Clinical and Microbial Determinants of Upper Respiratory Colonization with Streptococcus pneumoniae and Native Microbiota in People with HIV-1 and Control Adults.

BACKGROUND: The substantial risk for respiratory and invasive infections with Streptococcus pneumoniae (Spn) among people with HIV-1 (PWH) begins with asymptomatic colonization. The frequency of Spn colonization among U.S. adults with and without HIV-1 infection is not well-characterized in the conjugate vaccine era. METHODS: We determined Spn colonization frequency by culture and specific lytA gene QPCR and microbiota profile by 16S rRNA gene sequencing in nasopharyngeal (NP) and oropharyngeal (OP) DNA from 138 PWH and 93 control adults and associated clinical characteristics. RESULTS: The frequencies of Spn colonization among PWH and controls did not differ (11.6% vs 8.6%, respectively; p=0.46) using combined results of culture and PCR, independent of vaccination or behavioral risks. PWH showed altered microbiota composition (i.e., beta-diversity. NP: p=0.0028, OP: p=0.0098), decreased alpha-diversity (NP: p=0.024, OP: p=0.0045), and differences in the relative abundance of multiple bacterial taxa. Spn colonization was associated with altered beta-diversity in the NP (p=0.011), but not OP (p=0.21). CONCLUSIONS: Despite widespread conjugate vaccine and antiretroviral use, frequencies of Spn colonization among PWH and controls are currently consistent with those reported in the pre-conjugate era. The persistently increased risk of pneumococcal disease despite ART may relate to behavioral and immunologic variables other than colonization.

Gut Microbiota and Other Factors Associated With Increased Regulatory T Cells in Hiv-exposed Uninfected Infants.

HIV-exposed uninfected infants (HEU) have higher infectious morbidity than HIV-unexposed infants (HUU). HEU have multiple immune defects of unknown origin. We hypothesized that HEU have higher regulatory T cells (Treg) than HUU, which may dampen their immune defenses against pathogens. We compared 25 Treg subsets between HEU and HUU and sought the factors that may affect Treg frequencies. At birth, 3 Treg subsets, including CD4 + FOXP3 + and CD4 + FOXP3 + CD25+, had higher frequencies in 123 HEU than 117 HUU and 3 subsets were higher in HUU. At 28 and 62 weeks of life, 5 Treg subsets were higher in HEU, and none were higher in HUU. The frequencies of the discrepant Treg subsets correlated at birth with differential abundances of bacterial taxas in maternal gut microbiome and at subsequent visits in infant gut microbiomes. In vitro, bacterial taxa most abundant in HEU expanded Treg subsets with higher frequencies in HEU, recapitulating the in vivo observations. Other factors that correlated with increased Treg were low maternal CD4 + T cells in HEU at birth and male sex in HUU at 28 weeks. We conclude that maternal and infant gut dysbiosis are central to the Treg increase in HEU and may be targeted by mitigating interventions.

Microbiota-dependent indole production stimulates the development of collagen-induced arthritis in mice.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model, we identified alterations in tryptophan metabolism, and specifically indole, that correlated with disease. We demonstrated that both bacteria and dietary tryptophan were required for disease and that indole supplementation was sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colonic lymphocytes to indole increased the expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a unique therapeutic pathway for RA and SpA.

Microbiota-dependent indole production is required for the development of collagen-induced arthritis.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model we identify alterations in tryptophan metabolism, and specifically indole, that correlate with disease. We demonstrate that both bacteria and dietary tryptophan are required for disease, and indole supplementation is sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA.

Otitis Media in Children with Down Syndrome Is Associated with Shifts in the Nasopharyngeal and Middle Ear Microbiotas.

Background: Otitis media (OM) is defined as middle ear (ME) inflammation that is usually due to infection. Globally, OM is a leading cause of hearing loss and is the most frequently diagnosed disease in young children. For OM, pediatric patients with Down syndrome (DS) demonstrate higher incidence rates, greater severity, and poorer outcomes. However, to date, no studies have investigated the bacterial profiles of children with DS and OM. Method: We aimed to determine if there are differences in composition of bacterial profiles or the relative abundance of individual taxa within the ME and nasopharyngeal (NP) microbiotas of pediatric OM patients with DS (n = 11) compared with those without DS (n = 84). We sequenced the 16S rRNA genes and analyzed the sequence data for diversity indices and relative abundance of individual taxa. Results: Individuals with DS demonstrated increased biodiversity in their ME and NP microbiotas. In children with OM, DS was associated with increased biodiversity and higher relative abundance of specific taxa in the ME. Conclusion: Our findings suggest that dysbioses in the NP of DS children contributes to their increased susceptibility to OM compared with controls. These findings suggest that DS influences regulation of the mucosal microbiota and contributes to OM pathology.

The Gut Microbiota Differ in Exclusively Breastfed and Formula-Fed United States Infants and are Associated with Growth Status.

BACKGROUND: Evidence regarding the effects of infant feeding type (exclusive breastfeeding compared with exclusive formula feeding) on the gut microbiota and how it impacts infant growth status is limited. OBJECTIVES: The primary objective was to compare gut microbiota by feeding type and characterize the associations between gut microbiota and infant growth status. METHODS: Stool samples from healthy, full-term infants (4-5 mo-old) who were either exclusively breastfed (BF) or exclusively formula-fed (FF) in Denver, CO, United States were collected, and fecal 16S ribosomal ribonucleic acid gene-based profiling was conducted. Length and weight were measured at the time of stool collection. Length-for-age z-score, weight-for-age z-scores (WAZ), and weight-for-length z-scores were calculated based on the World Health Organization standards. Associations between gut microbial taxa and anthropometric z-scores were assessed by Spearman's rank correlation test. RESULTS: A total of 115 infants (BF n = 54; FF n = 61) were included in this study. Feeding type (BF compared with FF) was the most significant tested variable on gut microbiota composition (P < 1 × 10-6), followed by mode of delivery and race. Significant differences were observed in α-diversity, ß-diversity, and relative abundances of individual taxa between BF and FF. BF infants had lower α-diversity than FF infants. Abundances of Bifidobacterium and Lactobacillus were greater in the breastfeeding group. FF infants had a higher relative abundance of unclassified Ruminococcaceae (P < 0.001), which was associated with a higher WAZ (P < 0.001) and length-for-age z-score (P < 0.01). Lactobacillus was inversely associated with WAZ (P < 0.05). CONCLUSIONS: Feeding type is the main driver of gut microbiota differences in young infants. The gut microbiota differences based on feeding type (exclusive breast- or formula feeding) were associated with observed differences in growth status. This trial was registered at clinicaltrials.gov as NCT02142647, NCT01693406, and NCT04137445.

Effects of Extended Cage Component Sanitation Interval on the Microenvironment, Health, and Gastrointestinal Microbiome of Rats (Rattus norvegicus).

Washing and sanitizing rodent cage components requires costly equipment, significant personnel effort, and use of natural resources. The benchmark frequency for sanitation of individually ventilated caging (IVC) has traditionally been every 2 wk. In this study, we investigated the effects of extending this interval on the cage microenvironment, basic markers of health, and the gastrointestinal microbiota of rats. We compared our institutional standard of changing the sanitation interval for rat cage lids, box feeders, and enrichment devices from every 4 wk to an interval of 12 wk. The cage bottom and bedding continued to be changed every 2 wk for both groups. We hypothesized that we would find no significant difference between our current practice of 4 wks and continuous use for 12 wk. Our data showed that intracage ammonia levels remained below 5 ppm for most cages in both groups, with the exception of cages that experienced a cage flood. We found no significant difference between groups in bacterial colony forming units (CFU) on cage components. We used 3 novel methods of assessing cleanliness of enrichment devices and found no significant effect of continuous use for 12 wk on the number of CFU. In addition, we found no significant differences between groups for animal weight, routine blood work, or fecal and cecal microbiomes. These data indicate that a sanitation interval of up to 12 wk for components of rat IVC caging has no significant effects on the microenvironment or health of rats. Using the longer interval will improve efficiency, reduce the use of natural resources, and decrease costs while maintaining high-quality animal care.

Aging-Associated Augmentation of Gut Microbiome Virulence Capability Drives Sepsis Severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition but also an overabundance of genomic virulence factors that have functional consequence on host immune evasion. IMPORTANCE Older adults suffer more frequent and worse outcomes from sepsis, a critical illness secondary to infection. The reasons underlying this unique susceptibility are incompletely understood. Prior work in this area has focused on how the immune response changes with age. The current study, however, focuses instead on alterations in the community of bacteria that humans live with within their gut (i.e., the gut microbiome). The central concept of this paper is that the bacteria in our gut evolve along with the host and "age," making them more efficient at causing sepsis.

Aging-associated augmentation of gut microbiome virulence capability drives sepsis severity.

Prior research has focused on host factors as mediators of exaggerated sepsis-associated morbidity and mortality in older adults. This focus on the host, however, has failed to identify therapies that improve sepsis outcomes in the elderly. We hypothesized that the increased susceptibility of the aging population to sepsis is not only a function of the host, but also reflects longevity-associated changes in the virulence of gut pathobionts. We utilized two complementary models of gut microbiota-induced experimental sepsis to establish the aged gut microbiome as a key pathophysiologic driver of heightened disease severity. Further murine and human investigations into these polymicrobial bacterial communities demonstrated that age was associated with only subtle shifts in ecological composition, but an overabundance of genomic virulence factors that have functional consequence on host immune evasion. One Sentence Summary: The severity of sepsis in the aged host is in part mediated by longevity-associated increases in gut microbial virulence.

Evolution of the Gut Microbiome in HIV-Exposed Uninfected and Unexposed Infants during the First Year of Life.

HIV-exposed uninfected infants (HEU) have abnormal immunologic functions and increased infectious morbidity in the first 6 months of life, which gradually decreases thereafter. The mechanisms underlying HEU immune dysfunctions are unknown. We hypothesized that unique characteristics of the HEU gut microbiota associated with maternal HIV status may underlie the HEU immunologic dysfunctions. We characterized the infant gut, maternal gut, and breast milk microbiomes of mother-infant pairs, including 123 with HEU and 117 with HIV-uninfected infants (HUU), from South Africa. Pan-bacterial 16S rRNA gene sequencing was performed on (i) infant stool at 6, 28, and 62 weeks; (ii) maternal stool at delivery and 62 weeks; and (iii) breast milk at 6 weeks. Infant gut alpha and beta diversities were similar between groups. Microbial composition significantly differed, including 12 genera, 5 families and 1 phylum at 6 weeks; 12 genera and 2 families at 28 weeks; and 2 genera and 2 families at 62 weeks of life. Maternal gut microbiomes significantly differed in beta diversity and microbial composition, and breast milk microbiomes differed in microbial composition only. Infant gut microbiotas extensively overlapped with maternal gut and minimally with breast milk microbiotas. Nevertheless, exclusively breastfed HEU and HUU had less divergent microbiomes than nonexclusively breastfed infants. Feeding pattern and maternal gut microbiome imprint the HEU gut microbiome. Compared to HUU, the HEU gut microbiome prominently differs in early infancy, including increased abundance of taxa previously observed to be present in excess in adults with HIV. The HEU and HUU gut microbiome compositions converge over time, mirroring the kinetics of HEU infectious morbidity risk. IMPORTANCE HIV-exposed uninfected infants (HEU) are highly vulnerable to infections in the first 6 months of life, and this vulnerability decreases to the age of 24 months. Because the microbiome plays a critical role in the education of the infant immune system, which protects them against infections, we characterized the gut microbiomes of HEU and HIV-unexposed infants (HUU) in the first year of life. The HEU and HUU gut microbiomes showed prominent differences at 6 and 28 weeks of life but converged at 62 weeks of life, mirroring the time course of the HEU excess infectious morbidity and suggesting a potential association between the infant gut microbiome structure and susceptibility to infections. Infant gut microbiotas extensively overlapped with maternal gut and minimally with breast milk microbiotas. Moreover, exclusively breastfed HEU and HUU had less divergent microbiomes at 6 and 28 weeks than nonexclusively breastfed HEU and HUU. The factors that affect the HEU gut microbiome, maternal gut microbiome and exclusive breastfeeding, may be targeted by interventions.

A maternal higher-complex carbohydrate diet increases bifidobacteria and alters early life acquisition of the infant microbiome in women with gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is associated with considerable imbalances in intestinal microbiota that may underlie pathological conditions in both mothers and infants. To more definitively identify these alterations, we evaluated the maternal and infant gut microbiota through the shotgun metagenomic analysis of a subset of stool specimens collected from a randomized, controlled trial in diet-controlled women with GDM. The women were fed either a CHOICE diet (60% complex carbohydrate/25% fat/15% protein, n=18) or a conventional diet (CONV, 40% complex carbohydrate/45% fat/15% protein, n=16) from 30 weeks' gestation through delivery. In contrast to other published studies, we designed the study to minimize the influence of other dietary sources by providing all meals, which were eucaloric and similar in fiber content. At 30 and 37 weeks' gestation, we collected maternal stool samples; performed the fasting measurements of glucose, glycerol, insulin, free fatty acids, and triglycerides; and administered an oral glucose tolerance test (OGTT) to measure glucose clearance and insulin response. Infant stool samples were collected at 2 weeks, 2 months, and 4-5 months of age. Maternal glucose was controlled to conventional targets in both diets, with no differences in Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). No differences in maternal alpha or beta diversity between the two diets from baseline to 37 weeks' gestation were observed. However, women on CHOICE diet had higher levels of Bifidobacteriaceae, specifically Bifidobacterium adolescentis, compared with women on CONV. Species-level taxa varied significantly with fasting glycerol, fasting glucose, and glucose AUC after the OGTT challenge. Maternal diet significantly impacted the patterns of infant colonization over the first 4 months of life, with CHOICE infants showing increased microbiome alpha diversity (richness), greater Clostridiaceae, and decreased Enterococcaceae over time. Overall, these results suggest that an isocaloric GDM diet containing greater complex carbohydrates with reduced fat leads to an ostensibly beneficial effect on the maternal microbiome, improved infant gut microbiome diversity, and reduced opportunistic pathogens capable of playing a role in obesity and immune system development. These results highlight the critical role a maternal diet has in shaping the maternal and infant microbiome in women with GDM.

Microbiota Associated With Cholesteatoma Tissue in Chronic Suppurative Otitis Media.

Otitis media (OM), defined as infection or inflammation of the middle ear (ME), remains a major public health problem worldwide. Cholesteatoma is a non-cancerous, cyst-like lesion in the ME that may be acquired due to chronic OM and cause disabling complications. Surgery is required for treatment, with high rates of recurrence. Current antibiotic treatments have been largely targeted to previous culturable bacteria, which may lead to antibiotic resistance or treatment failures. For this study, our goal was to determine the microbiota of cholesteatoma tissue in comparison with other ME tissues in patients with long-standing chronic OM. ME samples including cholesteatoma, granulation tissue, ME mucosa and discharge were collected from patients undergoing tympanomastoidectomy surgery for chronic OM. Bacteria were profiled by 16S rRNA gene sequencing in 103 ME samples from 53 patients. Respiratory viruses were also screened in 115 specimens from 45 patients. Differences in bacterial profiles (beta-diversity) and the relative abundances of individual taxa were observed between cholesteatoma and ME sample-types. Additionally, patient age was associated with differences in overall microbiota composition while numerous individual taxa were differentially abundant across age quartiles. No viruses were identified in screened ME samples. Biodiversity was moderately lower in cholesteatoma and ME discharge compared to ME mucosal tissues. We also present overall bacterial profiles of ME tissues by sample-type, age, cholesteatoma diagnosis and quinolone use, including prevalent bacterial taxa. Our findings will be useful for fine-tuning treatment protocols for cholesteatoma and chronic OM in settings with limited health care resources.

A dysbiotic microbiome promotes head and neck squamous cell carcinoma.

Recent studies have reported dysbiotic oral microbiota and tumor-resident bacteria in human head and neck squamous cell carcinoma (HNSCC). We aimed to identify and validate oral microbial signatures in treatment-naïve HNSCC patients compared with healthy control subjects. We confirm earlier reports that the relative abundances of Lactobacillus spp. and Neisseria spp. are elevated and diminished, respectively, in human HNSCC. In parallel, we examined the disease-modifying effects of microbiota in HNSCC, through both antibiotic depletion of microbiota in an induced HNSCC mouse model (4-Nitroquinoline 1-oxide, 4NQO) and reconstitution of tumor-associated microbiota in a germ-free orthotopic mouse model. We demonstrate that depletion of microbiota delays oral tumorigenesis, while microbiota transfer from mice with oral cancer accelerates tumorigenesis. Enrichment of Lactobacillus spp. was also observed in murine HNSCC, and activation of the aryl-hydrocarbon receptor was documented in both murine and human tumors. Together, our findings support the hypothesis that dysbiosis promotes HNSCC development.

Different gut microbiota in U.S. formula-fed infants consuming a meat vs. dairy-based complementary foods: A randomized controlled trial.

Objective: This project aimed to evaluate the impact of meat- vs. dairy-based complementary foods on gut microbiota and whether it relates to growth. Design: Full-term, formula-fed infants were recruited from the metro Denver area (Colorado, US) and randomized to a meat- or dairy-based complementary diet from 5 to 12 months of age. Infant's length and weight were measured, and stool samples were collected at 5, 10, and 12 months for 16S rRNA gene sequencing and short-chain fatty acids (SCFAs) quantification. Results: Sixty-four infants completed the dietary intervention (n = 32/group). Weight-for-age Z (WAZ) scores increased in both groups and length-for-age Z scores (LAZ) increased in the meat group only, which led to a significant group-by-time interaction (P = 0.02) of weight-for-length Z (WLZ) score. Microbiota composition (Beta-diversity) differed between groups at 12 months (weighted PERMANOVA P = 0.01) and had a group-by-time interaction of P = 0.09. Microbial community richness (Chao1) increased in the meat group only. Genus Akkermansia had a significant group-by-time interaction and increased in the dairy group and decreased in the meat group. A significant fold change of butyric acid from 5 to 12 months was found in the meat group (+1.75, P = 0.011) but not in the dairy group. Regression analysis showed that Chao1 had a negative association with WLZ and WAZ. Several genera also had significant associations with all growth Z scores. Conclusion: Complementary feeding not only impacts infant growth but also affects gut microbiota maturation. Complementary food choices can affect both the gut microbiota diversity and structures and these changes in gut microbiota are associated with infant growth.

Specialized pro-resolving mediator lipidome and 16S rRNA bacterial microbiome data associated with human chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a clinical syndrome defined by symptoms including nasal congestion, facial pain and pressure, anosmia, and rhinorrhea lasting more than 12 weeks. Several mechanistically distinct processes lead to the development of clinical symptoms in CRS including innate immune dysfunction, dysregulated eicosanoid metabolism and perturbations in host-microbiome interactions [1]. We developed a database comprised of patient demographic information, lipid mediator metabolomic profiles, and 16S bacterial rRNA gene sequence data from 66 patients undergoing endoscopic sinus surgery. Briefly, ethmoid sinus tissue and middle meatal swabs were collected from patients, including non-CRS controls, CRS with polyps (CRSwNP), and CRS without polyps (CRSsNP). Lipid mediator pathways from arachidonic acid (AA) and docosahexanoic acid (DHA) were analyzed by liquid chromatography/tandem mass spectrometry. Bacterial taxa were profiled in parallel by 16S rRNA gene sequencing. This database provides a useful compendium of AA/DHA metabolomic profiles and associated bacterial microbiota in patients with varying disease subtypes, demographics, and risk factors/comorbidities.

The FUT2 Variant c.461G>A (p.Trp154*) Is Associated With Differentially Expressed Genes and Nasopharyngeal Microbiota Shifts in Patients With Otitis Media.

Otitis media (OM) is a leading cause of childhood hearing loss. Variants in FUT2, which encodes alpha-(1,2)-fucosyltransferase, were identified to increase susceptibility to OM, potentially through shifts in the middle ear (ME) or nasopharyngeal (NP) microbiotas as mediated by transcriptional changes. Greater knowledge of differences in relative abundance of otopathogens in carriers of pathogenic variants can help determine risk for OM in patients. In order to determine the downstream effects of FUT2 variation, we examined gene expression in relation to carriage of a common pathogenic FUT2 c.461G>A (p.Trp154*) variant using RNA-sequence data from saliva samples from 28 patients with OM. Differential gene expression was also examined in bulk mRNA and single-cell RNA-sequence data from wildtype mouse ME mucosa after inoculation with non-typeable Haemophilus influenzae (NTHi). In addition, microbiotas were profiled from ME and NP samples of 65 OM patients using 16S rRNA gene sequencing. In human carriers of the FUT2 variant, FN1, KMT2D, MUC16 and NBPF20 were downregulated while MTAP was upregulated. Post-infectious expression in the mouse ME recapitulated these transcriptional differences, with the exception of Fn1 upregulation after NTHi-inoculation. In the NP, Candidate Division TM7 was associated with wildtype genotype (FDR-adj-p=0.009). Overall, the FUT2 c.461G>A variant was associated with transcriptional changes in processes related to response to infection and with increased load of potential otopathogens in the ME and decreased commensals in the NP. These findings provide increased understanding of how FUT2 variants influence gene transcription and the mucosal microbiota, and thus contribute to the pathology of OM.

Altered tissue specialized pro-resolving mediators in chronic rhinosinusitis.

Current literature implicates arachidonic acid-derived leukotrienes and prostaglandins in the pathogenesis of chronic rhinosinusitis. However, other omega-3 and omega-6 derived lipid mediators, such as specialized pro-resolving mediators (SPMs), may also be important in chronic inflammatory disorders of the upper airway. We hypothesize that SPMs differ among CRS subtypes compared to controls and in relation to sinonasal microbiota. Ethmoid sinus tissue and middle meatal swabs were collected from a convenience sample of 66 subjects, including non-CRS controls, CRS with polyps (CRSwNP), and CRS without polyps (CRSsNP). Lipid mediator pathways were analyzed by liquid chromatography/tandem mass spectrometry. Bacterial taxa were profiled in parallel by 16S rRNA gene sequencing. Resolvin D2 was elevated in both CRSwNP (p = 0.00076) and CRSsNP (p = 0.030) compared with non-CRS controls. Lipoxin A4 was significantly increased in CRSwNP compared with CRSsNP (p = 0.000033) and controls (p = 0.044). Cigarette smoking was associated with significantly lower concentrations of several 15-lipoxygenase metabolites including resolvin D1 (p = 0.0091) and resolvin D2 (p = 0.0097), compared with never-smokers. Several of the lipid compounds also correlated with components of the sinonasal mucosal microbiota, including bacterial pathogens such as Pseudomonas aeruginosa. These data suggest that dysfunctional lipid mediator pathways in CRS extend beyond the traditional descriptions of leukotrienes and prostaglandins and include SPMs. Furthermore, dysregulated SPM signaling may contribute to persistent inflammation and bacterial colonization in CRS.

Development of a Novel and Rapid Antibody-Based Diagnostic for Chronic Staphylococcus aureus Infections Based on Biofilm Antigens.

Prosthetic joint infections are difficult to diagnose and treat due to biofilm formation by the causative pathogens. Pathogen identification relies on microbial culture that requires days to weeks, and in the case of chronic biofilm infections, lacks sensitivity. Diagnosis of infection is often delayed past the point of effective treatment such that only the removal of the implant is curative. Early diagnosis of an infection based on antibody detection might lead to less invasive, early interventions. Our study examined antibody-based assays against the Staphylococcus aureus biofilm-upregulated antigens SAOCOL0486 (a lipoprotein), glucosaminidase (a domain of SACOL1062), and SACOL0688 (the manganese transporter MntC) for detection of chronic S. aureus infection. We evaluated these antigens by enzyme-linked immunosorbent assay (ELISA) using sera from naive rabbits and rabbits with S. aureus-mediated osteomyelitis, and then we validated a proof of concept for the lateral flow assay (LFA). The SACOL0688 LFA demonstrated 100% specificity and 100% sensitivity. We demonstrated the clinical diagnostic utility of the SACOL0688 antigen using synovial fluid (SF) from humans with orthopedic implant infections. Elevated antibody levels to SACOL0688 in clinical SF specimens correlated with 91% sensitivity and 100% specificity for the diagnosis of S. aureus infection by ELISA. We found measuring antibodies levels to SACOL0688 in SF using ELISA or LFA provides a tool for the sensitive and specific diagnosis of S. aureus prosthetic joint infection. Development of the LFA diagnostic modality is a desirable, cost-effective option, potentially providing rapid readout in minutes for chronic biofilm infections.

Expression of immunoglobulin D is increased in chronic rhinosinusitis.

BACKGROUND: Immunoglobulin (Ig) D is largely localized to the upper airway and reacts with colonizing respiratory pathogens. OBJECTIVE: To determine whether chronic rhinosinusitis (CRS) is associated with increased IgD expression. METHODS: We performed immunofluorescent staining for cytoplasmic IgD, IgA, IgM, and surface plasma cell marker CD138 (syndecan-1) in sinus tissue of patients with CRS with and without nasal polyps (CRSwNP and CRSsNP, respectively) and control subjects without CRS (n = 6 each). Sinonasal mucus antibody levels of patients with CRSwNP or CRSsNP and control subjects were measured by enzyme-linked immunosorbent assay (n = 13, 11, and 9 subjects, respectively). Cells per square millimeter and antibody levels were compared by analysis of variance. Histopathology was performed with sinus tissue from subjects in the 3 groups (n = 6, 8, and 13 subjects respectively). RESULTS: Cells expressing cytoplasmic IgD exceeded those with cytoplasmic IgA and IgM and represented most CD138+ plasma cells in the lamina propria. The frequencies of IgD+ plasma cells were significantly higher in patients with CRSsNP and CRSwNP compared with control subjects (P < .01). Only patients with CRSwNP showed increased frequencies of IgM and IgA plasma cells (P < .01). In contrast to high plasma cell frequencies in tissues, the levels of secreted IgD were lower than those of IgA, IgM, and IgG but were highest in the CRSwNP group compared with the other groups (P < .05). CONCLUSION: IgD plasma cells are prominent in sinus tissues and are increased in CRS. That IgD protein also shows the lowest concentration of antibodies in secretions suggests that its activity might be targeted to the tissue rather than secretions.

Characterization of Sinus Microbiota by 16S Sequencing from Swabs.

New culture-independent microbiology methods are leading to a paradigm shift in our understanding of how the microbial community at the mucosal surface impacts sinonasal health and disease. Whereas traditional culture-based protocols were designed to identify specific pathogens in order to direct antibiotic therapies and eradicate bacteria, newer molecular techniques allow for the identification of both culturable and nonculturable bacteria in diverse communities. As a result of the recent explosion in the use of molecular techniques, we are gaining an understanding of how commensal bacteria may help modulate the host immune response and promote homeostasis. Here, we describe the general workflow of microbiome sequencing including the detailed methods for extracting mixed-community genomic DNA from sinonasal swabs, amplifying bacterial 16S rRNA genes using quantitative PCR, and preparing the samples for next-generation sequencing on the most commonly used sequencing platforms.