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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.

Early childhood caries (ECC) is the most common chronic disease of childhood, with especially high prevalence in Arizona and Hawaii where several racial and ethnic groups experience disproportionate burden. Although ECC is a polymicrobial disease, plays a central role in its development, and evidence suggests that strain-level genetic variation influences its cariogenic potential. Understanding whether specific lineages are associated with higher ECC risk and whether these lineages are more common in disproportionately affected groups is an important step toward identifying biological contributors to ECC outcomes. We conducted a cross-sectional study of 408 preschool-aged children (1-6 years) from Arizona and Hawaii. Saliva samples were tested for using quantitative polymerase chain reaction (qPCR), and positive samples were genotyped using a custom amplicon sequencing assay. Logistic regression was used to evaluate associations between demographic factors (race, ethnicity, age, and sex), colonization, and ECC status. To assess whether certain genotypes were associated with ECC risk, we calculated a K-nearest-neighbor-smoothed risk score for each genotype based on patristic distances. Genetic markers of ECC risk were identified using a pseudo-genome-wide association approach. ECC odds increased with age and were higher among Native Hawaiian/Pacific Islander, Asian, American Indian, and Hispanic children compared to non-Hispanic White children, although estimates for some groups were imprecise due to limited subgroups sizes. colonization increased ECC odds by 361%, but colonization rates did not significantly differ across groups. Genotypes from Arizona and Hawaii showed no evidence of geographic clustering but ECC risk was non-randomly distributed across the phylogeny with multiple localized regions of higher risk genotypes. Native Hawaiian/Pacific Islander children were significantly more likely to carry higher-risk strains. Genetic markers linked with ECC risk mapped to genes involved in cariogenic processes-many of which were previously shown to be upregulated in caries-active plaque. The observed correlation between genotype and ECC risk, together with the finding that higher-risk genotypes were more prevalent among at least one disproportionately affected group, suggests that strain-level variation may contribute to population-level disparities. The identification of functional markers linked to ECC risk further supports biologically meaningful strain-specific effects and warrants further investigation. These findings highlight the value of incorporating microbial genetic diversity into ECC risk frameworks, while recognizing that fully disentangling microbial contributions will require studies that integrate social, behavioral, and dietary determinants.

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.

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 , , , , and . Weekly average positivity rates were 17.75% for (0% - 39.39%), 9.95% for (0% - 37.74%), 31.89% for (1.79% - 41.67%), and for 28.00% for (0% - 55.36%). was not detected. and predominantly colonized the oral cavity, while and predominantly colonized the nasal cavity. and colonizations were significantly more persistent than and , 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.

Leptospirosis (caused by pathogenic bacteria in the genus ) 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, , 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 . 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 . 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 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 genomes from rats. We also generated an enriched 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 among rats, with specific clades tightly linked to distinct rat populations. This pattern suggests 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.

A significant proportion of people are asymptomatic carriers of Staphylococcus aureus (SA), an important risk factor for 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 9 participants every two weeks for up to 18 weeks and qPCR positive samples were analyzed 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 that had positive oral and nasal samples had no fixed SNP differences between the two body sites, suggesting likely within-person spread. In these cases, we were able to infer the most likely direction of spread (nasal to oral sites) by analyzing segregating rare variants. While WGS can be used to provide higher resolution to colonization patterns and validate these findings, our amplicon sequencing approach offers a rapid, cost-effective, direct-from-sample method for species-specific screening intended for population-level characterization that allows researchers to characterize strain types, identify or eliminate likely transmission cases, and identify potential reservoirs before resorting to more expensive WGS methods. Colonizing opportunistic pathogens like Staphylococcus aureus present a unique challenge for disease study because rather than causing acute infections upon transmission, they persist asymptomatically for long periods of time allowing the bacterial population to evolve and differentiate. Characterizing the diversity within these populations is important for choosing correct treatments, quantifying the risk of horizontal gene transfer, and understanding paths of transmission between people and spread to different body sites. The gold-standard approach for characterizing population diversity is through culturing and whole-genome sequencing of multiple colonies per sample which is labor-intensive and expensive for any large-scale study. Using a custom-designed species-specific amplicon sequencing assay, we offer a cost-effective method for characterizing the diversity in Staphylococcus aureus populations directly from samples without the need for labor-intensive culturing or whole-genome sequencing. Our small-scale study highlights how this method provides a scalable tool for large epidemiological studies ideal for systematically exploring broader patterns of carriage and transmission.