Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumor growth.

The gut microbiota's function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria, Faecalibaculum rodentium, rescued this increased tumor growth. Single-cell transcriptomics identified an increased number of cells with a stromal signature in tumors, and subsequent histology revealed an increased abundance of mast cells in the tumor stromal regions. We show that administration of a mast cell stabilizer, cromolyn, rescues increased tumor growth in antibiotic treated animals but has no influence on tumors from control cohorts. These findings highlight that BrCa-microbiota interactions are different from other cancers studied to date and suggest new research avenues for therapy development.

A polyphenol-rich dietary pattern improves intestinal permeability, evaluated as serum zonulin levels, in older subjects: The MaPLE randomised controlled trial.

BACKGROUND & AIM: Increased intestinal permeability (IP) can occur in older people and contribute to the activation of the immune system and inflammation. Dietary interventions may represent a potential strategy to reduce IP. In this regard, specific food bioactives such as polyphenols have been proposed as potential IP modulator due to their ability to affect several critical targets and pathways that control IP. The trial aimed to test the hypothesis that a polyphenol-rich dietary pattern can decrease serum zonulin levels, an IP surrogate marker involved in tight junction modulation, and can beneficially alter the intestinal microbiota, and IP-associated biochemical and clinical markers in older subjects. METHODS: A randomised, controlled, cross-over intervention trial was performed. Sixty-six subjects (aged ≥ 60 y) with increased IP based on serum zonulin levels, were randomly allocated to one of the two arms of the intervention consisting of a control diet (C-diet) vs. a polyphenol-rich diet (PR-diet). Each intervention was 8-week long and separated by an 8-week wash out period. At the beginning and at the end of each intervention period, serum samples were collected for the quantification of zonulin and other biological markers. Faecal samples were also collected to investigate the intestinal microbial ecosystem. In addition, anthropometrical/physical/biochemical parameters and food intake were evaluated. RESULTS: Fifty-one subjects successfully completed the intervention and a high compliance to the dietary protocols was demonstrated. Overall, polyphenol intake significantly increased from a mean of 812 mg/day in the C diet to 1391 mg/day in the PR-diet. Two-way analysis of variance showed a significant effect of treatment (p = 0.008) and treatment × time interaction (p = 0.025) on serum zonulin levels, which decreased after the 8-week PR-diet. In addition, a treatment × time interaction was observed showing a reduction of diastolic blood pressure (p = 0.028) following the PR-diet, which was strongest in those not using antihypertensive drugs. A decrease in both diastolic (p = 0.043) and systolic blood pressure (p = 0.042) was observed in women. Interestingly, a significant increase in fibre-fermenting and butyrate-producing bacteria such as the family Ruminococcaceae and members of the genus Faecalibacterium was observed following the PR intervention. The efficacy of this dietary intervention was greater in subjects with higher serum zonulin at baseline, who showed more pronounced alterations in the markers under study. Furthermore, zonulin reduction was also stronger among subjects with higher body mass index and with insulin resistance at baseline, thus demonstrating the close interplay between IP and metabolic features. CONCLUSIONS: These data show, for the first time, that a PR-diet can reduce serum zonulin levels, an indirect marker of IP. In addition, PR-diet reduced blood pressure and increased fibre-fermenting and butyrate-producing bacteria. These findings may represent an initial breakthrough for further intervention studies evaluating possible dietary treatments for the management of IP, inflammation and gut function in different target populations. THIS STUDY WAS REGISTERED AT WWW.ISRCTN. ORG AS: ISRCTN10214981.

Estimated Intakes of Nutrients and Polyphenols in Participants Completing the MaPLE Randomised Controlled Trial and Its Relevance for the Future Development of Dietary Guidelines for the Older Subjects.

The evaluation of food intake in older subjects is crucial in order to be able to verify adherence to nutritional recommendations. In this context, estimation of the intake of specific dietary bioactives, such as polyphenols, although particularly challenging, is necessary to plan possible intervention strategies to increase their intake. The aims of the present study were to: (i) evaluate the nutritional composition of dietary menus provided in a residential care setting; (ii) estimate the actual intake of nutrients and polyphenols in a group of older subjects participating in the MaPLE study; and (iii) investigate the impact of an eight-week polyphenol-rich dietary pattern, compared to an eight-week control diet, on overall nutrient and polyphenol intake in older participants. The menus served to the participants provided ~770 mg per day of total polyphenols on average with small variations between seasons. The analysis of real consumption, measured using weighed food diaries, demonstrated a lower nutrient (~20%) and polyphenol intake (~15%) compared to that provided by the menus. The feasibility of dietary patterns that enable an increase in polyphenol intake with putative health benefits for age-related conditions is discussed, with a perspective to developing dietary guidelines for this target population.

Effect of a polyphenol-rich dietary pattern on intestinal permeability and gut and blood microbiomics in older subjects: study protocol of the MaPLE randomised controlled trial.

BACKGROUND: During aging, alterations of the intestinal microbial ecosystem can occur contributing to immunosenescence, inflamm-aging and impairment of intestinal barrier function (increased intestinal permeability; IP). In the context of a diet-microbiota-IP axis in older subjects, food bioactives such as polyphenols may play a beneficial modulatory role. METHODS: MaPLE is a project centered on a randomized, controlled cross-over dietary intervention trial [polyphenol-rich diet (PR-diet) versus control diet (C-diet)] targeted to older people (≥ 60 y) living in a well-controlled setting (i.e. nursing home). The 8-week interventions are separated by an 8-week wash-out period. Three small portions per day of selected polyphenol-rich foods are consumed during intervention in substitution of other comparable products within the C-diet. Biological samples are collected before and after each treatment period to evaluate markers related to IP, inflammation, vascular function, oxidative stress, gut and blood microbiomics, metabolomics. A sample size of 50 subjects was defined based on IP as primary outcome. DISCUSSION: Evidence that increasing the consumption of polyphenol-rich food products can positively affect intestinal microbial ecosystem resulting in reduced IP and decreased translocation of inflammogenic bacterial factors into the bloodstream will be provided. The integration of data from gut and blood microbiomics, metabolomics and other IP-related markers will improve the understanding of the beneficial effect of the intervention in the context of polyphenols-microbiota-IP interactions. Finally, findings obtained will provide a proof of concept of the reliability of the dietary intervention, also contributing to future implementations of dietary guidelines directed to IP management in the older and other at risk subjects. TRIAL REGISTRATION: The trial is registered at (ISRCTN10214981); April 28, 2017.

Polyphenols and Intestinal Permeability: Rationale and Future Perspectives.

Increasing evidence links intestinal permeability (IP), a feature of the intestinal barrier, to several pathological or dysfunctional conditions. Several host and environmental factors, including dietary factors, can affect the maintenance of normal IP. In this regard, food bioactives, such as polyphenols, have been proposed as potential IP modulators, even if the mechanisms involved are not yet fully elucidated. The aim of the present paper is to provide a short overview of the main evidence from in vitro and in vivo studies supporting the role of polyphenols in modulating IP and briefly discuss future perspectives in this research area.

Exploring the Molecular Pathways Behind the Effects of Nutrients and Dietary Polyphenols on Gut Microbiota and Intestinal Permeability: A Perspective on the Potential of Metabolomics and Future Clinical Applications.

The gut microbiota is involved in the regulation of the intestinal permeability (IP), whose disruption is a frequent condition in older people and is associated with the development of several diseases. The diet can affect the gut microbiota and IP, although the molecular mechanisms involved are unclear. Metabolomics is one of the suitable approaches to study the effects of diet on gut microbiota and IP, although, up to now, the research has focused only on a few dietary components. The aim here was to review the most recent literature concerning the application of metabolomics to the study of the diet-induced alterations of gut microbiota and the effects on IP, with a particular focus on the molecular pathways involved. An additional aim was to give a perspective on the future research involving dietary polyphenols, because despite their potential use in the management of increased IP, few studies have been reported to date.

Systematic Review on Polyphenol Intake and Health Outcomes: Is there Sufficient Evidence to Define a Health-Promoting Polyphenol-Rich Dietary Pattern?

Growing evidence support association between polyphenol intake and reduced risk for chronic diseases, even if there is a broad debate about the effective amount of polyphenols able to exert such protective effect. The present systematic review provides an overview of the last 10-year literature on the evaluation of polyphenol intake and its association with specific disease markers and/or endpoints. An estimation of the mean total polyphenol intake has been performed despite the large heterogeneity of data reviewed. In addition, the contribution of dietary sources was considered, suggesting tea, coffee, red wine, fruit and vegetables as the main products providing polyphenols. Total flavonoids and specific subclasses, but not total polyphenols, have been apparently associated with a low risk of diabetes, cardiovascular events and all-cause mortality. However, large variability in terms of methods for the evaluation and quantification of polyphenol intake, markers and endpoints considered, makes it still difficult to establish an evidence-based reference intake for the whole class and subclass of compounds. Nevertheless, the critical mass of data available seem to strongly suggest the protective effect of a polyphenol-rich dietary pattern even if further well targeted and methodologically sound research should be encouraged in order to define specific recommendations.

The ß3-integrin endothelial adhesome regulates microtubule-dependent cell migration.

Integrin ß3 is seen as a key anti-angiogenic target for cancer treatment due to its expression on neovasculature, but the role it plays in the process is complex; whether it is pro- or anti-angiogenic depends on the context in which it is expressed. To understand precisely ß3's role in regulating integrin adhesion complexes in endothelial cells, we characterised, by mass spectrometry, the ß3-dependent adhesome. We show that depletion of ß3-integrin in this cell type leads to changes in microtubule behaviour that control cell migration. ß3-integrin regulates microtubule stability in endothelial cells through Rcc2/Anxa2-driven control of active Rac1 localisation. Our findings reveal that angiogenic processes, both in vitro and in vivo, are more sensitive to microtubule targeting agents when ß3-integrin levels are reduced.

Complete Genome Sequences of Two Bioluminescent Vibrio campbellii Strains Isolated from Biofouling Communities in the Bay of Bengal.

Vibrio campbellii is a pathogen of aquatic animals and has been proposed as a bacterial partner in the formation of bioluminescent milky seas. We present here the complete genome sequences assembled from Illumina and Oxford Nanopore data for two bioluminescent Vibrio campbellii strains (BoB-53 and BoB-90) isolated from biofouled moorings in the Bay of Bengal.

Learning from losers.

Bacteria can overcome environmental challenges by killing nearby bacteria and incorporating their DNA.

Evaluation of Parameters for High Efficiency Transformation of Acinetobacter baumannii.

Acinetobacter baumannii is an emerging, nosocomial pathogen that is poorly characterized due to a paucity of genetic tools and methods. While whole genome sequence data from several epidemic and environmental strains have recently become available, the functional characterization of genes is significantly lagging. Efficient transformation is one of the first steps to develop molecular tools that can be used to address these shortcomings. Here we report parameters allowing high efficiency transformation of A. baumannii. Using a multi-factorial experimental design we found that growth phase, voltage, and resistance all significantly contribute to transformation efficiency. The highest efficiency (4.3 × 10(8) Transformants/µg DNA) was obtained at the stationary growth phase of the bacterium (OD 6.0) using 25 ng of plasmid DNA under 100 Ohms resistance and 1.7 kV/cm voltage. The optimized electroporation parameters reported here provide a useful tool for genetic manipulation of A. baumannii.

Suppression of ß3-integrin in mice triggers a neuropilin-1-dependent change in focal adhesion remodelling that can be targeted to block pathological angiogenesis.

Anti-angiogenic treatments against αvß3-integrin fail to block tumour growth in the long term, which suggests that the tumour vasculature escapes from angiogenesis inhibition through αvß3-integrin-independent mechanisms. Here, we show that suppression of ß3-integrin in mice leads to the activation of a neuropilin-1 (NRP1)-dependent cell migration pathway in endothelial cells via a mechanism that depends on NRP1's mobilisation away from mature focal adhesions following VEGF-stimulation. The simultaneous genetic targeting of both molecules significantly impairs paxillin-1 activation and focal adhesion remodelling in endothelial cells, and therefore inhibits tumour angiogenesis and the growth of already established tumours. These findings provide a firm foundation for testing drugs against these molecules in combination to treat patients with advanced cancers.

Airworthiness testing of medical maggots.

An investigation was conducted to test and certify medicinal maggots to facilitate rapid healing of traumatic and chronic wound infections in Wounded warriors being transported onboard military aircraft. Our specific aims included (1) to test the ability of medical grade larvae to withstand the rigors of U.S. Army aeromedical certification, including tolerance to change in pressure, temperature, and humidity inside ground-based chambers; (2) to evaluate the efficacy of the medical grade larvae during a high-vibration rotary-wing medical transport flight; and (3) to gain U.S. Army aeromedical certification and U.S. Air Force safe-to-fly approval and begin the steps needed to deploy/implement the use of medicinal maggots in patient care regimes for medical airlift standard operating procedures. This report outlines the ground-based and initial air-based tests performed during the study. Maggot mortality was very low during all tests, with a mortality rate of less than 1%. Maggot growth rates in wound arenas were mixed but generally depended on temperature. Overall, the results of these tests suggest that medicinal maggots can withstand the rigors of aeromedical evacuation flights in simulated flight environments and rotary- or fixed-wing aircraft.

Bacterial Strain Diversity Within Wounds.

Significance: Rare bacterial taxa (taxa of low relative frequency) are numerous and ubiquitous in virtually any sample-including wound samples. In addition, even the high-frequency genera and species contain multiple strains. These strains, individually, are each only a small fraction of the total bacterial population. Against the view that wounds contain relatively few kinds of bacteria, this newly recognized diversity implies a relatively high rate of migration into the wound and the potential for diversification during infection. Understanding the biological and medical importance of these numerous taxa is an important new element of wound microbiology. Recent Advances: Only recently have these numerous strains been discovered; the technology to detect, identify, and characterize them is still in its infancy. Multiple strains of both gram-negative and gram-positive bacteria have been found in a single wound. In the few cases studied, the distribution of the bacteria suggests microhabitats and biological interactions. Critical Issues: The distribution of the strains, their phenotypic diversity, and their interactions are still largely uncharacterized. The technologies to investigate this level of genomic detail are still developing and have not been largely deployed to investigate wounds. Future Directions: As advanced metagenomics, single-cell genomics, and advanced microscopy develop, the study of wound microbiology will better address the complex interplay of numerous individually rare strains with both the host and each other.

Microbial profiling of combat wound infection through detection microarray and next-generation sequencing.

Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden.

Proteomic sample preparation for blast wound characterization.

BACKGROUND: Blast wounds often involve diverse tissue types and require substantial time and treatment for appropriate healing. Some of these subsequent wounds become colonized with bacteria requiring a better understanding of how the host responds to these bacteria and what proteomic factors contribute wound healing outcome. In addition, using reliable and effective proteomic sample preparation procedures can lead to novel biomarkers for improved diagnosis and therapy. RESULTS: To address this need, suitable sample preparation for 2-D DIGE proteomic characterization of wound effluent and serum samples from combat-wounded patients was investigated. Initial evaluation of crude effluent and serum proved the necessity of high abundant protein depletion. Subsequently, both samples were successfully depleted using Agilent Multiple Affinity Removal system and showed greatly improved 2-D spot maps, comprising 1,800 and 1,200 protein spots, respectively. CONCLUSION: High abundant protein removal was necessary for both wound effluent and serum. This is the first study to show a successful method for high abundant protein depletion from wound effluent which is compatible with downstream 2-D DIGE analysis. This development allows for improved biomarker discovery in wound effluent and serum samples.

Validation of a novel murine wound model of Acinetobacter baumannii infection.

Patients recovering from traumatic injuries or surgery often require weeks to months of hospitalization, increasing the risk for wound and surgical site infections caused by ESKAPE pathogens, which include A. baumannii (the ESKAPE pathogens are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). As new therapies are being developed to counter A. baumannii infections, animal models are also needed to evaluate potential treatments. Here, we present an excisional, murine wound model in which a diminutive inoculum of a clinically relevant, multidrug-resistant A. baumannii isolate can proliferate, form biofilms, and be effectively treated with antibiotics. The model requires a temporary, cyclophosphamide-induced neutropenia to establish an infection that can persist. A 6-mm-diameter, full-thickness wound was created in the skin overlying the thoracic spine, and after the wound bed was inoculated, it was covered with a dressing for 7 days. Uninoculated control wounds healed within 13 days, whereas infected, placebo-treated wounds remained unclosed beyond 21 days. Treated and untreated wounds were assessed with multiple quantitative and qualitative techniques that included gross pathology, weight loss and recovery, wound closure, bacterial burden, 16S rRNA community profiling, histopathology, peptide nucleic acid-fluorescence in situ hybridization, and scanning electron microscopy assessment of biofilms. The range of differences that we are able to identify with these measures in antibiotic- versus placebo-treated animals provides a clear window within which novel antimicrobial therapies can be assessed. The model can be used to evaluate antimicrobials for their ability to reduce specific pathogen loads in wounded tissues and clear biofilms. Ultimately, the mouse model approach allows for highly powered studies and serves as an initial multifaceted in vivo assessment prior to testing in larger animals.

Antimicrobial resistance determinants in Acinetobacter baumannii isolates taken from military treatment facilities.

Multidrug-resistant (MDR) Acinetobacter baumannii infections are of particular concern within medical treatment facilities, yet the gene assemblages that give rise to this phenotype remain poorly characterized. In this study, we tested 97 clinical A. baumannii isolates collected from military treatment facilities (MTFs) from 2003 to 2009 by using a molecular epidemiological approach that enabled for the simultaneous screening of 236 antimicrobial resistance genes. Overall, 80% of the isolates were found to be MDR, each strain harbored between one and 17 resistant determinants, and a total of 52 unique resistance determinants or gene families were detected which are known to confer resistance to ß-lactam (e.g., blaGES-11, blaTEM, blaOXA-58), aminoglycoside (e.g., aphA1, aacC1, armA), macrolide (msrA, msrB), tetracycline [e.g., tet(A), tet(B), tet(39)], phenicol (e.g., cmlA4, catA1, cat4), quaternary amine (qacE, qacEΔ1), streptothricin (sat2), sulfonamide (sul1, sul2), and diaminopyrimidine (dfrA1, dfrA7, dfrA19) antimicrobial compounds. Importantly, 91% of the isolates harbored blaOXA-51-like carbapenemase genes (including six new variants), 40% harbored the blaOXA-23 carbapenemase gene, and 89% contained a variety of aminoglycoside resistance determinants with up to six unique determinants identified per strain. Many of the resistance determinants were found in potentially mobile gene cassettes; 45% and 7% of the isolates contained class 1 and class 2 integrons, respectively. Combined, the results demonstrate a facile approach that supports a more complete understanding of the genetic underpinnings of antimicrobial resistance to better assess the load, transmission, and evolution of MDR in MTF-associated A. baumannii.

Wound outcome in combat injuries is associated with a unique set of protein biomarkers.

BACKGROUND: The ability to forecast whether a wound will heal after closure without further debridement(s), would provide substantial benefits to patients with severe extremity trauma. METHODS: Wound effluent is a readily available material which can be collected without disturbing healthy tissue. For analysis of potential host response biomarkers, forty four serial combat wound effluent samples from 19 patients with either healing or failing traumatic- and other combat-related wounds were examined by 2-D DIGE. Spot map patterns were correlated to eventual wound outcome (healed or wound failure) and analyzed using DeCyder 7.0 and differential proteins identified via LC-MS/MS. RESULTS: This approach identified 52 protein spots that were differentially expressed and thus represent candidate biomarkers for this clinical application. Many of these proteins are intimately involved in inflammatory and immune responses. Furthermore, discriminate analysis further refined the 52 differential protein spots to a smaller subset of which successfully differentiate between wounds that will heal and those that will fail and require further surgical intervention with greater than 83% accuracy. CONCLUSION: These results suggest candidates for a panel of protein biomarkers that may aid traumatic wound care prognosis and treatment. We recommend that this strategy be refined, and then externally validated, in future studies of traumatic wounds.

The Hospital Microbiome Project: Meeting Report for the 1st Hospital Microbiome Project Workshop on sampling design and building science measurements, Chicago, USA, June 7th-8th 2012.

This report details the outcome of the 1st Hospital Microbiome Project workshop held on June 7th-8th, 2012 at the University of Chicago, USA. The workshop was arranged to determine the most appropriate sampling strategy and approach to building science measurement to characterize the development of a microbial community within a new hospital pavilion being built at the University of Chicago Medical Center. The workshop made several recommendations and led to the development of a full proposal to the Alfred P. Sloan Foundation as well as to the creation of the Hospital Microbiome Consortium.