Nursing home acquired pneumonia (NHAP) is a leading cause of mortality in long-term care facilities (LTCFs), primarily resulting from the macro-aspiration of opportunistic pathogens colonizing the upper respiratory tract. While multiple bacterial species can cause NHAP, Streptococcus pneumoniae is the most common, and pneumococcal vaccination remains the primary preventative measure. Oral hygiene interventions are also increasingly explored as a complementary strategy to reduce pathogen colonization, although their reported effectiveness has been mixed, likely due to differences in how individual pathogens respond. To better understand these discrepancies, we examined whether pathogen-specific colonization patterns vary in response to oral health behaviors in elderly LTCF residents. We collected longitudinal oral and nasal colonization data for four key NHAP-associated pathogens, along with oral health survey responses, and pneumococcal vaccination status. Better oral health behaviors predicted reduced colonization with S. pneumoniae and Haemophilus influenzae, both of which were more commonly found in oral samples in this population. There was no relationship with Staphylococcus aureus and Pseudomonas aeruginosa, which were more commonly found in the nasal cavity. Our findings suggest that oral hygiene interventions will only impact NHAP risk for pathogens primarily colonizing the oral cavity, potentially explaining the mixed outcomes in prior studies.

Summary:MetaTracer is a nucleotide alignment-based tool for metatranscriptomic analysis of complex bacterial communities that assigns sequence reads to both taxonomic groups and expressed genes in a single pass. Full nucleotide-level alignment improves accuracy relative to k-mer-based classifiers and preserves species-level resolution that is often lost in protein-based approaches. By retaining alignment coordinates and mapping reads directly to annotated genomic features, MetaTracer enables direct attribution of gene expression to specific microbial species. On simulated datasets, MetaTracer achieves high accuracy for both taxonomic and gene assignment. Applied to real dental plaque metatranscriptomic datasets, MetaTracer resolves species-specific transcriptional activity and detects reproducible differences in microbial gene expression between children with early childhood caries and healthy controls. Availability and implementation:MetaTracer is implemented as a Python-based workflow wrapper (metatracer v0.1.0) that depends on the mtsv-tools core engine (v2.1.0), which is written in Rust. The required functionality is supported by the v2.1.0 release of mtsv-tools. Both packages are open source under the MIT license and are available at github.com/FofanovLab/metatracer and github.com/FofanovLab/mtsv-tools. Versioned releases are archived at Zenodo (DOI: 10.5281/zenodo.18665766 and DOI: 10.5281/zenodo.18718002). Installation is supported via Bioconda.

National surveillance data show persistent racial and ethnic disparities in early childhood caries (ECC), but the underlying causes of these differences have not been determined. This study examined both functional and taxonomic differences in ECC-related microbial activity between two high-risk groups of children: African American (AA) and Latin American Hispanic (LAH). Metatranscriptomic profiling of paired non-caries and caries plaque revealed consistent population-level shifts in gene expression and enabled species-level attribution of metabolically active microbes in caries lesions. A core set of well-established cariogenic organisms was consistently present and highly over-expressed in caries of both groups, including Streptococcus mutans, Veillonella parvula, Propionibacterium acidifaciens, and Lactobacillus rhamnosus. Beyond identifying the core organisms and functions active in lesions, we have also made two significant observations. First, the active communities in the two groups have substantially diverged: 4,900+ genes across 413 Kyoto Encyclopedia of Genes and Genomes Orthology (KO) groups were consistently (25%+ of samples) over-expressed in AA children, and 6,500+ genes across 382 KOs were consistently (57% of samples) over-expressed in LAH children. This reproducibility across multiple samples indicates robust group-level differences rather than random variation or single-sample effects. Second, although AA and LAH children exhibited similar functional responses to caries (sharing 1,392 KOs), these shifts were expressed by different bacterial species, indicating that distinct taxa may occupy similar metabolic niches in different groups. Taken together, these findings suggest that there is no single universal caries-associated microbiome; instead, a shared cariogenic core is necessary, but differences among the non-core taxa and their functional activity may be key to understanding ECC disparities.IMPORTANCEThe disparity in tooth decay among young children has long been demonstrated in national surveillance data. While various factors including family, culture, access to health insurance, and medical infrastructure have been studied, the global transcriptomic perspective remains underexplored. Employing RNA-Seq technology, we examine functional and taxonomic differences in caries-associated microbial activity between two high-risk populations. Besides a core set of well-established cariogenic organisms, we observed significant and consistent differences in the active microbial communities between these two high-risk populations, African American (AA) and Latin American Hispanic (LAH) children. In AA children, Pseudopropionibacterium propionicum and Cardiobacterium hominis consistently showed the highest caries-related gene expression. In contrast, among LAH children, Propionibacterium acidifaciens, Selenomonas sp., Rothia dentocariosa, Atopobium parvulum, and Streptococcus sanguinis were the primary drivers of gene expression in caries lesions. By identifying the unique microbial mechanisms and pathways active in each population, we can better define the core factors required for caries development and uncover how differences in microbial function contribute to persistent disparities.

Nursing home acquired pneumonia (NHAP), and its subset - aspiration-associated pneumonia, is a leading cause of morbidity and mortality among residents in long-term care facilities (LTCFs). Understanding colonization dynamics of respiratory pathogens in LTCF residents is essential for effective infection control. This study examines the longitudinal trends in prevalence, persistence, bacterial load, and co-colonization patterns of five respiratory pathogens in three LTCFs in Phoenix, Arizona. Anterior nares and oral swabs were collected every other week and tested using qPCR for Haemophilus influenzae, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, and Chlamydia pneumoniae. Weekly average positivity rates were 17.75% for H. influenzae (0% - 39.39%), 9.95% for P. aeruginosa (0% - 37.74%), 31.89% for S. pneumoniae (1.79% - 41.67%), and for 28.00% for S. aureus (0% - 55.36%). C. pneumoniae was not detected. H. influenzae and S. pneumoniae predominantly colonized the oral cavity, while P. aeruginosa and S. aureus predominantly colonized the nasal cavity. Colonization by S. pneumoniae and S. aureus was significantly more persistent than H. influenzae and P. aeruginosa, with persistence correlating with significantly higher bacterial loads. Co-colonization did occur in ~20% of positive samples but appeared to be due to random chance. This study reveals distinct colonization patterns among respiratory pathogens in LTCF residents, highlighting differences in site-specific prevalence, persistence, and bacterial load. These findings underscore the importance of longitudinal monitoring to inform targeted infection control strategies in LTCFs.

Leptospirosis (caused by pathogenic bacteria in the genus Leptospira) is prevalent worldwide but more common in tropical and subtropical regions. Transmission can occur following direct exposure to infected urine from reservoir hosts, or a urine-contaminated environment, which then can serve as an infection source for additional rats and other mammals, including humans. The brown rat, Rattus norvegicus, is an important reservoir of Leptospira spp. in urban settings. We investigated the presence of Leptospira spp. among brown rats in Boston, Massachusetts and hypothesized that rat population dynamics in this urban setting influence the transportation, persistence, and diversity of Leptospira spp. We analyzed DNA from 328 rat kidney samples collected from 17 sites in Boston over a seven-year period (2016-2022); 59 rats representing 12 of 17 sites were positive for Leptospira spp. We used 21 neutral microsatellite loci to genotype 311 rats and utilized the resulting data to investigate genetic connectivity among sampling sites. We generated whole genome sequences for 28 Leptospira spp. isolates obtained from frozen and fresh tissue from some of the 59 positive rat kidneys. When isolates were not obtained, we attempted genomic DNA capture and enrichment, which yielded 14 additional Leptospira spp. genomes from rats. We also generated an enriched Leptospira spp. genome from a 2018 human case in Boston. We found evidence of high genetic structure among rat populations that is likely influenced by major roads and/or other dispersal barriers, resulting in distinct rat population groups within the city; at certain sites these groups persisted for multiple years. We identified multiple distinct phylogenetic clades of L. interrogans among rats that were tightly linked to distinct rat populations. This pattern suggests L. interrogans persists in local rat populations and its transportation is influenced by rat population dynamics. Finally, our genomic analyses of the Leptospira spp. detected in the 2018 human leptospirosis case in Boston suggests a link to rats as the source. These findings will be useful for guiding rat control and human leptospirosis mitigation efforts in this and other similar urban settings.

Early childhood caries (ECC) is the most prevalent chronic childhood disease, disproportionately affecting children from specific racial/ethnic groups. While ECC is a multifactorial disease influenced by demographic, behavioral, and environmental factors, it is also widely associated with the presence of Streptococcus mutans in the oral microbiome. To better understand the interplay between demographic and microbial factors and ECC risk, we collected saliva samples from 408 preschool children aged 1-6 years, including 266 from northern Arizona and 142 from Hawaii, representing racially and geographically diverse populations. Logistic regression showed that the odds of developing ECC increased 80.09% with each year of age, and that compared to White children, the odds were significantly higher for Native Hawaiian/Pacific Islander (330.94%), Native American (282.35%), and Hispanic (245.22%) children. Oral S. mutans colonization increased odds by 360.49%. The S. mutans-positive samples were genotyped using a multiplexed targeted amplicon sequencing assay. Phylogenetic analysis showed high genetic diversity in S. mutans between and within populations, with no geographic clustering. Some S. mutans clades were associated with up to a 33-fold increase in ECC odds. Only Native Hawaiian/Pacific Islander children were more likely to carry S. mutans strains from higher-risk clades. Markers associated with high risk S. mutans strains were identified in genes involved in carbohydrate metabolism, pH regulation, and biofilm formation. This study underscores the multifaceted nature of ECC, linking demographic disparities with strain-specific features of S. mutans and identifies new genetic markers that may play a role in S. mutans virulence. ### Competing Interest Statement The authors have declared no competing interest.

A significant proportion of people are asymptomatic carriers of Staphylococcus aureus (SA), an important risk factor for the development of opportunistic infections. SA colonization is dynamic, appearing and disappearing, with strains evolving and potentially shifting in composition over time and between body sites. These changes make detection challenging, and the numerous potential sources of reintroduction from other people and even other body site reservoirs preclude efficient efforts to prevent transmission and spread. Identifying typical sources is therefore critical for mitigation. Whole-genome sequencing (WGS), ideally of multiple colonies from multiple body sites, is the gold standard for characterizing SA strains and confirming transmission. However, this is often too resource-intensive for initial assessments of transmission and not feasible for large-scale studies involving various body sites from multiple individuals over time. To address these challenges, we developed a low-cost, custom, species-specific amplicon sequencing (AmpSeq) assay optimized to provide high-resolution discrimination of SA genotypes directly from samples. We tested this approach on a subset of samples that were a part of a large-scale longitudinal study of SA carriage. Oral and nasal samples were collected from nine participants every 2 weeks for up to 18 weeks and qPCR positive samples were analysed using our AmpSeq assay directly from the sample without culturing. The longitudinal sampling strategy enabled us to characterize changes in SA colonization patterns over time, detect potential strain mixtures and identify rare variants that may serve as signatures of transmission between different body sites or among individuals. Without using WGS, we were able to rapidly eliminate the possibility of transmission between sampled residents. Participants who had positive oral and nasal samples had no fixed SNP differences between the two body sites, suggesting likely within-person spread. Analysis of rare variants segregating in the oral and nasal populations suggests that the nasal populations were the likely source of the spread because the nasal samples had higher diversity and most of the variants identified in the oral samples were shared with the nasal samples. While WGS can be used to provide higher resolution to colonization patterns and validate these findings, our AmpSeq approach offers a rapid, cost-effective, direct-from-sample method for species-specific screening intended for population-level characterization that allows researchers to strain type, identify or eliminate likely transmission cases and identify potential reservoirs before resorting to more expensive WGS methods.

National surveillance data has long shown a significant disparity in tooth decay among young children (early childhood caries, ECC). While factors including household poverty level, culture, health insurance, and infrastructure have been studied, the biomedical perspective is less explored. Using RNASeq technology, our findings show that, besides Streptococcus mutans, which is most commonly associated with caries, several additional dental plaque bacteria are significantly overexpressed in caries lesions. Notably, the bacterial species and functional profiles differ markedly between African American and Latin American Hispanic children. In African American children, gene expression profiles linked to Pseudopropionibacterium propionicum and Cardiobacterium hominis; in contrast, in Latin American Hispanic children, gene expression profiles were dominated by Propionibacterium acidifaciens, Selenomonas sp., Rothia dentocariosa, Atopobium parvulum, and Streptococcus sanguinis. This study underscores the diverse metabolic pathways in plaque bacteria contributing to ECC in minority populations, identifying significant bacterial species beyond common cariogenic bacteria. Main Text:

Leptospirosis (caused by pathogenic bacteria in the genus Leptospira ) is prevalent worldwide but more common in tropical and subtropical regions. Transmission can occur following direct exposure to infected urine from reservoir hosts, such as rats, or a urine-contaminated environment, which then can serve as an infection source for additional rats and other mammals, including humans. The brown rat, Rattus norvegicus , is an important reservoir of leptospirosis in urban settings. We investigated leptospirosis among brown rats in Boston, Massachusetts and hypothesized that rat dispersal in this urban setting influences the movement, persistence, and diversity of Leptospira . We analyzed DNA from 328 rat kidney samples collected from 17 sites in Boston over a seven-year period (2016-2022); 59 rats representing 12 of 17 sites were positive for Leptospira . We used 21 neutral microsatellite loci to genotype 311 rats and utilized the resulting data to investigate genetic connectivity among sampling sites. We generated whole genome sequences for 28 Leptospira isolates obtained from frozen and fresh tissue from some of the 59 Leptospira -positive rat kidneys. When isolates were not obtained, we attempted Leptospira genomic DNA capture and enrichment, which yielded 14 additional Leptospira genomes from rats. We also generated an enriched Leptospira genome from a 2018 human case in Boston. We found evidence of high genetic structure and limited dispersal among rat populations that is likely influenced by major roads and/or other unknown dispersal barriers, resulting in distinct rat population groups within the city; at certain sites these groups persisted for multiple years. We identified multiple distinct phylogenetic clades of L. interrogans among rats, with specific clades tightly linked to distinct rat populations. This pattern suggests L. interrogans persists in local rat populations and movement of leptospirosis in this urban rat community is driven by rat dispersal. Finally, our genomic analyses of the 2018 human leptospirosis case in Boston suggests a link to rats as the source. These findings will be useful for guiding rat control and human leptospirosis mitigation efforts in this and other urban settings.

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.

Genomic diversity in a pathogen population is the foundation for evolution and adaptations in virulence, drug resistance, pathogenesis, and immune evasion. Characterizing, analyzing, and understanding population-level diversity is also essential for epidemiological and forensic tracking of sources and revealing detailed pathways of transmission and spread. For bacteria, culturing, isolating, and sequencing the large number of individual colonies required to adequately sample diversity can be prohibitively time-consuming and expensive. While sequencing directly from a mixed population will show variants among reads, they cannot be linked to reveal allele combinations associated with particular traits or phylogenetic inheritance patterns. Here, we describe the theory and method of how population sequencing directly from a mixed sample can be used in conjunction with sequencing a very small number of colonies to describe the phylogenetic diversity of a population without haplotype reconstruction. To demonstrate the utility of population sequencing in capturing phylogenetic diversity, we compared isogenic clones to population sequences of Burkholderia pseudomallei from the sputum of a single patient. We also analyzed population sequences of Staphylococcus aureus derived from different people and different body sites. Sequencing results confirm our ability to capture and characterize phylogenetic diversity in our samples. Our analyses of B. pseudomallei populations led to the surprising discovery that the pathogen population is highly structured in sputum, suggesting that for some pathogens, sputum sampling may preserve structuring in the lungs and thus present a non-invasive alternative to understanding colonization, movement, and pathogen/host interactions. Our analyses of S. aureus samples show how comparing phylogenetic diversity across populations can reveal directionality of transmission between hosts and across body sites, demonstrating the power and utility for characterizing the spread of disease and identification of reservoirs at the finest levels. We anticipate that population sequencing and analysis can be broadly applied to accelerate research in a broad range of fields reliant on a foundational understanding of population diversity.

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.

The New Mexico Jumping Mouse (Zapus luteus) is a federally endangered granivore active for only 3 to 5 months annually. Knowledge of diet can help guide habitat recovery and yet despite calls for restoration of its riparian habitat, the diet of Z. luteus remains largely unknown. To date, only 8 plant species have been described in the diet-whereas insectivory, mycophagy, and dietary shifts have only been hypothesized. In the late summer, Z. luteus rapidly accumulates fat for a 9-month hibernation and restoration may fail if seasonal variation in the diet is overlooked. We used DNA metabarcoding on fecal DNA (n = 165) to resolve dietary taxa within multiple trophic levels of the diet and investigate monthly patterns of consumption. Seldom studied in metabarcoding frameworks, we also investigated exogenous contamination in Sherman live-capture traps. Potential contamination among feces, pelage, and trap surfaces was detectable but low (median = 0% to 2%), with fecal communities distinct from exogenous sources. The diet of Z. luteus was varied and most frequently (frequency of occurrence) of graminoids, forbs, lepidopterans, dipterids, and false-truffles. For plant foods, dietary diversity increased throughout their season of activity, shifting from sedges and woody vegetation in the early season (June to July) to forbs and grasses in the late season (August to September). Insect consumption was consistently detected throughout the season, whereas mycophagy was more frequently detected in August, when hypogeous fungi are typically more abundant. The breadth of dietary taxa suggests plasticity in resource use, potentially accommodating diverse patterns of seed availability throughout their active period. Shifts in plant consumption may reflect a dietary adaptation to the phenological patterns of their plant communities, a synchrony that might benefit both pre- and post-hibernation survival. Our work substantially improves our understanding of the diet of Z. luteus (241 dietary taxa) and will be useful for guiding habitat recovery. It also offers scalable methods to further investigate the diet under disturbance contexts (e.g., grazing, wildfire, drought). Although known as granivores, the diet of the federally endangered New Mexico jumping mouse remains largely unknown regarding the species they feed on and whether the diet shifts during the year. Using fecal DNA, we describe a varied diet of grasses, sedges, forbs, moths, flies, and false-truffles. Seasonal variation in diversity of the plant diet suggests that New Mexico Jumping Mice follow patterns of seed maturity from mid-spring into the late summer, which may play a role in pre- and post-hibernation survival. This new knowledge can aid in the identification, maintenance, and recovery of its riparian habitat in the southwestern United States.

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.

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.

ABSTRACTStaphylococcus aureus is a frequent cause of mild and severe infections that occur when these commensal bacteria penetrate the outer layers of skin or mucosa. As most S. aureus infections are the result of autoinfection, and community-acquired infections are increasingly common, it is important to better understand S. aureus colonization characteristics in the community setting. Using standard culture technique and a quantitative PCR assay (SaQuant), we detected and quantified S. aureus across the nares, throat, and palm of 548 community-dwelling individuals in southwestern Arizona. Using culture-based methods, we detected S. aureus colonization in the nares of 26.3% of individuals (n = 144); however, the combination of two detection methods across multiple body sites resulted in much higher prevalence than has been reported previously. Overall, 65.9% of participants were colonized, with significantly higher prevalence in males (compared to females) and non-Hispanics (compared to Hispanics), with this pattern especially evident in nares and throat samples. Colonizing quantities in the nares were slightly higher in males and significantly greater among non-Hispanics. The clear sex and ethnicity patterns warrant further investigation in order to identify and leverage protective factors that may drive these disparities. In the nares, S. aureus density was the highest, most variable, and correlates with colonization in other body sites such as throat and palm. Our results demonstrate that screening by culture-based methods only can miss individuals colonized by S. aureus and that previous carriage statistics are likely underestimates. By including a highly sensitive quantitative assay, this work provides a roadmap towards more comprehensive and accurate characterization of S. aureus carriage and the potential for more effective mitigation.AUTHOR SUMMARYEffective disease control and prevention is tied to pathogen identification and understanding reservoirs. Staphylococcus aureus infection prevention efforts and protocols are based upon decades of research on colonization patterns and associated links to subsequent infection. Unfortunately, efforts to prevent S. aureus infections have been met with diminishing returns, suggesting significant gaps in fundamental knowledge of colonization. However, this knowledge and resulting protocols, are founded upon culture-based detection. By employing a new quantitative PCR assay on samples from three body sites in 548 individuals, we can characterize colonization more comprehensively than previous studies by describing both prevalence and pathogen quantity. Our highly sensitive detection resulted in an overall prevalence of 65.9%. Higher quantities were associated with the nares and were highest among non-Hispanic males (86.9%). Overall prevalence was much higher than has been previously documented. Common research practices, such as culture-based detection from a single body site, may misclassify over half of colonized persons. Future studies incorporating quantitative data, especially with longitudinal sampling at more body sites will provide a more wholistic understanding of community carriage, colonization dynamics, and likelihood of autoinfection and transmission.

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 alignmentbased 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.

Since the reemergence of St. Louis Encephalitis (SLE) Virus (SLEV) in the Southwest United States, identified during the 2015 outbreak in Arizona, SLEV has been seasonally detected within Culex spp. populations throughout the Southwest United States. Previous work revealed the 2015 outbreak was caused by an importation of SLEV genotype III, which had only been detected previously in Argentina. However, little is known about when the importation occurred or the transmission and genetic dynamics since its arrival into the Southwest. In this study, we sought to determine whether the annual detection of SLEV in the Southwest is due to enzootic cycling or new importations. To address this question, we analyzed 174 SLEV genomes (142 sequenced as part of this study) using Bayesian phylogenetic analyses to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Maricopa and Riverside counties form two distinct populations with little evidence of inter-county transmission since the onset of the outbreak. Alternatively, it appears that in 2019, Yuma and Clark counties experienced annual importations of SLEV that originated in Riverside and Maricopa counties. Finally, the earliest representatives of SLEV genotype III in the Southwest form a polytomy that includes both California and Arizona samples. We propose that the initial outbreak most likely resulted from the importation of a population of SLEV genotype III variants, perhaps in multiple birds, possibly multiple species, migrating north in 2013, rather than a single variant introduced by one bird.

A large portion of activity in soil microbial communities occurs in short time frames in response to an increase in C availability, affecting the biogeochemical cycling of nitrogen. These changes are of particular importance as nitrogen represents both a limiting nutrient for terrestrial plants as well as a potential pollutant.

The Late Quaternary extinctions of megafauna (defined as animal species >44.5 kg) reduced the dispersal of seeds and nutrients, and likely also microbes and parasites. Here we use body-mass based scaling and range maps for extinct and extant mammal species to show that these extinctions led to an almost seven-fold reduction in the movement of gut-transported microbes, such as Escherichia coli (3.3 km2/day to 0.5 km2/day). Similarly, the extinctions led to a seven-fold reduction in the mean home ranges of vector-borne pathogens (7.8 km2 to 1.1 km2). To understand the impact of this, we created an individual-based model where an order of magnitude decrease in home range increased maximum aggregated microbial mutations 4-fold after 20,000 years. We hypothesize that pathogen speciation and hence endemism increased with isolation, as global dispersal distances decreased through a mechanism similar to the theory of island biogeography. To investigate if such an effect could be found, we analysed where 145 zoonotic diseases have emerged in human populations and found quantitative estimates of reduced dispersal of ectoparasites and fecal pathogens significantly improved our ability to predict the locations of outbreaks (increasing variance explained by 8%). There are limitations to this analysis which we discuss in detail, but if further studies support these results, they broadly suggest that reduced pathogen dispersal following megafauna extinctions may have increased the emergence of zoonotic pathogens moving into human populations.