ColiSeq: a multiplex amplicon assay that provides strain level resolution of Escherichia coli directly from clinical specimens.

Escherichia coli is a diverse pathogen, causing a range of disease in humans, from self-limiting diarrhea to urinary tract infections (UTIs). Uropathogenic E. coli (UPEC) is the most frequently observed uropathogen in UTIs, a common disease in high-income countries, incurring billions of dollars yearly in treatment costs. Although E. coli is easily grown and identified in the clinical laboratory, genotyping the pathogen is more complicated, yet critical for reducing the incidence of disease. These goals can be achieved through whole-genome sequencing of E. coli isolates, but this approach is relatively slow and typically requires culturing the pathogen in the laboratory. To genotype E. coli rapidly and inexpensively directly from clinical samples, including but not limited to urine, we developed and validated a multiplex amplicon sequencing assay, called ColiSeq. The assay consists of targets designed for E. coli species confirmation, high resolution genotyping, and mixture deconvolution. To demonstrate its utility, we screened the ColiSeq assay against 230 clinical urine samples collected from a hospital system in Flagstaff, Arizona, USA. A limit of detection analysis demonstrated the ability of ColiSeq to identify E. coli at a concentration of ~2 genomic equivalent (GEs)/mL and to generate high-resolution genotyping at a concentration of 1 × 105 GEs/mL. The results of this study suggest that ColiSeq could be a valuable method to understand the source of UPEC strains and guide infection mitigation efforts. As sequence-based diagnostics become accepted in the clinical laboratory, workflows such as ColiSeq will provide actionable information to improve patient outcomes.IMPORTANCEUrinary tract infections (UTIs), caused primarily by Escherichia coli, create an enormous health care burden in the United States and other high-income countries. The early detection of E. coli from clinical samples, including urine, is important to target therapy and prevent further patient complications. Additionally, understanding the source of E. coli exposure will help with future mitigation efforts. In this study, we developed, tested, and validated an amplicon sequencing assay focused on direct detection of E. coli from urine. The resulting sequence data were demonstrated to provide strain level resolution of the pathogen, not only confirming the presence of E. coli, which can focus treatment efforts, but also providing data needed for source attribution and contact tracing. This assay will generate inexpensive, rapid, and reproducible data that can be deployed by public health agencies to track, diagnose, and potentially mitigate future UTIs caused by E. coli.

Pan-Enterovirus Characterization Reveals Cryptic Circulation of Clinically Relevant Subtypes in Arizona Wastewater.

Background: Most seasonally circulating enteroviruses result in asymptomatic or mildly symptomatic infections. In rare cases, however, infection with some subtypes can result in paralysis or death. Of the 300 subtypes known, only poliovirus is reportable, limiting our understanding of the distribution of other enteroviruses that can cause clinical disease. Objective: The overarching objectives of this study were to: 1) describe the distribution of enteroviruses in Arizona during the late summer and fall of 2022, the time of year when they are thought to be most abundant, and 2) demonstrate the utility of viral pan-assay approaches for semi-agnostic discovery that can be followed up by more targeted assays and phylogenomics. Methods: This study utilizes pooled nasal samples collected from school-aged children and long-term care facility residents, and wastewater from multiple locations in Arizona during July-October of 2022. We used PCR to amplify and sequence a region common to all enteroviruses, followed by species-level bioinformatic characterization using the QIIME 2 platform. For Enterovirus-D68 (EV-D68), detection was carried out using RT-qPCR, followed by confirmation using near-complete whole EV-D68 genome sequencing using a newly designed tiled amplicon approach. Results: In the late summer and early fall of 2022, multiple enterovirus species were identified in Arizona wastewater, with Coxsackievirus A6, EV-D68, and Coxsackievirus A19 composing 86% of the characterized reads sequenced. While EV-D68 was not identified in pooled human nasal samples, and the only reported acute flaccid myelitis case in Arizona did not test positive for the virus, an in-depth analysis of EV-D68 in wastewater revealed that the virus was circulating from August through mid-October. A phylogenetic analysis on this relatively limited dataset revealed just a few importations into the state, with a single clade indicating local circulation. Significance: This study further supports the utility of wastewater-based epidemiology to identify potential public health threats. Our further investigations into EV-D68 shows how these data might help inform healthcare diagnoses for children presenting with concerning neurological symptoms.

Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition.

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles.

A Quantitative Assessment of Staphylococcus aureus Community Carriage in Yuma, Arizona.

BACKGROUND: Disease control relies on pathogen identification and understanding reservoirs. Staphylococcus aureus infection prevention is based upon decades of research on colonization and infection, but diminishing returns from mitigation efforts suggest significant knowledge gaps. Existing knowledge and mitigation protocols are founded upon culture-based detection, with almost no information about pathogen quantities. METHODS: We used culture and a quantitative polymerase chain reaction assay on samples from 3 body sites to characterize colonization more comprehensively than previous studies by describing both prevalence and pathogen quantity. RESULTS: We show a much higher overall prevalence (65.9%) than previously documented, with higher quantities and prevalence associated with the nares, non-Hispanic males (86.9%), and correlating with colonization in other body sites. These results suggest that research and clinical practices likely misclassify over half of colonized persons, limiting mitigation measures and their impact. CONCLUSIONS: This work begins the process of rebuilding foundational knowledge of S aureus carriage with more accurate and wholistic approaches.

MTSv: rapid alignment-based taxonomic classification and high-confidence metagenomic analysis.

As the size of reference sequence databases and high-throughput sequencing datasets continue to grow, it is becoming computationally infeasible to use traditional alignment to large genome databases for taxonomic classification of metagenomic reads. Exact matching approaches can rapidly assign taxonomy and summarize the composition of microbial communities, but they sacrifice accuracy and can lead to false positives. Full alignment tools provide higher confidence assignments and can assign sequences from genomes that diverge from reference sequences; however, full alignment tools are computationally intensive. To address this, we designed MTSv specifically for alignment-based taxonomic assignment in metagenomic analysis. This tool implements an FM-index assisted q-gram filter and SIMD accelerated Smith-Waterman algorithm to find alignments. However, unlike traditional aligners, MTSv will not attempt to make additional alignments to a TaxID once an alignment of sufficient quality has been found. This improves efficiency when many reference sequences are available per taxon. MTSv was designed to be flexible and can be modified to run on either memory or processor constrained systems. Although MTSv cannot compete with the speeds of exact k-mer matching approaches, it is reasonably fast and has higher precision than popular exact matching approaches. Because MTSv performs a full alignment it can classify reads even when the genomes share low similarity with reference sequences and provides a tool for high confidence pathogen detection with low off-target assignments to near neighbor species.

Sample pooling methods for efficient pathogen screening: Practical implications.

Due to the large number of negative tests, individually screening large populations for rare pathogens can be wasteful and expensive. Sample pooling methods improve the efficiency of large-scale pathogen screening campaigns by reducing the number of tests and reagents required to accurately categorize positive and negative individuals. Such methods rely on group testing theory which mainly focuses on minimizing the total number of tests; however, many other practical concerns and tradeoffs must be considered when choosing an appropriate method for a given set of circumstances. Here we use computational simulations to determine how several theoretical approaches compare in terms of (a) the number of tests, to minimize costs and save reagents, (b) the number of sequential steps, to reduce the time it takes to complete the assay, (c) the number of samples per pool, to avoid the limits of detection, (d) simplicity, to reduce the risk of human error, and (e) robustness, to poor estimates of the number of positive samples. We found that established methods often perform very well in one area but very poorly in others. Therefore, we introduce and validate a new method which performs fairly well across each of the above criteria making it a good general use approach.

Selective whole genome amplification and sequencing of Coxiella burnetii directly from environmental samples.

Whole genome sequencing (WGS) is a widely available, inexpensive means of providing a wealth of information about an organism's diversity and evolution. However, WGS for many pathogenic bacteria remain limited because they are difficult, slow and/or dangerous to culture. To avoid culturing, metagenomic sequencing can be performed directly on samples, but the sequencing effort required to characterize low frequency organisms can be expensive. Recently developed methods for selective whole genome amplification (SWGA) can enrich target DNA to provide efficient sequencing. We amplified Coxiella burnetii (a bacterial select agent and human/livestock pathogen) from 3 three environmental samples that were overwhelmed with host DNA. The 68- to 147-fold enrichment of the bacterial sequences provided enough genome coverage for SNP analyses and phylogenetic placement. SWGA is a valuable tool for the study of difficult-to-culture organisms and has the potential to facilitate high-throughput population characterizations as well as targeted epidemiological or forensic investigations.

Phylogenetic analysis of West Nile Virus in Maricopa County, Arizona: Evidence for dynamic behavior of strains in two major lineages in the American Southwest.

West Nile Virus (WNV) has been detected annually in Maricopa County, Arizona, since 2003. With this in mind, we sought to determine if contemporary strains are endemic to the county or are annually imported. As part of this effort, we developed a new protocol for tiled amplicon sequencing of WNV to efficiently attain greater than 99% coverage of 14 WNV genomes collected directly from positive mosquito pools distributed throughout Maricopa County between 2014 and 2017. Bayesian phylogenetic analyses revealed that contemporary genomes fall within two major lineages; NA/WN02 and SW/WN03. We found that all of the Arizona strains possessed an amino acid substitution known to be under positive selection, which has arisen independently at least four times in Arizona. The SW/WN03 strains exhibited transient behavior, with at least 10 separate introductions into Arizona when considering both historical and contemporary strains. However, NA/WN02 strains are geographically differentiated and appear to be endemic in Arizona, with two clades that have been circulating for four and seven years. This establishment in Maricopa County provides the first evidence of local overwintering by a WNV strain over the course of several years in Arizona. Within a national context, the placement of eleven contemporary Arizona strains in the NA/WN02 lineage indicates while WNV first entered the northeastern United States in 1999, the most ancestral extant strains of WNV are now circulating in the American southwest.

Variant site strain typer (VaST): efficient strain typing using a minimal number of variant genomic sites.

BACKGROUND: Targeted PCR amplicon sequencing (TAS) techniques provide a sensitive, scalable, and cost-effective way to query and identify closely related bacterial species and strains. Typically, this is accomplished by targeting housekeeping genes that provide resolution down to the family, genera, and sometimes species level. Unfortunately, this level of resolution is not sufficient in many applications where strain-level identification of bacteria is required (biodefense, forensics, clinical diagnostics, and outbreak investigations). Adding more genomic targets will increase the resolution, but the challenge is identifying the appropriate targets. VaST was developed to address this challenge by finding the minimum number of targets that, in combination, achieve maximum strain-level resolution for any strain complex. The final combination of target regions identified by the algorithm produce a unique haplotype for each strain which can be used as a fingerprint for identifying unknown samples in a TAS assay. VaST ensures that the targets have conserved primer regions so that the targets can be amplified in all of the known strains and it also favors the inclusion of targets with basal variants which makes the set more robust when identifying previously unseen strains. RESULTS: We analyzed VaST's performance using a number of different pathogenic species that are relevant to human disease outbreaks and biodefense. The number of targets required to achieve full resolution ranged from 20 to 88% fewer sites than what would be required in the worst case and most of the resolution is achieved within the first 20 targets. We computationally and experimentally validated one of the VaST panels and found that the targets led to accurate phylogenetic placement of strains, even when the strains were not a part of the original panel design. CONCLUSIONS: VaST is an open source software that, when provided a set of variant sites, can find the minimum number of sites that will provide maximum resolution of a strain complex, and it has many different run-time options that can accommodate a wide range of applications. VaST can be an effective tool in the design of strain identification panels that, when combined with TAS technologies, offer an efficient and inexpensive strain typing protocol.

Guano exposed: Impact of aerobic conditions on bat fecal microbiota.

Bats and their associated guano microbiota provide important terrestrial and subterranean ecosystem services and serve as a reservoir for a wide range of epizootic and zoonotic diseases. Unfortunately, large-scale studies of bats and their guano microbiotas are limited by the time and cost of sample collection, which requires specially trained individuals to work at night to capture bats when they are most active. Indirectly surveying bat gut microbiota through guano deposits could be a more cost-effective alternative, but it must first be established whether the postdefecation exposure to an aerobic environment has a large impact on the guano microbial community. A number of recent studies on mammalian feces have shown that the impact of aerobic exposure is highly species specific; therefore, it is difficult to predict how exposure will affect the bat guano microbiota without empirical data. In our study, we collected fresh guano samples from 24 individuals of 10 bat species that are common throughout the arid environments of the American southwest and subjected the samples to 0, 1, and 12 hr of exposure. The biodiversity decreased rapidly after the shift from an anaerobic to an aerobic environment-much faster than previously reported in mammalian species. However, the relative composition of the core guano microbiota remained stable and, using highly sensitive targeted PCR methods, we found that pathogens present in the original, non-exposed samples could still be recovered after 12 hr of exposure. These results suggest that with careful sample analysis protocols, a more efficient passive collection strategy is feasible; for example, guano could be collected on tarps placed near the roost entrance. Such passive collection methods would greatly reduce the cost of sample collection by allowing more sites or roosts to be surveyed with a fraction of trained personnel, time, and effort investments needed.

The impact of self-incompatibility systems on the prevention of biparental inbreeding.

Inbreeding in hermaphroditic plants can occur through two different mechanisms: biparental inbreeding, when a plant mates with a related individual, or self-fertilization, when a plant mates with itself. To avoid inbreeding, many hermaphroditic plants have evolved self-incompatibility (SI) systems which prevent or limit self-fertilization. One particular SI system-homomorphic SI-can also reduce biparental inbreeding. Homomorphic SI is found in many angiosperm species, and it is often assumed that the additional benefit of reduced biparental inbreeding may be a factor in the success of this SI system. To test this assumption, we developed a spatially-explicit, individual-based simulation of plant populations that displayed three different types of homomorphic SI. We measured the total level of inbreeding avoidance by comparing each population to a self-compatible population (NSI), and we measured biparental inbreeding avoidance by comparing to a population of self-incompatible plants that were free to mate with any other individual (PSI). Because biparental inbreeding is more common when offspring dispersal is limited, we examined the levels of biparental inbreeding over a range of dispersal distances. We also tested whether the introduction of inbreeding depression affected the level of biparental inbreeding avoidance. We found that there was a statistically significant decrease in autozygosity in each of the homomorphic SI populations compared to the PSI population and, as expected, this was more pronounced when seed and pollen dispersal was limited. However, levels of homozygosity and inbreeding depression were not reduced. At low dispersal, homomorphic SI populations also suffered reduced female fecundity and had smaller census population sizes. Overall, our simulations showed that the homomorphic SI systems had little impact on the amount of biparental inbreeding in the population especially when compared to the overall reduction in inbreeding compared to the NSI population. With further study, this observation may have important consequences for research into the origin and evolution of homomorphic self-incompatibility systems.

The effect of the dispersal kernel on isolation-by-distance in a continuous population.

Under models of isolation-by-distance, population structure is determined by the probability of identity-by-descent between pairs of genes according to the geographic distance between them. Well established analytical results indicate that the relationship between geographical and genetic distance depends mostly on the neighborhood size of the population which represents a standardized measure of gene flow. To test this prediction, we model local dispersal of haploid individuals on a two-dimensional landscape using seven dispersal kernels: Rayleigh, exponential, half-normal, triangular, gamma, Lomax and Pareto. When neighborhood size is held constant, the distributions produce similar patterns of isolation-by-distance, confirming predictions. Considering this, we propose that the triangular distribution is the appropriate null distribution for isolation-by-distance studies. Under the triangular distribution, dispersal is uniform over the neighborhood area which suggests that the common description of neighborhood size as a measure of an effective, local panmictic population is valid for popular families of dispersal distributions. We further show how to draw random variables from the triangular distribution efficiently and argue that it should be utilized in other studies in which computational efficiency is important.

Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning.

Phloem loading is a critical process in plant physiology. The potential of regulating the translocation of photoassimilates from source to sink tissues represents an opportunity to increase crop yield. Pyrophosphate homeostasis is crucial for normal phloem function in apoplasmic loaders. The involvement of Arabidopsis (Arabidopsis thaliana) type I proton-pumping pyrophosphatase (AVP1) in phloem loading was analyzed at genetic, histochemical, and physiological levels. A transcriptional AVP1 promoter::GUS fusion revealed phloem activity in source leaves. Ubiquitous AVP1 overexpression (35S::AVP1 cassette) enhanced shoot biomass, photoassimilate production and transport, rhizosphere acidification, and expression of sugar-induced root ion transporter genes (POTASSIUM TRANSPORTER2 [KUP2], NITRATE TRANSPORTER2.1 [NRT2.1], NRT2.4, and PHOSPHATE TRANSPORTER1.4 [PHT1.4]). Phloem-specific AVP1 overexpression (Commelina Yellow Mottle Virus promoter [pCOYMV]::AVP1) elicited similar phenotypes. By contrast, phloem-specific AVP1 knockdown (pCoYMV::RNAiAVP1) resulted in stunted seedlings in sucrose-deprived medium. We also present a promoter mutant avp1-2 (SALK046492) with a 70% reduction of expression that did not show severe growth impairment. Interestingly, AVP1 protein in this mutant is prominent in the phloem. Moreover, expression of an Escherichia coli-soluble pyrophosphatase in the phloem (pCoYMV::pyrophosphatase) of avp1-2 plants resulted in severe dwarf phenotype and abnormal leaf morphology. We conclude that the Proton-Pumping Pyrophosphatase AVP1 localized at the plasma membrane of the sieve element-companion cell complexes functions as a synthase, and that this activity is critical for the maintenance of pyrophosphate homeostasis required for phloem function.