG detection at E. coli relative abundances of 1% would require assembly of approximately 30 million reads to achieve 15x target coverage. The minimum sequencing depths were validated using public data sets of 948 E. coli genomes and 10 metagenomic rectal swab samples. A read-based approach using k-mer alignment (KMA) for ARG prediction did not substantially improve minimum sequencing depths for ARG detection compared to assembly of the E. coli ST38 genome or the combined metagenomic samples. Analysis of sequencing depths from recent studies assessing ARG content in metagenomic samples demonstrated that sequencing depths had a median estimated detection frequency of 84% (interquartile range: 30%-92%) for a relative abundance of 1%. IMPORTANCE Systematically determining Illumina sequencing performance characteristics for detection of ARGs in metagenomic samples is essential to inform study design and appraisal of human, animal, and environmental metagenomic antimicrobial resistance studies. In this study, we quantified the performance characteristics of ARG detection in E. coli genomes and metagenomes and established a benchmark of ~15x coverage for ARG detection for E. coli in metagenomes. We demonstrate that for low relative abundances, sequencing depths of ~30 million reads or more may be required for adequate sensitivity for many applications

Mitochondrial phylogeography and demographic history of the Vicuña: implications for conservation

The vicuña (Vicugna vicugna; Miller, 1924) is a conservation success story, having recovered from near extinction in the 1960s to current population levels estimated at 275 000. However, lack of information about its demographic history and genetic diversity has limited both our understanding of its recovery and the development of science-based conservation measures. To examine the evolution and recent demographic history of the vicuña across its current range and to assess its genetic variation and population structure, we sequenced mitochondrial DNA from the control region (CR) for 261 individuals from 29 populations across Peru, Chile and Argentina. Our results suggest that populations currently designated as Vicugna vicugna vicugna and Vicugna vicugna mensalis comprise separate mitochondrial lineages. The current population distribution appears to be the result of a recent demographic expansion associated with the last major glacial event of the Pleistocene in the northern (18 to 22°S) dry Andes 14–12 000 years ago and the establishment of an extremely arid belt known as the 'Dry Diagonal' to 29°S. Within the Dry Diagonal, small populations of V. v. vicugna appear to have survived showing the genetic signature of demographic isolation, whereas to the north V. v. mensalis populations underwent a rapid demographic expansion before recent anthropogenic impacts

Sporormiella as a tool for detecting the presence of large herbivores in the Neotropics

The reliability of using the abundance of Sporormiella spores as a proxy for the presence and abundance of megaherbivores was tested in southern Brazil. Mud-water interface samples from nine lakes, in which cattle-use was categorized as high, medium, or low, were assayed for Sporormiella representation. The sampling design allowed an analysis of both the influence of the number of animals using the shoreline and the distance of the sampling site from the nearest shoreline. Sporormiella was found to be a reliable proxy for the presence of large livestock. The concentration and abundance of spores declined from the edge of the lake toward the center, with the strongest response being in sites with high livestock use. Consistent with prior studies in temperate regions, we find that Sporormiella spores are a useful proxy to study the extinction of Pleistocene megafauna or the arrival of European livestock in Neotropical landscapes

Ancient Nursery Area for the Extinct Giant Shark Megalodon from the Miocene of Panama

BACKGROUND: As we know from modern species, nursery areas are essential shark habitats for vulnerable young. Nurseries are typically highly productive, shallow-water habitats that are characterized by the presence of juveniles and neonates. It has been suggested that in these areas, sharks can find ample food resources and protection from predators. Based on the fossil record, we know that the extinct Carcharocles megalodon was the biggest shark that ever lived. Previous proposed paleo-nursery areas for this species were based on the anecdotal presence of juvenile fossil teeth accompanied by fossil marine mammals. We now present the first definitive evidence of ancient nurseries for C. megalodon from the late Miocene of Panama, about 10 million years ago. METHODOLOGY/PRINCIPAL FINDINGS: We collected and measured fossil shark teeth of C. megalodon, within the highly productive, shallow marine Gatun Formation from the Miocene of Panama. Surprisingly, and in contrast to other fossil accumulations, the majority of the teeth from Gatun are very small. Here we compare the tooth sizes from the Gatun with specimens from different, but analogous localities. In addition we calculate the total length of the individuals found in Gatun. These comparisons and estimates suggest that the small size of Gatun's C. megalodon is neither related to a small population of this species nor the tooth position within the jaw. Thus, the individuals from Gatun were mostly juveniles and neonates, with estimated body lengths between 2 and 10.5 meters. CONCLUSIONS/SIGNIFICANCE: We propose that the Miocene Gatun Formation represents the first documented paleo-nursery area for C. megalodon from the Neotropics, and one of the few recorded in the fossil record for an extinct selachian. We therefore show that sharks have used nursery areas at least for 10 millions of years as an adaptive strategy during their life histories

The Co-Morbidity Burden of Children and Young Adults with Autism Spectrum Disorders

Objectives: Use electronic health records Autism Spectrum Disorder (ASD) to assess the comorbidity burden of ASD in children and young adults. Study Design: A retrospective prevalence study was performed using a distributed query system across three general hospitals and one pediatric hospital. Over 14,000 individuals under age 35 with ASD were characterized by their co-morbidities and conversely, the prevalence of ASD within these comorbidities was measured. The comorbidity prevalence of the younger (Age<18 years) and older (Age 18–34 years) individuals with ASD was compared. Results: 19.44% of ASD patients had epilepsy as compared to 2.19% in the overall hospital population (95% confidence interval for difference in percentages 13.58–14.69%), 2.43% of ASD with schizophrenia vs. 0.24% in the hospital population (95% CI 1.89–2.39%), inflammatory bowel disease (IBD) 0.83% vs. 0.54% (95% CI 0.13–0.43%), bowel disorders (without IBD) 11.74% vs. 4.5% (95% CI 5.72–6.68%), CNS/cranial anomalies 12.45% vs. 1.19% (95% CI 9.41–10.38%), diabetes mellitus type I (DM1) 0.79% vs. 0.34% (95% CI 0.3–0.6%), muscular dystrophy 0.47% vs 0.05% (95% CI 0.26–0.49%), sleep disorders 1.12% vs. 0.14% (95% CI 0.79–1.14%). Autoimmune disorders (excluding DM1 and IBD) were not significantly different at 0.67% vs. 0.68% (95% CI −0.14-0.13%). Three of the studied comorbidities increased significantly when comparing ages 0–17 vs 18–34 with p<0.001: Schizophrenia (1.43% vs. 8.76%), diabetes mellitus type I (0.67% vs. 2.08%), IBD (0.68% vs. 1.99%) whereas sleeping disorders, bowel disorders (without IBD) and epilepsy did not change significantly. Conclusions: The comorbidities of ASD encompass disease states that are significantly overrepresented in ASD with respect to even the patient populations of tertiary health centers. This burden of comorbidities goes well beyond those routinely managed in developmental medicine centers and requires broad multidisciplinary management that payors and providers will have to plan for

Cheek Tooth Morphology and Ancient Mitochondrial DNA of Late Pleistocene Horses from the Western Interior of North America: Implications for the Taxonomy of North American Late Pleistocene Equus

Horses were a dominant component of North American Pleistocene land mammal communities and their remains are well represented in the fossil record. Despite the abundant material available for study, there is still considerable disagreement over the number of species of Equus that inhabited the different regions of the continent and on their taxonomic nomenclature. In this study, we investigated cheek tooth morphology and ancient mtDNA of late Pleistocene Equus specimens from the Western Interior of North America, with the objective of clarifying the species that lived in this region prior to the end-Pleistocene extinction. Based on the morphological and molecular data analyzed, a caballine (Equus ferus) and a non-caballine (E. conversidens) species were identified from different localities across most of the Western Interior. A second non-caballine species (E. cedralensis) was recognized from southern localities based exclusively on the morphological analyses of the cheek teeth. Notably the separation into caballine and non-caballine species was observed in the Bayesian phylogenetic analysis of ancient mtDNA as well as in the geometric morphometric analyses of the upper and lower premolars. Teeth morphologically identified as E. conversidens that yielded ancient mtDNA fall within the New World stilt-legged clade recognized in previous studies and this is the name we apply to this group. Geographic variation in morphology in the caballine species is indicated by statistically different occlusal enamel patterns in the specimens from Bluefish Caves, Yukon Territory, relative to the specimens from the other geographic regions. Whether this represents ecomorphological variation and/or a certain degree of geographic and genetic isolation of these Arctic populations requires further study

The multi-peak adaptive landscape of crocodylomorph body size evolution

Background: Little is known about the long-term patterns of body size evolution in Crocodylomorpha, the > 200-million-year-old group that includes living crocodylians and their extinct relatives. Extant crocodylians are mostly large-bodied (3–7 m) predators. However, extinct crocodylomorphs exhibit a wider range of phenotypes, and many of the earliest taxa were much smaller ( Results: Crocodylomorphs reached an early peak in body size disparity during the Late Jurassic, and underwent an essentially continual decline since then. A multi-peak Ornstein-Uhlenbeck model outperforms all other evolutionary models fitted to our data (including both uniform and non-uniform), indicating that the macroevolutionary dynamics of crocodylomorph body size are better described within the concept of an adaptive landscape, with most body size variation emerging after shifts to new macroevolutionary regimes (analogous to adaptive zones). We did not find support for a consistent evolutionary trend towards larger sizes among lineages (i.e., Cope’s rule), or strong correlations of body size with climate. Instead, the intermediate to large body sizes of some crocodylomorphs are better explained by group-specific adaptations. In particular, the evolution of a more aquatic lifestyle (especially marine) correlates with increases in average body size, though not without exceptions. Conclusions: Shifts between macroevolutionary regimes provide a better explanation of crocodylomorph body size evolution on large phylogenetic and temporal scales, suggesting a central role for lineage-specific adaptations rather than climatic forcing. Shifts leading to larger body sizes occurred in most aquatic and semi-aquatic groups. This, combined with extinctions of groups occupying smaller body size regimes (particularly during the Late Cretaceous and Cenozoic), gave rise to the upward-shifted body size distribution of extant crocodylomorphs compared to their smaller-bodied terrestrial ancestors.</p

Bone histology provides insights into the life history mechanisms underlying dwarfing in hipparionins

Size shifts may be a by-product of alterations in life history traits driven by natural selection. Although this approach has been proposed for islands, it has not yet been explored in continental faunas. The trends towards size decrease experienced by some hipparionins constitute a good case study for the application of a life history framework to understand the size shifts on the continent. Here, we analysed bone microstructure to reconstruct the growth of some different-sized hipparionins from Greece and Spain. The two dwarfed lineages studied show different growth strategies. The Greek hipparions ceased growth early at a small size thus advancing maturity, whilst the slower-growing Spanish hipparion matured later at a small size. Based on predictive life history models, we suggest that high adult mortality was the likely selective force behind early maturity and associated size decrease in the Greek lineage. Conversely, we infer that resource limitation accompanied by high juvenile mortality triggered decrease in growth rate and a relative late maturity in the Spanish lineage. Our results provide evidence that different selective pressures can precipitate different changes in life history that lead to similar size shifts