Search

Little is known about the relationship of airway microbiota with bronchiolitis in infants. We aimed to identify nasal airway microbiota profiles and to determine their association with the likelihood of bronchiolitis in infants.A case-control study was conducted. As a part of a multicenter prospective study, we collected nasal airway samples from 40 infants hospitalized with bronchiolitis. We concurrently enrolled 110 age-matched healthy controls. By applying 16S ribosomal RNA gene sequencing and an unbiased clustering approach to these 150 nasal samples, we identified microbiota profiles and determined the association of microbiota profiles with likelihood of bronchiolitis.Overall, the median age was 3 months and 56% were male. Unbiased clustering of airway microbiota identified 4 distinct profiles: Moraxella-dominant profile (37%), Corynebacterium/Dolosigranulum-dominant profile (27%), Staphylococcus-dominant profile (15%) and mixed profile (20%). Proportion of bronchiolitis was lowest in infants with Moraxella-dominant profile (14%) and highest in those with Staphylococcus-dominant profile (57%), corresponding to an odds ratio of 7.80 (95% confidence interval, 2.64-24.9; P0.001). In the multivariable model, the association between Staphylococcus-dominant profile and greater likelihood of bronchiolitis persisted (odds ratio for comparison with Moraxella-dominant profile, 5.16; 95% confidence interval, 1.26-22.9; P = 0.03). By contrast, Corynebacterium/Dolosigranulum-dominant profile group had low proportion of infants with bronchiolitis (17%); the likelihood of bronchiolitis in this group did not significantly differ from those with Moraxella-dominant profile in both unadjusted and adjusted analyses.In this case-control study, we identified 4 distinct nasal airway microbiota profiles in infants. Moraxella-dominant and Corynebacterium/Dolosigranulum-dominant profiles were associated with low likelihood of bronchiolitis, while Staphylococcus-dominant profile was associated with high likelihood of bronchiolitis.

BACKGROUND: Bronchiolitis, the leading cause of hospitalization among infants in the United States, is most commonly caused by respiratory syncytial virus (RSV), followed by rhinovirus (RV). Conventional perception is that bronchiolitis is a single entity, albeit with different viral etiologies and degrees of severity. METHODS: We conducted a cross-sectional study of nasopharyngeal aspirates from 106 infants hospitalized with bronchiolitis due to either RSV only (80 patients) or RV only (26 patients). We performed metabolomics analysis and 16S ribosomal RNA gene sequencing on all samples and metagenomic sequencing on 58 of 106 samples. RESULTS: Infants with RSV-only and RV-only infections had significantly different nasopharyngeal metabolome profiles (P < .001) and bacterial metagenome profiles (P < .05). RSV-only infection was associated with metabolites from a range of pathways and with a microbiome dominated by Streptococcus pneumoniae. By contrast, RV-only infection was associated with increased levels of essential and nonessential N-acetyl amino acids and with a high relative abundance of Haemophilus influenzae. These co-occurring species were associated with driving the bacterially derived metabolic pathways. Multi-omic analysis showed that both the virus and the microbiome were significantly associated with metabolic function in infants hospitalized with bronchiolitis. CONCLUSION: Although replication of these findings is necessary, they highlight that bronchiolitis is not a uniform disease between RSV and RV infections, a result with future implications for prevention and treatment.

Accurate classification of the human virome is critical to a full understanding of the role viruses play in health and disease. This implies the need for sensitive, specific, and practical pipelines that return precise outputs while still enabling case-specific post hoc analysis. Viral taxonomic characterization from metagenomic data suffers from high background noise and signal crosstalk that confounds current methods. Here we develop VirMAP that overcomes these limitations using techniques that merge nucleotide and protein information to taxonomically classify viral reconstructions independent of genome coverage or read overlap. We validate VirMAP using published data sets and viral mock communities containing RNA and DNA viruses and bacteriophages. VirMAP offers opportunities to enhance metagenomic studies seeking to define virome-host interactions, improve biosurveillance capabilities, and strengthen molecular epidemiology reporting., Viral taxonomic characterization from metagenomic data suffers from high background noise and signal crosstalk. Here, the authors develop VirMAP, a novel pipeline for analyses of metagenomic data that classifies viral reconstructions independent of genome coverage or read overlap.

BackgroundTo optimise fecal sampling and analysis yielding reproducible microbiome data, and gain further insight into sources of its variation, we compared different collection conditions and 16S rRNA gene sequencing protocols in two centers. Fecal samples were collected on three sequential days from six healthy adults and placed in commercial collection tubes (OMNIgeneGut OMR-200) at room temperature or in sterile 5 ml screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer at 4°C to the laboratory and storage at - 80°C within 24 hours. Replicate samples were shipped on dry ice to centers in Australia and the USA for DNA extraction and sequencing of the V4 region of the 16S rRNA gene, using different PCR protocols. Sequences were analysed with the QIIME pipeline and Greengenes database at the Australian center and with an in-house pipeline and SILVA database at the USA center.ResultsVariation in gut microbiome composition and diversity was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection. Larger differences were evident between the two centers, including in the relative abundances of genus Akkermansia, in phylum Verrucomicrobiales, and Bifidobacteria in Actinobacteria.ConclusionsCollection with storage and transport at 4°C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. However, variation between sequencing centers suggests that cohort samples should be sequenced by the same method in one center. Differences in handling, shipping and methods of PCR gene amplification and sequence analysis in different centers introduce variation in ways that are not fully understood. These findings are particularly relevant as microbiome studies shift towards larger population-based and multicenter studies.

Vibrio gazogenes ATCC 43942 has the potential to synthesize a plethora of metabolites which are of clinical and agricultural significance in response to environmental triggers. The complete genomic sequence of Vibrio gazogenes ATCC 43942 is reported herein, contributing to the knowledge base of strains in the Vibrio genus.

Objective Emerging evidence demonstrated that the structure of fecal microbiome is associated with the likelihood of bronchiolitis in infants. However, no study has examined functional profiles of fecal microbiome in infants with bronchiolitis. In this context, we conducted a case–control study. As a part of multicenter prospective study, we collected stool samples from 40 infants hospitalized with bronchiolitis (cases). We concurrently enrolled 115 age-matched healthy controls. Results First, by applying 16S rRNA gene sequencing to these 155 fecal samples, we identified the taxonomic profiles of fecal microbiome. Next, based on the taxonomy data, we inferred the functional capabilities of fecal microbiome and tested for differences in the functional capabilities between cases and controls. Overall, the median age was 3 months and 45% were female. Among 274 metabolic pathways surveyed, there were significant differences between bronchiolitis cases and healthy controls for 37 pathways, including lipid metabolic pathways (false discovery rate [FDR]

Recent trials suggest fecal microbiota transplantation (FMT) with repeated enemas and high-diversity FMT donors is a promising treatment to induce remission in ulcerative colitis.We designed a prospective, open-label pilot study to assess the safety, clinical efficacy, and microbial engraftment of single FMT delivery by colonoscopy for active ulcerative colitis using a 2-donor fecal microbiota preparation (FMP). Safety and clinical endpoints of response, remission, and mucosal healing at week 4 were assessed. Fecal DNA and rectal biopsies were used to characterize the microbiome and mucosal CD4 T cells, respectively, before and after FMT.Of the 20 patients enrolled in this study, 7 patients (35%) achieved a clinical response by week 4. Three patients (15%) were in remission at week 4 and 2 of these patients (10%) achieved mucosal healing. Three patients (15%) required escalation of care. No serious adverse events were observed. Microbiome analysis revealed that restricted diversity of recipients pre-FMT was significantly increased by high-diversity 2-donor FMP. The microbiome of recipients post-transplant was more similar to the donor FMP than the pretransplant recipient sample in both responders and nonresponders. Notably, donor composition correlated with clinical response. Mucosal CD4 T-cell analysis revealed a reduction in both Th1 and regulatory T-cells post-FMT.High-diversity, 2-donor FMP delivery by colonoscopy seems safe and effective in increasing fecal microbial diversity in patients with active ulcerative colitis. Donor composition correlated with clinical response and further characterization of immunological parameters may provide insight into factors influencing clinical outcome.

© 2017, Association of Biomolecular Resource Facilities. All rights reserved. The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the “Door to Hell” crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.

Pregnancy characteristics may influence the infant fecal microbiota during early life. We aimed to examine associations of maternal gestational weight gain with infant fecal microbiota composition, bacterial community richness, and Shannon diversity index.We analyzed data from a prospective cohort study of healthy infants. We collected prenatal data, including report of mother's gestational weight gain, and infant fecal samples from 84 infant-mother dyads. By applying 16S rRNA gene sequencing and an unbiased clustering by partitioning around medoids using Bray-Curtis distances, we identified 4 fecal microbiota profiles, and examined the associations of maternal gestational weight gain with the 4 fecal microbiota profiles, bacterial community richness, and Shannon diversity index.Overall, the median age of infants was 4.0 months and 43% were girls. The mothers of the 84 infants gained a mean of 14.2 kg (standard deviation, 5.4 kg) during pregnancy. We identified 4 distinct microbiota profiles: Bifidobacterium-dominant (42%), Enterobacter/Veillonella-dominant (23%), Bacteroides-dominant (19%), and Escherichia-dominant (17%). Infants whose mothers had higher gestational weight gain were less likely to have a Bacteroides-dominant profile, corresponding to a relative risk ratio of 0.83 (95% confidence interval, 0.71-0.96; P = 0.01) per 1 kg increase in weight. In addition, higher gestational weight gain was also associated with lower bacterial community richness and Shannon diversity index (P 0.05).In this prospective cohort study of healthy infants, maternal gestational weight gain was associated with the infant fecal microbiota profiles, bacterial community richness, and Shannon diversity index.

Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. However, a mechanistic understanding of the link between intestinal inflammation and SpA has yet to emerge. We evaluated and functionally characterized the fecal microbiome of IBD patients with or without peripheral SpA. Coupling the sorting of immunoglobulin A (IgA)-coated microbiota with 16S ribosomal RNA-based analysis (IgA-seq) revealed a selective enrichment in IgA-coated Escherichia coli in patients with Crohn's disease-associated SpA (CD-SpA) compared to CD alone. E. coli isolates from CD-SpA-derived IgA-coated bacteria were similar in genotype and phenotype to an adherent-invasive E. coli (AIEC) pathotype. In comparison to non-AIEC E. coli, colonization of germ-free mice with CD-SpA E. coli isolates induced T helper 17 cell (TH17) mucosal immunity, which required the virulence-associated metabolic enzyme propanediol dehydratase (pduC). Modeling the increase in mucosal and systemic TH17 immunity we observed in CD-SpA patients, colonization of interleukin-10-deficient or K/BxN mice with CD-SpA-derived E. coli lead to more severe colitis or inflammatory arthritis, respectively. Collectively, these data reveal the power of IgA-seq to identify immunoreactive resident pathosymbionts that link mucosal and systemic TH17-dependent inflammation and offer microbial and immunophenotype stratification of CD-SpA that may guide medical and biologic therapy.

The importance of a healthy gut microbiota on host physiology is becoming increasingly evident. Recent studies suggest that alterations to the microbiota can have adverse effects beyond the GI tract, and has been linked to hypertension and stroke. Thus we hypothesized that gut dysbiosis could contribute to the development of cerebral small vessel disease (CSVD). Giving merit to this hypothesis, we found that the microbiota of the spontaneously hypertensive stroke prone rat (SHRSP) is significantly different than that of WKY controls. Using 16s rRNA sequencing of bacterial DNA we found that SHRSP animals had decreases in measures of bacterial richness (p=.005) and diversity (p=.028), indicative of gut dysbiosis. Phenotypically, CSVD includes vessel remodeling, BBB breakdown and neuroinflammation. Gut dysbiosis is often associated with a leaky gut barrier that allows bacteria to enter the systemic circulation. We observed significantly greater permeability of the SHRSP colon barrier when compared to WKY (p=.026). We next sought to determine if impaired colon barrier function in SHRSP could lead to increased bacterial translocation to the periphery and ultimately to the brain. Brains were harvested from 24 week old SHRSP and WKY animals and qRT-PCR of the 16s rRNA gene was performed to detect the presence of bacteria in the brain. We discovered that SHRSP animals displayed a 50% increase in bacterial 16S rRNA load in the brain compared to WKY animals (p=.0063) confirming that bacteria are not only present in CSVD rats, but also in greater abundance than WKYs. Similarly, we found that SHRSP animals displayed a near 2-fold increase of the bacterial endotoxin LPS in the brain, as compared to WKY brain LPS levels (p=.01). Finally, to further confirm the presence of bacterial components in the brain we used immunofluorescence imaging to visualize peptidoglycan (PG), a molecule found solely in bacterial cell walls. We discovered that we could visualize the presence of PG in the brains of SHRSP animals, and note that PG was commonly observed inside microglia. We conclude from this study that SHRSP rats exhibit gut dysbiosis, gut barrier breakdown, and bacterial products in the brain. Further studies will examine how this bacterial presence contributes to the CSVD phenotypes.

Background LL-37 is a host defense peptide with antimicrobial and immunomodulatory properties. We examined the relation of serum LL-37 levels to the severity of bronchiolitis and viral etiology. Methods We performed a 17-center prospective cohort study in infants hospitalized with bronchiolitis over 3 winters (2011-2014). Site teams collected clinical data, nasopharyngeal aspirates and serum. We used real-time polymerase chain reaction to test nasopharyngeal aspirates for 16 viruses. We tested serum for LL-37. Severity of bronchiolitis was defined by intensive care use and hospital length of stay. Viral etiology was defined as respiratory syncytial virus (RSV) or rhinovirus (RV), including coinfections with other viruses. Results The median age of the 1005 enrolled infants was 3 months (interquartile range, 2-6 months). After adjustment for 12 variables, LL-37 levels in the lowest quartile, compared with the highest, were associated both with intensive care use (adjusted odds ratio [aOR], 1.97; P = .01) and longer hospital stay (1.34; P < .001). In separate multivariable models, infants with LL-37 levels in the lowest 3 quartiles, compared with the highest, were more likely to have RSV (eg, aOR, 2.6 [lowest quartile]; P < .001 [all quartiles]). By contrast, infants with the lowest 3 LL-37 quartiles were less likely to have RV (eg, aOR, 0.5 [lowest quartile]; Pall quartiles ≤ .03 [all quartiles]). Conclusions In a large multicenter study of infants hospitalized with bronchiolitis, lower levels of serum LL-37 were associated with increased severity of illness. There was also an inverse relationship between LL-37 levels and the most common virus causing bronchiolitis, RSV. These findings highlight the role of LL-37 in the pathogenesis of bronchiolitis.

The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/β) and type III IFN (IFN-λ) function at the epithelial level, and their respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures from multiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine.

Recent discoveries on the role of commensal microbiota have significantly changed our understanding of human physiology. The host-microbiota interplay is now an important aspect to take into account to understand immune responses and immunological diseases. Autoimmune uveitis is a sight-threatening disease that arises without a known infectious etiology. It is unknown where and how autoreactive T cells become primed to trigger disease in the eye, which is an immune privileged site. We recently reported data supporting the notion that retina-specific T cells receive a signal in the gut from commensal microbiota-derived cross-reactive antigen(s) and trigger autoimmune uveitis in the R161H mouse model. Here we discuss our published findings, as well as our recent attempts to identify the responsible microbe(s) by using different antibiotic treatments, 16S rDNA sequencing and homology searches for candidate antigenic mimic(s) of the retinal antigen.

Background Neonatal sepsis in preterm infants is often due to organisms that colonize the skin including Staphylococcus spp. and Candida spp. Development and maturation of the skin microbiome in the neonatal period, especially in preterm infants, may be critical in preventing colonization with pathogens and subsequent progression to neonatal sepsis. Development of the skin microbiome in preterm infants or its determinants in the first 4 weeks of life has not been evaluated. Methods We evaluated the skin microbiome from three body sites, antecubital fossa, forehead and gluteal region, in a prospective cohort of 15 preterm (birth weight < 1500 g and < 32 weeks of gestation) and 15 term neonates. The microbiome community membership and relative abundance were evaluated by amplification and sequencing the bacterial V3-V5 region of the16S rRNA gene on the 454 GS FLX platform. We used linear mixed effects models to analyze longitudinal data. Results The structure and composition of the skin microbiome did not differ between the three sampling sites for term and preterm infants in the neonatal period. However, skin bacterial richness was positively associated with gestational age in the first four weeks of life. Intravenous antibiotics negatively impacted the bacterial diversity of the skin but we did not see differences with respect to feeding or mode of delivery. Conclusions Gestational age, which influences the maturity of skin structure and function, is associated with the development of the preterm cutaneous microbiome. Understanding the maturation of a healthy skin microbiome, prevention of pathogen colonization and its role in the development of immunity will be pivotal in the development of novel interventions to prevent infections in critically ill preterm infants.

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

Gut dysbiosis has been linked to cardiovascular diseases including hypertension. We tested the hypothesis that hypertension could be induced in a normotensive strain of rats or attenuated in a hypertensive strain of rats by exchanging the gut microbiota between the two strains. Cecal contents from spontaneously hypertensive stroke prone rats (SHRSP) were pooled. Similarly, cecal contents from normotensive WKY rats were pooled. Four-week-old recipient WKY and SHR rats, previously treated with antibiotics to reduce the native microbiota, were gavaged with WKY or SHRSP microbiota, resulting in four groups; WKY with WKY microbiota (WKY g-WKY), WKY with SHRSP microbiota (WKY g-SHRSP), SHR with SHRSP microbiota (SHR g-SHRSP), and SHR with WKY microbiota (SHR g-WKY). Systolic blood pressure (SBP) was measured weekly using tail-cuff plethysmography. At 11.5 wk of age systolic blood pressure increased 26 mmHg in WKY g-SHRSP compared with that in WKY g-WKY (182 ± 8 vs. 156 ± 8 mmHg, P = 0.02). Although the SBP in SHR g-WKY tended to decrease compared with SHR g-SHRSP, the differences were not statistically significant. Fecal pellets were collected at 11.5 wk of age for identification of the microbiota by sequencing the 16S ribosomal RNA gene. We observed a significant increase in the Firmicutes:Bacteroidetes ratio in the hypertensive WKY g-SHRSP, as compared with the normotensive WKY g-WKY ( P = 0.042). Relative abundance of multiple taxa correlated with SBP. We conclude that gut dysbiosis can directly affect SBP. Manipulation of the gut microbiota may represent an innovative treatment for hypertension.

ummary Background Faecal microbiota transplantation (FMT) has become routine in managing recurrent C. difficile infection (CDI) refractory to antibiotics. Aim To compare clinical response and improvements in colonic microbiota diversity in subjects with recurrent CDI using different donor product. Methods Seventy-two subjects with ≥3 bouts of CDI were randomised in a double-blind study to receive fresh, frozen or lyophilised FMT product via colonoscopy from 50 g of stool per treatment from eight healthy donors. Recipients provided stools pre- and 7, 14 and 30 days post-FMT for C. difficile toxin and, in a subset, microbiome composition by 16S rRNA gene profiling. Results Overall resolution of CDI was 87% during 2 months of follow-up after FMT. Stool samples before FMT had significantly decreased bacterial diversity with a high proportion of Proteobacteria compared to donors. Cure rates were highest for the group receiving fresh product seen in 25/25 (100%), lowest for the lyophilised product 16/23 (78%; P = 0.022 vs. fresh and 0.255 vs. frozen) and intermediate for frozen product 20/24 (P = 0.233 vs. fresh). Microbial diversity was reconstituted by day 7 in the subjects receiving fresh or frozen product. Improvement in diversity was seen by day 7 in those randomised to lyophilised material with reconstitution by 30 days. Conclusions Comparative efficacy in faecal microbiota transplantation was observed in subjects receiving fresh or frozen faecal product from the same donors. The lyophilised product had a slightly lowered efficacy compared with fresh product, but it resembled other treatments in microbial restoration 1 month after faecal microbiota transplantation.

Most patients diagnosed with pancreatic adenocarcinoma (PDAC) survive less than 5 years, but a very small subset of patients survive longer. The factors that determine the long-term survivorship remain elusive. Recently, studies have shown that bacteria can be found in PDAC which may influence therapy responses. In this study, we aimed to determine if the tumor microbiome, and its associated immune responses, can guide long-term survivorship in resected PDAC patients. Using 16S rRNA gene sequencing, we analyzed the tumor microbiome composition and immunoprofile in PDAC patients who survived less than 5 years (short term survivors, STS) versus those who survived more than 5 years (long term survivors, LTS) in two independent cohorts of patients from two institutions (MD Anderson Cancer Center and Johns Hopkins University). We found higher alpha-diversity in the tumor microbiome from LTS compared to STS PDAC patients. Additionally, we found greater densities of immune cells in the LTS compared to STS, with significant correlation with alpha-diversity. Taken together, our study demonstrates that the PDAC microbiome composition may influence the host immune response and the natural history of the disease. E.R. and Y.Z. contributed equally to this work. Citation Format: Erick M. Riquelme, Yu Zhang, Liangliang Zhang, Montiel Maria, Zoltan Michelle, Wenli Dong, Pompeyo Quesada, Ismet Sahin, Vidhi Chandra, Anthony San Lucas, Paul Scheet, Hanwen Xu, Samir M. Hanash, Lei Feng, Nadim Ajami, Joseph Petrosino, Christine B. Peterson, Deborah Nejman, Michael P. Kim, Cynthia L. Sears, Laura D. Wood, Anirban Maitra, Ravid Straussman, Matthew Katz, James Robert White, Robert Jenq, Jennifer Wargo, Florencia McAllister. Pancreatic tumor microbiome and associated immune responses determine clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2829.

One carbon (1C) metabolism nutrients influence epigenetic regulation and they are supplied by diet and synthesized by gut microbiota. We examined the association between dietary consumption of methyl donors (methionine, betaine and choline) and B vitamins (folate, B2, B6, and B12) and the community composition and structure of the colonic mucosa-associated gut microbiota determined by 16S rRNA gene sequencing in 97 colonic biopsies of 35 men. We used the food frequency questionnaire to assess daily consumption of nutrients, and the UPARSE and SILVA databases for operational taxonomic unit classification. The difference in bacterial diversity and taxonomic relative abundance were compared between low versus high consumption of these nutrients. False discover rate (FDR) adjusted p value &lt, 0.05 indicated statistical significance. The bacterial richness and composition differed significantly by the consumption of folate and B vitamins (p &lt, 0.001). Compared with higher consumption, a lower consumption of these nutrients was associated with a lower abundance of Akkermansia (folate), Roseburia (vitamin B2), and Faecalibacterium (vitamins B2, B6, and B12) but a higher abundance of Erysipelatoclostridium (vitamin B2) (FDR p values &lt, 0.05). The community composition and structure of the colonic bacteria differed significantly by dietary consumption of folate and B vitamins.

Bacteriophages are the most abundant members of the microbiota and have the potential to shape gut bacterial communities. Changes to bacteriophage composition are associated with disease, but how phages impact mammalian health remains unclear. We noted an induction of host immunity when experimentally treating bacterially driven cancer, leading us to test whether bacteriophages alter immune responses. Treating germ-free mice with bacteriophages leads to immune cell expansion in the gut. Lactobacillus, Escherichia, and Bacteroides bacteriophages and phage DNA stimulated IFN-γ via the nucleotide-sensing receptor TLR9. The resultant immune responses were both phage and bacteria specific. Additionally, increasing bacteriophage levels exacerbated colitis via TLR9 and IFN-γ. Similarly, ulcerative colitis (UC) patients responsive to fecal microbiota transplantation (FMT) have reduced phages compared to non-responders, and mucosal IFN-γ positively correlates with bacteriophage levels. Bacteriophages from active UC patients induced more IFN-γ compared to healthy individuals. Collectively, these results indicate that bacteriophages can alter mucosal immunity to impact mammalian health.

The evolution of human microbiome research has lead to a systems biology approach that encompasses multidisciplinary investigations. The implementation of next generation sequencing technologies has allowed researchers to study unculturable organisms, discover novel ones, and provide insights into the role of the human microbiome in health and disease. When these approaches are applied to large-scale longitudinal studies designed to interrogate the association of the microbiome with specific clinical outcomes, the development of new therapeutics and diagnostics intended to modulate or detect changes in microbiome composition to improve human health are born. We are just starting to unravel the role of the microbiome in a wide-variety of diseases, and while some of it appears to be related to causation and provide opportunities for intervention, a good dose of pragmatism is warranted as the field is still in its infancy.

An assay was developed to detect antibodies against two norovirus proteases among participants in a Norwalk virus (GI.1) challenge study. Prechallenge seroprevalence was lower against the protease from the homologous GI.1 virus than against protease from a heterologous GII.4 strain. Seroresponses were detected for 14 of 19 (74%) infected persons.

Background Previously rare A2ML1 variants were identified to confer otitis media susceptibility in an indigenous Filipino community and in otitis-prone US children. The goal of this study is to describe differences in the middle ear microbiome between carriers and non-carriers of an A2ML1 duplication variant that increases risk for chronic otitis media among indigenous Filipinos with poor health care access. Methods Ear swabs were obtained from 16 indigenous Filipino individuals with chronic otitis media, of whom 11 carry the A2ML1 duplication variant. Ear swabs were submitted for 16S rRNA gene sequencing. Results Genotype-based differences in microbial richness, structure, and composition were identified, but were not statistically significant. Taxonomic analysis revealed that the relative abundance of the phyla Fusobacteria and Bacteroidetes, and genus Fusobacterium were nominally increased in carriers compared to non-carriers, but were non-significant after correction for multiple testing. We also detected rare bacteria including Oligella that was reported only once in the middle ear. Conclusions These findings suggest that A2ML1-related otitis media susceptibility may be mediated by changes in the middle ear microbiome. Knowledge of middle ear microbial profiles according to genetic background can be potentially useful for therapeutic and prophylactic interventions for otitis media and can guide public health interventions towards decreasing otitis media prevalence within the indigenous Filipino community. Electronic supplementary material The online version of this article (doi:10.1186/s40249-016-0189-7) contains supplementary material, which is available to authorized users.

Rice farmers are pressured to grow rice using less water. The impacts of water-saving rice cultivation methods on rice methylmercury concentrations are uncertain. Rice (Oryza sativa L. cv. Nipponbare) was cultivated in fields using four water management treatments, including flooded (no dry-downs), alternating wetting and drying (AWD) (with one or three dry-downs), and furrow-irrigated fields (nine dry-downs) (n = 16 fields). Anoxic bulk soil was collected from rice roots during the rice maturation phase, and rice grain was harvested after fields were dried. Total mercury and methylmercury concentrations were determined in soil and polished rice samples, and the soil microbiome was analyzed using 16S (v4) rRNA gene profiling. Soil total mercury did not differ between fields. However, compared to continuously flooded fields, soil and rice methylmercury concentrations averaged 51% and 38% lower in the AWD fields, respectively, and 95% and 96% lower in the furrow-irrigated fields, respectively. Compared to flooded fields, grain yield was reduced on average by

Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. However, a mechanistic understanding of the link between intestinal inflammation and SpA has yet to emerge. We evaluated and functionally characterized the fecal microbiome of IBD patients with or without peripheral SpA. Coupling the sorting of immunoglobulin A (IgA)-coated microbiota with 16

Streptococcus mitis frequently causes invasive infections in neutropenic cancer patients, with a subset of patients developing viridans group streptococcal (VGS) shock syndrome. We report here the first complete genome sequence of S. mitis strain SVGS_061, which caused VGS shock syndrome, to help elucidate the pathogenesis of severe VGS infection.

Turicibacter bacteria are commonly detected in the gastrointestinal tracts and feces of humans and animals, but their phylogeny, ecological role, and pathogenic potential remain unclear. We present here the first complete genome sequence of Turicibacter sp. strain H121, which was isolated from the feces of a mouse line contaminated following germ-free derivation.

There is mounting evidence that the microbiome has potent immunoregulatory functions. We assessed the effects of intestinal dysbiosis in a model of Sjögren syndrome (SS) by subjecting mice to desiccating stress (DS) and antibiotics (ABX). We characterized the conjunctival, tongue and fecal microbiome profiles of patients with SS. Severity of ocular surface and systemic disease was graded. 16S ribosomal RNA gene sequencing characterized the microbiota. ABX + DS mice had a significantly worse dry eye phenotype compared to controls, a decrease in Clostridium and an increase in Enterobacter, Escherichia/Shigella, and Pseudomonas in stool after ABX + DS for 10 days. Goblet cell density was significantly lower in ABX treated groups compared to controls. Stool from SS subjects had greater relative abundances of Pseudobutyrivibrio, Escherichia/Shigella, Blautia, and Streptococcus, while relative abundance of Bacteroides, Parabacteroides, Faecalibacterium, and Prevotella was reduced compared to controls. The severity of SS ocular and systemic disease was inversely correlated with microbial diversity. These findings suggest that SS is marked by a dysbiotic intestinal microbiome driven by low relative abundance of commensal bacteria and high relative abundance of potentially pathogenic genera that is associated with worse ocular mucosal disease in a mouse model of SS and in SS patients.

BACKGROUND: Little is known about the association of gut microbiota, a potentially modifiable factor, with bronchiolitis in infants. We aimed to determine the association of fecal microbiota with bronchiolitis in infants. METHODS: We conducted a case–control study. As a part of multicenter prospective study, we collected stool samples from 40 infants hospitalized with bronchiolitis. We concurrently enrolled 115 age-matched healthy controls. By applying 16S rRNA gene sequencing and an unbiased clustering approach to these 155 fecal samples, we identified microbiota profiles and determined the association of microbiota profiles with likelihood of bronchiolitis. RESULTS: Overall, the median age was 3 months, 55% were male, and 54% were non-Hispanic white. Unbiased clustering of fecal microbiota identified 4 distinct profiles: Escherichia-dominant profile (30%), Bifidobacterium-dominant profile (21%), Enterobacter/Veillonella-dominant profile (22%), and Bacteroides-dominant profile (28%). The proportion of bronchiolitis was lowest in infants with the Enterobacter/Veillonella-dominant profile (15%) and highest in the Bacteroides-dominant profile (44%), corresponding to an odds ratio of 4.59 (95% confidence interval, 1.58–15.5; P = .008). In the multivariable model, the significant association between the Bacteroides-dominant profile and a greater likelihood of bronchiolitis persisted (odds ratio for comparison with the Enterobacter/Veillonella-dominant profile, 4.24; 95% confidence interval, 1.56–12.0; P = .005). In contrast, the likelihood of bronchiolitis in infants with the Escherichia-dominant or Bifidobacterium-dominant profile was not significantly different compared with those with the Enterobacter/Veillonella-dominant profile. CONCLUSIONS: In this case–control study, we identified 4 distinct fecal microbiota profiles in infants. The Bacteroides-dominant profile was associated with a higher likelihood of bronchiolitis.

Recent advances in sequencing technology have made it feasible to characterize human gut microbial communities (or microbiota) and examine their associations with disease outcomes, including cardiovascular disease (CVD) related traits. However, there is a paucity of research examining how the human gut microbiome may influence overall CVD risk in population based studies. The objective of the current study was to identify composite measures of microbiota diversity and specific microbial genera that associate with CVD risk among participants of the Bogalusa Heart Study (BHS). The current BHS cohort includes 1,257 participants who have been screened at least two times in childhood and two times in adulthood for CVD risk factors. For the microbiome study, we selected the 55 participants at highest CVD risk and 57 participants at lowest CVD risk using a z-score based data reduction technique, leveraging longitudinal mean measures of systolic BP, low-density lipoprotein cholesterol, and fasting plasma glucose levels. Next generation 16S rRNA sequencing of the V4 hypervariable region was conducted using genomic bacterial DNA extracted from stool samples of the 112 participants. The Wilcoxon rank sum test was used to examine differences in measures of alpha diversity, including observed operational taxonomic units (OTUs) and the Chao1 index, along with specific microbial taxa between CVD risk groups. Permutation multivariate analysis of variance (PERMANOVA) was used to test compositional differences based on unweighted Unifrac distance as a measure of beta diversity. We observed significantly less alpha diversity in those at high versus those at low CVD risk (P=0.04 and 0.02 for observed OTU and Chao1 index measures, respectively). The PERMANOVA test indicated marginal significance of compositional differences between the two groups (P=0.07). In addition, 10 microbial genera significantly differed between high and low CVD risk participants, including Faecalibacterium (P=0.0014), Subdoligranulum (P=0.01), Turicibacter (P=0.01), Rothia (P=0.01), Lachnospira (P=0.01), Haemophilus (P=0.03), Dialister (P=0.03), Bifidobacterium (P=0.03), Oscillibacter (P=0.04), and Megamonas (P=0.05). In summary, we provide empirical evidence of lower microbial enrichment among BHS participants at high versus those at low CVD risk, along with promising findings of differences in specific microbial genera between CVD risk groups.

Background Protein-energy undernutrition during early development confers a lifelong increased risk of obesity-related metabolic disease. Mechanisms by which metabolic abnormalities persist despite catch-up growth are poorly understood. Objective We sought to determine whether abnormal metabolomic and intestinal microbiota profiles from undernourished neonatal mice remain altered during catch-up growth. Methods Male and female CD1 mouse pups were undernourished by timed separation from lactating dams for 4 h at 5 d of age, 8 h at 6 d of age, and 12 h/d from 7 to 15 d of age, then resumed ad libitum nursing, whereas controls fed uninterrupted. Both groups were weaned simultaneously to a standard unpurified diet. At 3 time points (0, 1, and 3 wk after ending feed deprivation), metabolites in urine, plasma, and stool were identified with the use of mass spectrometry, and fecal microbes were identified with the use of 16S metagenomic sequencing. Results Undernourished mice completely recovered deficits of 36% weight and 9% length by 3 wk of refeeding, at which time they had 1.4-fold higher plasma phenyllactate and 2.0-fold higher urinary p-cresol sulfate concentrations than did controls. Plasma serotonin concentrations in undernourished mice were 25% lower at 0 wk but 1.5-fold higher than in controls at 3 wk. Whereas most urine and plasma metabolites normalized with refeeding, 117 fecal metabolites remained altered at 3 wk, including multiple N-linked glycans. Microbiota profiles from undernourished mice also remained distinct, with lower mean proportions of Bacteroidetes (67% compared with 83%) and higher proportions of Firmicutes (26% compared with 16%). Abundances of the mucolytic organisms Akkermansia muciniphila and Mucispirillum schaedleri were altered at 0 and 1 wk. Whereas microbiota from undernourished mice at 0 wk contained 11% less community diversity (P = 0.015), refed mice at 3 wk harbored 1.2-fold greater diversity (P = 0.0006) than did controls. Conclusion Microbial-derived metabolites and intestinal microbiota remain altered during catch-up growth in undernourished neonatal mice.

Non-steroidal anti-inflammatory drugs (NSAIDs) are routinely used in both veterinary and human medicine. Gastrointestinal injury is a frequent adverse event associated with NSAID use and evidence suggests that NSAIDs induce gastrointestinal microbial imbalance (i.e., dysbiosis) in both animals and people. It is unknown, however, whether cyclooxygenase (COX)-2-selective NSAIDs induce dysbiosis, or if this phenomenon occurs in horses administered any class of NSAIDs. Therefore, our objectives were to determine whether the composition and diversity of the fecal microbiota of adult horses were altered by NSAID use, and whether these effects differed between non-selective and COX-2-selective NSAIDs. Twenty-five adult horses were randomly assigned to 1 of 3 groups: control (n = 5); phenylbutazone (n = 10); or, firocoxib (n = 10). Treatments were administered for 10 days. Fecal samples were collected every 5 days for 25 days. DNA was extracted from feces and the 16S rRNA gene amplified and sequenced to determine the composition of the microbiota and the inferred metagenome. While the fecal microbiota profile of the control group remained stable over time, the phenylbutazone and firocoxib groups had decreased diversity, and alteration of their microbiota profiles was most pronounced at day 10. Similarly, there were clear alterations of the inferred metagenome at day 10 compared to all other days, indicating that use of both non-selective and selective COX inhibitors resulted in temporary alterations of the fecal microbiota and inferred metagenome. Dysbiosis associated with NSAID administration is clinically relevant because dysbiosis has been associated with several important diseases of horses including abdominal pain (colic), colitis, enteric infections, and laminitis.

Background: Diet is a modifiable risk factor for multiple cancers. It is also known to modulate gut microbial composition and metabolic activity, and plays an important role in maintaining gut homeostasis. However, our understanding of dietary quality and mucosa-associated microbiota in the large intestine in humans is limited. We examined the association between diet quality and gut microbiota in adults underwent routine colonoscopy in a cross-sectional study. Methods: We enrolled 21 men (50-75 years old, 71% White) who were found to have grossly normal colons at colonoscopy completed between July 2013 and April 2016. We obtained 98 colonic mucosa biopsies, with microbial DNA extracted from snap frozen biopsy. The 16S rRNA V4 region was amplified and sequenced on the Illumina MiSeq platform. The UPARSE and SILVA were used for operational taxonomic unit (OTU) classification. A self-administered BLOCK Food Frequency Questionnaire was used to assess dietary intake in the past year. Dietary quality was defined using the Healthy Eating Index (HEI)-2005, and further categorized as low or high using the median of total HEI or 12 individual component scores. We compared alpha-diversity (OTU and Shannon index), beta-diversity (Weighted UniFrac principal coordinates analysis), and relative abundance of bacterial phylum and genus by total HEI and all 12 individual HEI components using Mann-Whitney test. Reported P values were adjusted for multiple testing using false discovery rate. Results: The most abundant bacterial phyla observed were Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, and Fusobacteria. High-quality diet (total HEI score ≥ 63) was not associated with higher richness and evenness of gut microbiome (P = 0.12), but was significantly associated with bacterial composition (P = 0.046). Compared to men who had low-quality diet (total HEI score < 63), those with high-quality diet had a higher abundance of Proteobacteria (12.2% vs. 8.1%, P = 0.006), and lower abundance of Fusobacteria (0.13% vs. 3.37%, P = 0.004). At the genus level, those with high-quality diet had significantly lower abundance of Fusobacterium than those with low-quality diet (0.11% vs. 3.77%, P = 0.004). At the HEI component level, individuals with a higher consumption of solid fruits and milk had higher richness and evenness of gut microbiome (P < 0.05) as well as higher abundance of phylum Verrucomicrobia (P < 0.05) and genus Akkermansia (P < 0.001). Consuming more solid and saturated fat, alcohol, and added sugar were associated with a higher abundance of phylum Fusobacteria (P < 0.0001) and genus Fusobacterium (P = 0.0001). Conclusions: The colonic microbiome of men consuming a low-quality diet exhibited increased levels of Fusobacteria, which has been associated with colorectal cancer risk. If replicated in prospective research, our findings suggest low quality diet may contribute to colorectal cancer by modulating gut microbiome. Citation Format: Yanhong Liu, Nadim J. Ajami, Diane Hutchinson, David Graham, Sarah Plew, Ashley Johnson, Preksha Shah, Liang Chen, Kathryn Royse, Donna L. White, Jennifer Kramer, Matthew C. Wong, Rhonda Cole, Clark Hair, Jason Hou, Nisreen Husain, Maria Jarbrink-Sehgal, Fasiha Kanwal, Gyanprakash Ketwaroo, Rajesh Shah, Maria Velez, Melissa L. Bondy, Hashem B. El-Serag, Joseph F. Petrosino, Li Jiao. Healthy eating index 2005 and the mucosa associated gut microbiome in healthy individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3265.

Traveler's diarrhea is the most common medical complaint of international visitors to developing regions. Previous findings suggested that noroviruses (NoVs) are an underappreciated cause of traveler's diarrhea. METHODS. In the present study, we sought to define the presence of NoVs in 320 acute diarrheic stool samples collected from 299 US students who traveled to Guadalajara, Cuernavaca, or Puerto Vallarta, Mexico, during the period from 2007 through 2008. Conventional and quantitative real-time polymerase chain reaction assays were used to detect and determine NoV loads in stool samples. NoV strains were characterized by purification of viral RNA followed by sequencing of the viral capsid protein 1 gene. Sequences were compared using multiple sequence alignment, and phylogenetic trees were generated to evaluate the evolutionary relatedness of the viral strains associated with cases of traveler's diarrhea.NoV RNA was detected in 30 (9.4%) of 320 samples. Twelve strains belonged to genogroup I, and 18 strains belonged to genogroup II. NoV prevalence was higher in the winter season than in the summer season (23% vs 7%, respectively; P = .001). The cDNA viral loads of genogroup I viruses were found to be 500-fold higher than those of genogroup II strains. Phylogenetic analysis revealed a diverse population of NoV strains over different locations and years.NoV strains are important causes of traveler's diarrhea in Mexico, especially during the wintertime, and US students in Mexico may represent a suitable group for future NoV vaccine efficacy trials.

BACKGROUND Molecular characterization of Escherichia coli with use of the random amplified polymorphic DNA (RAPD) assay allows the determination of clonal origin and geographic clustering. METHODS Presumed enterotoxigenic Escherichia coli (ETEC) from 213 adults with travelers' diarrhea acquired in Mexico during the summer months of 2004-2007 were studied. Biochemical testing strips determined a 7-digit fingerprint on the basis of 21 biochemical reactions. E. coli producing enterotoxin were evaluated for clonality by RAPD assay. Dendrograms were developed using Pearson correlations with 80% similarity to determine clonal groups. RESULTS Of the presumed ETEC, 85% were confirmed to be E. coli on the basis of biochemical analysis. Other enterotoxigenic bacteria included Citrobacter species (9%) and other coliforms (all 2%). RAPD analysis with primers 1247 and 1254 determined 24 ETEC clonal groups containing 2-9 subjects each, of which 15 spanned the 4 years and 8 spanned both cities. CONCLUSIONS Complete biochemical evaluation of E. coli-like, enterotoxigenic organisms is crucial in ETEC identification. In addition, other enterotoxigenic organisms identified should be studied further for their role in enteric disease. Travelers to Mexico are exposed to a large pool of different ETEC strains from multiple sources, with a small number of dominant types showing a widespread and persistent reservoir of infection.

BackgroundThe use of electronic cigarettes (ECs) has increased drastically over the past five years, primarily as an alternative to smoking tobacco cigarettes. However, the adverse effects of acute and long-term use of ECs on the microbiota have not been explored. In this pilot study, we sought to determine if ECs or tobacco smoking alter the oral and gut microbiota in comparison to non-smoking controls.MethodsWe examined a human cohort consisting of 30 individuals: 10 EC users, 10 tobacco smokers, and 10 controls. We collected cross-sectional fecal, buccal swabs, and saliva samples from each participant. All samples underwent V4 16S rRNA gene sequencing.ResultsTobacco smoking had a significant effect on the bacterial profiles in all sample types when compared to controls, and in feces and buccal swabs when compared to EC users. The most significant associations were found in the gut, with an increased relative abundance ofPrevotella(P= 0.006) and decreasedBacteroides(P= 0.036) in tobacco smokers. The Shannon diversity was also significantly reduced (P= 0.009) in fecal samples collected from tobacco smokers compared to controls. No significant difference was found in the alpha diversity, beta-diversity or taxonomic relative abundances between EC users and controls.DiscussionFrom a microbial ecology perspective, the current pilot data demonstrate that the use of ECs may represent a safer alternative compared to tobacco smoking. However, validation in larger cohorts and greater understanding of the short and long-term impact of EC use on microbiota composition and function is warranted.

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

8 Background: The composition of the vaginal microbiome has been shown to affect clearance of HPV virus and transformation to invasive cancer. Clinical studies correlating the vaginal microbiome with immune activation and response to cancer treatment are lacking. We profiled intratumoral T-cell clonality during radiation therapy and correlated it with the diversity of the vaginal flora. Methods: Thirty patients with newly diagnosed locally advanced cervical cancer were enrolled on a prospective study to characterize changes in the cervical microbiome during chemoradiation. Cervical samples were obtained before radiation therapy and during the 1st, 3rd, and 5th week of radiation therapy. The vaginal microbiome was characterized using 16 sRNA gene sequencing to produce operational taxonomic units (OTU’s) representing individual bacterial species. Disease response was categorized as early response (ER), late response (LR), or nonresponse (NR) on the basis of clinical examination at brachytherapy and 3-month PET/CT. Twenty patients had T-cell receptor β sequencing of DNA performed using the ImmunoSEQ platform. The maximum productive frequency of the top three clones (MP3) was used to assess T-cell clonality. Results: Early response was associated with clonal T-cell expansion with an increase of MP3 of 11.1% during treatment as compared to a decline of 6.1% in patients with LR/NR (p = 0.05). Early response was also associated with lower quantity of observed OTU’s of vaginal microbiota (25.0 [SD 12.68]) vs patients with LR/NR (41.15 [SD = 23.3]) (p = 0·03). Increased MP3 was associated with increased abundance of Corynebacteriales (R = 0.90; p < .0001) , Actinomycetales (R = 0.83; p < .0001) and Bifidobacteriales (R = 0.82; p < .0001) . Decreased MP3 was associated with increased abundance of lactobacillus (R = -0.61; p < .0001). Conclusions: Increased diversity of the vaginal microbiome is negatively associated with outcome, supporting previous clinical studies in non-cancer settings. Specific vaginal bacterial species are associated with increased or decreased T-cell clonality at completion of radiation.

Fish has been an important source of proteins, essential vitamins, and low saturated fats for centuries. However, improperly handled fish can expose consumers to infectious bacteria, including difficult to treat multidrug-resistant pathogens. With the goal to investigate the existence of disease-causing and antibiotic-resistant bacteria, we examined bacterial communities present on various types of fish purchased from supermarkets in Houston, Texas, USA. The bacterial communities were characterized by selective phenotypic culture methods, 16S ribosomal RNA gene sequencing, and antibiotic susceptibility testing. The results revealed the presence of different bacterial communities on the fish samples examined. The bacterial communities were not significantly different between the supermarkets sampled. The following presumptive human pathogens were isolated on the fish samples: Escherichia coli (67%), enterohemorrhagic E. coli (31%), Shigella and Salmonella species (28%), Listeria species (29%), and Staphylococcus aureus (28%). Drug sensitivity assays showed resistance to commonly prescribed antibiotics ciprofloxacin, gentamicin, and vancomycin. Out of a total of 99 E. coli samples tested, 41.4% were resistant to ciprofloxacin, whereas 33.3% were resistant to gentamicin. Of the total of 31 S. aureus isolates tested, 87% were resistant to ciprofloxacin, whereas 61.3% were resistant to vancomycin. Moreover, some of the E. coli strains were resistant to both ciprofloxacin and gentamicin (28%), whereas 49% of the S. aureus isolates were resistant to both ciprofloxacin and vancomycin. These results highlight the prevalence of antimicrobial-resistant foodborne pathogens on fish purchased from the supermarkets and underscore the risk associated with improper handling of fish.