Joshua A. Granek

BACKGROUND: Obesity and weight loss in adults have been associated with distinct metabolome and gut microbiome features, but the extent to which those associations apply to adolescent stages remain unclear. METHODS: The Pediatric Obesity Microbiome and Metabolism Study (POMMS) enrolled 220 adolescents aged 10–18 with severe obesity (OB) and 67 individuals who were healthy weight controls (HWCs). Blood, stool, and clinical measures were collected at baseline and after a 6-month obesity intervention for the OB group. Metabolomic profiling in serum using targeted quantitative mass spectrometry and microbiome profiling in stool were performed, and those features were assessed for associations with BMI, insulin resistance, and inflammation. Fecal microbiome transplants (FMT) were performed on germ-free mice using samples from both groups to assess effects on weight gain and metabolic pathways. RESULTS: Adolescents with OB exhibited higher serum branched-chain amino acid (BCAA) but lower branched-chain ketoacid (BCKA) levels compared with HWC. This pattern was sex- and age-dependent and differed from adults with obesity who show elevated levels of both BCAA and BCKA. Longitudinal analysis identified metabolic and microbial features correlated with changes in health measures during the intervention. The fecal microbiomes of adolescents with OB and HWC had similar diversity but differed in membership and functional potential. FMT from both OB and HWC donors had similar effects on mouse body weight, but specific taxa were linked to weight gain in recipients of FMT. CONCLUSION: Adolescents with OB have unique metabolomic adaptations and microbiome signatures compared with their HWC counterparts and adults with OB. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03139877 (Observational Study) and NCT02959034 (Repository). FUNDING SUPPORT: American Heart Association Grants: 17SFRN33670990, 20PRE35180195; National Institute of Diabetes and Digestive and Kidney Diseases Grant: R24-DK110492.

Microbial habitats that receive repeated external input may not remain shaped by that input forever if local retention allows resident communities to build up over time. Here, we used a controlled bench-scale sink p-trap system to examine how community assembly unfolded during initial establishment in new, bleach-treated p-traps. Two p-traps received repeated handwashing-water input, while one received tap water as baseline. The treated p-traps, but not the control, showed clear successional change toward later resident-like states. Nested-model comparisons further showed that recent external input had its greatest influence early in succession, but the p-trap's own prior state remained the stronger predictor throughout. Final-day post-flush trajectories indicated short-term displacement from pre-flush positions, with later time points tending to move back toward late-stage resident centroids. Together, these results show that repeated inoculation does not necessarily keep communities under continued outside influence. Instead, retentive microbial habitats can shift over time from early sensitivity to external input toward persistence shaped more by local history.

Healthcare Associated Infections (HAIs) pose a significant public health risk. Hospital sinks and associated plumbing are one likely source. This study investigates bioaerosol emissions from sink P-traps during faucet operation to assess their potential role in pathogen transmission in the hospital context. We designed an aerosol chamber system with adjustable air change rate and a functioning sink meeting hospital standards. We quantified bioaerosol and large droplet emission factors from the sink, using FAmpEscherichia coli as a model for Enterobacteriaceae such as Klebsiella pneumoniae. We cultured Andersen Cascade Impactor and BioSpot sampler aerosol samples and deposition plates using MI agar, which is selective for E. coli. Across five experiments, deposition plate counts were substantial, while air samples were close to detection limits. We estimated PM5.0 bioaerosol emissions factors of 1.3 x 10-10 to 1.9 x 10-10 CFU emitted per CFU in P-trap. Large droplet emission factor was one order of magnitude larger, at 4.3 x 10-9 ± 2.8 x 10-9 CFU per CFU. Results were used to predict Klebsiella pneumoniae inhalation dose (0.78 to 1.12 CFU day-1) and inhalation infection probability (∼10-5) for 5.5 day hospital stay with colonized plumbing (air change rate 6 h-1). This translates to ∼1 infection per year for a hospital with a severe plumbing system colonization affecting ∼2000 continuously occupied rooms. For pathogens with different dose response curves, we demonstrate how infection probability via inhalation depends on dose response.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of birth defects worldwide, yet immune determinants of protection to inform maternal vaccine design remain elusive due to the lack of a translational animal model. Here, we characterized the outcome of primary rhesus CMV (RhCMV) infection in pregnant, immunocompetent, RhCMV-naïve rhesus macaques. RhCMV DNA was detected in amniotic fluid and/or fetal tissues in six of 12 (50% placental transmission) dams following early second trimester gestation RhCMV inoculation. Widespread tissue dissemination dominated by one of two inoculated RhCMV strains was present in one fetus (8.3% cCMV disease). RhCMV DNA detection in the amniotic fluid was associated with elevated fetal and maternal plasma TNF-alpha and reduced maternal brain-derived neurotrophic factor and IL-10 levels. Maternal RhCMV exposure during pregnancy had a broad impact on the placenta and fetus even in the absence of congenital infection, as evidenced by ubiquitous maternal-fetal interface infection, and reduced placental efficiency and small-for-gestation age fetuses compared to control pregnancies. This model provides new insights into the complexity of CMV vertical transmission and can be used to evaluate immune and viral determinants of protection against cCMV.

Barcode crosstalk is a potential source of error in indexed multiplex sequencing that can mimic cross-contamination and generate false-positive signals, particularly in experiments with low-biomass samples and imbalanced designs. Here we identify and quantify barcode crosstalk in multiplexed Oxford Nanopore sequencing and introduce post-ligation pooling (PLP). PLP is a drop-in library-preparation modification that prevents barcode crosstalk, rather than simply mitigating its effects. These results highlight the potential for barcode crosstalk to have detrimental effects in multiplexed long-read sequencing and establish PLP as an immediately adoptable mitigation, especially where negative controls and low-abundance signals inform biological interpretation.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of birth defects worldwide, yet immune determinants of protection to inform maternal vaccine design remain elusive due to the lack of a translational animal model. Here, we characterized the outcome of primary rhesus CMV (RhCMV) infection in pregnant, immunocompetent, CMV-naïve rhesus macaques. RhCMV DNA was detected in amniotic fluid and/or fetal tissues in six of 12 (50% placental transmission) dams following early second trimester gestation RhCMV inoculation. Widespread tissue dissemination dominated by one of two inoculated RhCMV strains was present in one fetus (8.3% cCMV disease). Placental transmission was associated with elevated fetal and maternal plasma TNF-alpha and reduced maternal brain-derived neurotrophic factor and IL-10 levels. CMV exposure during pregnancy had a broad impact on the placenta and fetus even in the absence of congenital infection, as evidenced by ubiquitous maternal-fetal interface infection, and reduced placental efficiency and small-for-gestation age fetuses compared to control pregnancies. This model recapitulates key aspects of human cCMV and provides new insights into the complexity of CMV vertical transmission.

BACKGROUND Towards improving outcomes for adolescents with obesity, we aimed to define metabolic and microbiome phenotypes at baseline and post-weight loss intervention. METHODS The Pediatric Obesity Microbiome and Metabolism Study enrolled 220 adolescents aged 10-18 with severe obesity (OB) and 67 healthy weight controls (HWC). Blood, stool, and clinical measures were collected at baseline and after a 6-month intervention for the OB group. Serum metabolomic and fecal microbiome data were analyzed for associations with BMI, insulin resistance, and inflammation. Fecal microbiome transplants were performed on germ-free mice using samples from both groups to assess weight gain and metabolomic changes. RESULTS Adolescents with OB exhibited elevated serum branched-chain amino acids (BCAA) but reduced ketoacid metabolites (BCKA) compared to HWC. This pattern was sex- and age-dependent, unlike adults with OB, who showed elevated levels of both. The fecal microbiomes of adolescents with OB and HWC had similar diversity but differed in membership and functional potential. FMT from OB and HWC donors had similar effects on mouse body weight, with specific taxa linked to weight gain in FMT recipients. Longitudinal analysis identified metabolic and microbial features correlated with changes in health measures during the intervention. CONCLUSION Adolescents with OB have unique metabolomic adaptations and microbiome signatures compared to their HWC counterparts and adults with OB. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT03139877 (Observational Study) and NCT02959034 (Repository) FUNDING SOURCES American Heart Association Grants: 17SFRN33670990, 20PRE35180195 National Institute of Diabetes and Digestive and Kidney Diseases Grant: R24-DK110492

Purpose: High body mass index (≥ 30 kg/m²) is associated with asthma severity, and nearly 40% of asthma patients exhibit obesity. Furthermore, over 40% of patients with obesity and asthma that receive bariatric surgery no longer require asthma medication. Increased levels of glucagon-like peptide 1 (GLP-1) occur after bariatric surgery, and recent studies suggest that GLP-1 receptor (GLP-1R) signaling may regulate the gut microbiome and have anti-inflammatory properties in the lung. Thus, we hypothesized that increased GLP-1R signaling following metabolic surgery in obese and allergen-challenged mice leads to gut/lung microbiome alterations, which together contribute to improved features of allergic airways disease. Methods: Male and female Glp1r-deficient (Glp1r^−/−) and replete (Glp1r^+/+) mice were administered high fat diet (HFD) to induce obesity with simultaneous intranasal challenge with house dust mite (HDM) allergen to model allergic airway disease with appropriate controls. Mice on HFD received either no surgery, sham surgery, or vertical sleeve gastrectomy (VSG) on week 10 and were sacrificed on week 13. Data were collected with regard to fecal and lung tissue microbiome, lung histology, metabolic markers, and respiratory inflammation. Results: HFD led to metabolic imbalance characterized by lower GLP-1 and higher leptin levels, increased glucose intolerance, and alterations in gut microbiome composition. Prevalence of bacteria associated with short chain fatty acid (SCFA) production, namely Bifidobacterium, Lachnospiraceae UCG-001, and Parasutterella, was reduced in mice fed HFD and positively associated with serum GLP-1 levels. Intranasal HDM exposure induced airway inflammation. While Glp1r^−/− genotype affected fecal microbiome beta diversity metrics, its effect was limited. Conclusion: Herein, GLP-1R deficiency had surprisingly little effect on host gut and lung microbiomes and health, despite recent studies suggesting that GLP-1 receptor agonists are protective against lung inflammation. Plain Language Summary: Asthma is a chronic lung disease that affects over 20 million Americans. In addition, obesity is a major worldwide healthcare problem whose impact is increasing each year. Asthma patients with obesity have worse symptoms, use more medications and have reduced asthma control than lean patients. Also, both asthma and obesity are linked to changes in the normal bacteria that live in the gut and the airways. Weight loss, either through bariatric surgery or through diet and exercise, improves asthma symptoms, through unknown mechanisms. One way that bariatric surgery may impact asthma is through the increase in beneficial gut hormones (GLP-1) and restoration of altered bacteria in the gut and airways. Our study shows that overall, obese mice experience changes in hormones involved in weight gain and glucose metabolism as well as gut bacteria, particularly those that may produce important factors that could improve health. We did not find any links between changes in the bacteria in the gut or lung and features of airway disease or effects from bariatric surgery in the mice. Mice that lacked GLP-1 activity did not differ from normal mice for weight, glucose tolerance, or prevalence of bacteria in the lung. Future studies will explore the timeline of these bacterial changes and the role of the gut and lung microbiomes in human obesity-associated asthma.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of birth defects worldwide, yet immune determinants of protection to inform design of a maternal vaccine remain elusive. Here, we characterized the outcome of primary rhesus CMV (RhCMV) infection during pregnancy in an immune competent nonhuman primate (NHP) model. RhCMV DNA was detected in amniotic fluid and/or fetal tissues in six of 12 (50% placental transmission) CMV-naive rhesus macaque dams inoculated intravenously with RhCMV in early second trimester gestation. Widespread tissue dissemination dominated by one of two inoculated RhCMV strains was present in one fetus (8.3% cCMV disease). Placental RhCMV transmission was associated with elevated fetal and maternal plasma TNF-alpha and reduced maternal brain-derived neurotrophic factor and IL-10 levels. CMV exposure during pregnancy had a broad impact on the placenta and fetus even in the absence of congenital infection as evidenced by RhCMV infection at the maternal-fetal interface in all 12 dams, along with significantly reduced placental efficiency and fetal growth metrics compared to gestation-matched control pregnancies. This NHP model recapitulates key aspects of human cCMV and provides new insight into barriers and biomarkers of successful vertical transmission. One sentence summary: The nonhuman primate model mirrors the epidemiology of human congenital CMV (cCMV) after primary infection and reveals its transmission bottlenecks.