Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins

Streptococcus pyogenes (group A Streptococcus; GAS) is always of clinical significance in wounds where it can initiate infection, destroy skin grafts and persist as a biofilm. Manuka honey has broad spectrum antimicrobial activity and its use in the clinical setting is beginning to gain acceptance with the continuing emergence of antibiotic resistance and the inadequacy of established systemic therapies; novel inhibitors may affect clinical practice. In this study, the effect of manuka honey on S. pyogenes (M28) was investigated in vitro with planktonic and biofilm cultures using MIC, MBC, microscopy and aggregation efficiency. Bactericidal effects were found in both planktonic cultures and biofilms, although higher concentrations of manuka honey were needed to inhibit biofilms. Abrogation of adherence and intercellular aggregation was observed. Manuka honey permeated 24 h established biofilms of S. pyogenes, resulting in significant cell death and dissociation of cells from the biofilm. Sublethal concentrations of manuka honey effectively prevented the binding of S. pyogenes to the human tissue protein fibronectin, but did not inhibit binding to fibrinogen. The observed inhibition of fibronectin binding was confirmed by a reduction in the expression of genes encoding two major fibronectin-binding streptococcal surface proteins, Sof and SfbI. These findings indicate that manuka honey has potential in the topical treatment of wounds containing S. pyogenes. INTRODUCTION Streptococcus pyogenes (group A Streptococcus) colonizes the nasopharynx and skin of healthy individuals, forming part of the commensal microbiota. Under appropriate conditions, S. pyogenes can be transmitted to wounds and is especially problematic after surgery, following skin grafting and for military personal with traumatic or puncture wounds. Wounds provide a route of entry to the host and damaged tissues display a matrix of proteins including collagen, albumin, fibronectin and fibrinogen, which collectively provide a plethora of ligands to which opportunistic pathogens, including streptococci, adhere Biofilms have been associated with persistent or chronic wound infections and are a major obstacle to healing Honey has had a valued place in traditional medicine for many centuries and was reintroduced into modern medicine during the 21st century. Honey exhibits broad spectrum antibacterial activities and has been reported to inhibit more than 80 species of bacteria, including meticillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Lancefield groups A, C and G streptococci, Pseudomonas aeruginosa and Actinomyces species METHODS Bacterial strains. S. pyogenes MGAS6180 (M28, invasive disease; Green et al., 2005) was grown in Todd-Hewitt (TH) broth (Oxoid) containing 0.5 % yeast extract; Bacto agar (Difco) was added to achieve a 1.4 % (w/v) final concentration in agar plates. Cultures were incubated at 37 uC in air supplemented with 5 % CO 2 . For analysis of aggregation properties, S. pyogenes was grown in C medium containing 0.2 % glucose (Lyon et al., 1998). Medical grade manuka honey. Sterile medical grade manuka honey (Medihoney) was kindly donated by Comvita. Medihoney was provided in sterile 50 g portions. Minimum inhibitory and bactericidal concentrations. The MIC for manuka honey against planktonically grown S. pyogenes MGAS6180 was determined by serial dilution (0-95 %; w/v) in a total volume of 5 ml iso-sensitest broth (Oxoid) (according to the British Society for Antimicrobial Chemotherapy methodology for determining MIC; Andrews, 2011). Cultures were incubated for 16 h at 37 uC in aerobic conditions with 5 % CO 2 . To establish the MBC, samples corresponding to the MIC and including three samples of a higher concentration were plated onto Todd-Hewitt agar and incubated for 16 h at 37 uC in aerobic conditions with 5 % CO 2 . Assays were done in triplicate on each of three separate occasions. Inhibition of bacterial growth. To determine the extent of growth inhibition by manuka honey, triplicate cultures of S. pyogenes MGAS6180 were grown for 8 h at 37 uC in 10 ml TH broth, in aerobic conditions with 5 % CO 2 , supplemented with manuka honey (0, 20 and 40 % w/v). Bacterial growth was monitored at OD 650 at hourly intervals. Growth experiments also utilized triplicate biological samples. Aggregation assays. S. pyogenes MGAS6180 was initially grown in C medium for 16 h (Lyon et al., 1998) and harvested by centrifugation at 5000 g for 10 min. Bacterial cell pellets were resuspended in either 1 ml C medium or an appropriate concentration of manuka honey (5 and 10 %; w/v) dissolved in C medium and the OD 600 of the culture was adjusted to 1.0 (±0.05) if necessary. In both cases, manuka honey at twice the desired concentration was dissolved in double strength TH and diluted to the required concentration using TH; cell pellets were resuspended in either 5 or 10 % (w/v) honey solutions. In an untreated control, manuka honey was replaced with PBS added to maintain the appropriate volume and concentration of TH media. Aggregation assays were carried out in triplicate as described previously Static biofilm model. S. pyogenes MGAS6180 was initially grown in C medium for 16 h and these stationary phase cultures were harvested by centrifugation and adjusted to OD 650 0.1. To determine whether manuka honey prevented biofilm formation, biofilms were established in 96-well microtitre plates (Greiner) in 50 ml TH, supplemented with manuka honey (0, 10, 20 and 40 % w/v), by inoculating each well with 5 ml harvested cells. Plates were incubated at 37 uC for 24 h, aerobically with 5 % CO 2 . To estimate biomass, unattached cells were gently aspirated and discarded, and adherent cells were washed twice with PBS and stained with crystal violet (0.25 %; w/v) for 10 min; following a further two washes with PBS, cell-bound crystal violet was resolubilized with 7 % acetic acid, and absorbance was measured at 595 nm (A 595 ) Live-Dead staining. Images of bacterial cells were collected for control cells (untreated) and for cells treated with 40 % (w/v) manuka honey for 2 h, to determine the effect of manuka honey on viability. Biofilms were grown in Petri dishes in 5 ml TH media, or 5 ml 40 % (w/v) honey dissolved in TH media, as previously described; liquid was aspirated from the plates and biofilms were washed with 1 ml PBS. Biofilms were scraped from the coverslips using a cell scraper, resuspended in 1 ml PBS and stained with Live-Dead BacLight bacterial viability kit (Invitrogen), following the manufacturer's instructions. Fluorescence microscopy images were obtained using a Nikon Eclipse 80i fluorescent microscope with oil immersion and 6100 lens. For detection of SYTO 9 (green channel) a 488 nm excitation and 520 nm emission filter was used. For propidium iodide detection (red channel) a 543 nm excitation and 572 nm emission filter was used. Image analysis used Volocity Software (PerkinElmer). Biofilm disruption. To determine whether manuka honey affected biofilm biomass by facilitating the dissociation of adherent cells from S. E. Maddocks and others 782 Microbiology 158 the biofilm, assays were conducted to monitor the numbers of viable planktonic cells that were released into the liquid phase, from established biofilms following treatment with manuka honey. Biofilms were grown for 24 h as described above. The liquid was aspirated from each well, biofilms were washed twice with PBS to remove any planktonic or loosely adherent cells, and manuka honey over a range of concentrations [0, 10, 20 and 40 % (w/v), respectively] was added to the 24 h established biofilms. Following the application of honey, biofilms were incubated for a further 2 h at 37 uC as above and samples of the liquid above the biofilm were collected at 30 min intervals. Bacterial cells were enumerated using the total viable cell (TVC) counting method described by 21 . Fibronectin-and fibrinogen-binding assays. To determine the effect of manuka honey on adherence of S. pyogenes cells to immobilized fibronectin and fibrinogen, a crystal violet assay was conducted as described previously, using 1 % BSA to block wells prior to assaying cell binding RNA extraction from S. pyogenes biofilms. Large scale, static biofilms of S. pyogenes were grown in duplicate in 5 ml C medium (with 20 % honey for the 'treated' biofilms) in sterile Petri dishes for 24 h at 37 uC, as for the small scale biofilms. The liquid was aspirated and the biofilm was scraped from the surface of the Petri dish using a sterile cell scraper. Biofilms were resuspended in 500 ml PBS and vortexed for 1 min to break up cell aggregates. Honey-treated and untreated cell suspensions were equilibrated (to approximately 2.5610 9 c.f.u.) prior to treatment with mutanolysin (100 mg) and lysozyme (100 mg) for 20 min at 37 uC. RNA extraction was carried out using the SV Wizard total RNA extraction kit (Promega) according the manufacturer's instructions. RNA quantification was performed by spectrophotometric measurement using a NanoDrop ND-1000 (NanoDrop Technologies) and each RNA sample was adjusted to give a final concentration of 10 ng ml 21 . End point RT-PCR to determine the relative expression of sof and sfbI. PCR primers were designed to amplify a 1100 bp fragment of the sof gene (sof-fwd: 59-ACTTAGAAAGTTATCTGTAGGG; sofrev: 59-TCTCTCGAGCTTTATGGATAG) and 1200 bp fragment of the sfbI gene (sbfI-fwd: 59-AACTGCTTTAGGAACAGCTTC; sbfI-rev: 59-CCACCATAGCCACAATGCT). The complete genome sequence for S. pyogenes MGAS6180 was obtained from the NCBI database (www.ncbi.nlm.nih.gov) and used as a basis to design the primers used in this study. Internal control primers were designed to amplify a 900 bp internal fragment of the glr (murI; glutamate racemase) gene (glr-fwd: 59-ATGGATACAAGACCAATTGGG; glr-rev: 59-TCATAA-GGTGACATGCTCCAC), a known housekeeping gene in S. pyogenes that is commonly used for MLST (http://spyogenes.mlst.net/misc/ info.asp) RESULTS Inhibition of planktonic S. pyogenes by manuka honey The MIC of manuka honey against S. pyogenes MGAS6180 was found to be 20 % (w/v) and the MBC was found to be 45 % (w/v). Growth curves with 20 % (w/v) manuka honey resulted in a reduced growth rate and reduction in overall cell number ( S. pyogenes aggregation and biofilm development are inhibited by sublethal concentrations of manuka honey To test the capacity for manuka honey to inhibit intercellular aggregation, suspensions of S. pyogenes MGAS6180 were mixed with 10 % (w/v) manuka honey. In the absence of manuka honey, cells strongly aggregated but in the presence of 10 % (w/v) honey, aggregation was completely inhibited (P,0.05) When biofilms of S. pyogenes were initiated in the presence of 10, 20 and 40 % (w/v) manuka honey, a statistically significant reduction in biomass was observed in each case. A reduction of 75 % (P50.03) was observed with 10 % (w/ v) manuka honey; 20 and 40 % (w/v) manuka honey resulted in between 90 % (P50.02) and 96 % (P50.01) reduction in biomass, respectively Manuka honey facilitates cell death and dissociation of bacterial cells from established S. pyogenes biofilms To determine the effect of manuka honey against established biofilms of S. pyogenes MGAS6180, biofilms were grown for 24 h prior to honey treatment. Following 2 h treatment with 10 and 20 % manuka honey (w/v), a 72 % (P50.32) and 77 % (P50.08) reduction in biomass was observed, respectively. Following treatment for 2 h with 40 % (w/v) manuka honey, the observed reduction in biofilm biomass was even greater, and statistically significant at 85 % (P50.01) Live-Dead viability staining (Invitrogen) of untreated biofilms grown for 24 h at 37 uC, honey-treated (40 % w/ v) biofilms grown for 24 h at 37 uC, and biofilms that were grown for 24 h at 37 uC, then treated for 2 h with 40 % (w/ v) manuka honey, showed that in both cases, the honey treatment resulted in cell death With time, bacterial cells may dissociate from a biofilm. To determine whether manuka honey facilitated the dissociation of S. pyogenes from biofilms, the levels of planktonic cells in the liquid phase were monitored for 2 h following the application of manuka honey (the biofilm data described above showed that the biomass was reduced following 2 h treatment, so this time period was deemed appropriate for these experiments). Immediately after application of the manuka honey (0 min) the c.f.u. was approximately the same for each condition. After 30 min and throughout the duration of the experiment (120 min), higher c.f.u. values were recovered from biofilms treated with 10 and 20 % (w/v) manuka honey (subinhibitory concentrations) compared with untreated biofilms, with a maximum recovery of 1.2610 6 c.f.u. S. E. Maddocks and others 784 Microbiology To establish whether manuka honey affected binding of S. pyogenes MGAS6180 to the wound-associated proteins fibronectin and fibrinogen, 1 mg of each of these two protein ligands was immobilized to the surface of a microtitre plate. The extent of binding inhibition using a sublethal dose (20 % w/v) of manuka honey was compared with adherence observed in the absence of manuka honey. Binding of S. pyogenes to fibronectin in the presence of 20 % (w/v) manuka honey was reduced by 74 %, which was found to be statistically significant (P50.01); conversely no such reduction in binding was observed for fibrinogen (P50.38) Genes encoding the surface adhesins Sof and SbfI are differentially expressed in response to exposure to a sublethal concentration of manuka honey To determine whether the decreased binding to fibronectin was the result of differential expression of two of the major fibronectin-binding proteins (Sof and SfbI) of S. pyogenes MGAS6180 in response to sublethal concentrations of honey, end point RT-PCR was employed. The PCR products were analysed by densitometry and quantified relative to the New England Biolabs 1 kb quantitative molecular mass Streptococcus pyogenes biofilms and honey http://mic.sgmjournals.org 785 marker (thresholds for detection ¢0.15 nmol). The results confirmed that both sfbI and sof were expressed to a lesser extent in the presence of 20 % (w/v) manuka hone

Factors Affecting Frequency Discrimination of Vibrotactile Stimuli: Implications for Cortical Encoding

BACKGROUND: Measuring perceptual judgments about stimuli while manipulating their physical characteristics can uncover the neural algorithms underlying sensory processing. We carried out psychophysical experiments to examine how humans discriminate vibrotactile stimuli. METHODOLOGY/PRINCIPAL FINDINGS: Subjects compared the frequencies of two sinusoidal vibrations applied sequentially to one fingertip. Performance was reduced when (1) the root mean square velocity (or energy) of the vibrations was equated by adjusting their amplitudes, and (2) the vibrations were noisy (their temporal structure was irregular). These effects were super-additive when subjects compared noisy vibrations that had equal velocity, indicating that frequency judgments became more dependent on the vibrations' temporal structure when differential information about velocity was eliminated. To investigate which areas of the somatosensory system use information about velocity and temporal structure, we required subjects to compare vibrations applied sequentially to opposite hands. This paradigm exploits the fact that tactile input to neurons at early levels (e.g., the primary somatosensory cortex, SI) is largely confined to the contralateral side of the body, so these neurons are less able to contribute to vibration comparisons between hands. The subjects' performance was still sensitive to differences in vibration velocity, but became less sensitive to noise. CONCLUSIONS/SIGNIFICANCE: We conclude that vibration frequency is represented in different ways by different mechanisms distributed across multiple cortical regions. Which mechanisms support the “readout” of frequency varies according to the information present in the vibration. Overall, the present findings are consistent with a model in which information about vibration velocity is coded in regions beyond SI. While adaptive processes within SI also contribute to the representation of frequency, this adaptation is influenced by the temporal regularity of the vibration

Obstacles to compassion-giving among nursing and midwifery managers: an international study

Aim: To explore nursing and midwifery managers’ views regarding obstacles to compassion-giving across country cultures. Background: The benefit of compassionate leadership is being advocated, but despite the fact that healthcare is invariably conducted within culturally diverse workplaces, the interconnection of culture, compassion and leadership is rarely addressed. Furthermore, evidence on how cultural factors hinder the expression of compassion among nursing and midwifery managers is lacking. Methods: Cross-sectional, exploratory, international online survey involving 1 217 participants from 17 countries. Managers’ responses on open-ended questions related to barriers for providing compassion were entered and thematically analysed through NVivo. Results: Three key themes related to compassion-giving obstacles emerged across countries: 1. related to the managers’ personal characteristics and experiences; 2. system-related; 3. staff-related. Conclusions: Obstacles to compassion-giving among managers vary across countries. An understanding of the variations across countries and cultures of what impedes compassion to flourish in healthcare is important. Implications for nursing practice and policy: Nursing mangers should wisely use their power by adopting leadership styles that promote culturally competent and compassionate workplaces with respect for human rights. Policymakers should identify training and mentoring needs to enable the development of managers’ practical wisdom. Appropriate national and international policies should facilitate the establishment of standards and guidelines for compassionate leadership, in the face of distorted organisational cultures and system-related obstacles to compassion-giving

Enactment of compassionate leadership by nursing and midwifery managers: results from an international online survey

Aim To explore the views of an international sample of nursing and midwifery managers concerning attributes that they associate with compassionate management. Method A cross- sectional online survey. Using a snowballing sampling method, 1217 responses were collected from nursing and midwifery managers in 17 countries. A total of complete 933 responses to a question related to which actions and behaviours indicated that a manager was exercising compassionate leadership were analysed for this paper. First, content analysis of the responses was conducted, and second, a relative distribution of the identified themes for the overall sample and for each participating country was calculated. Results Six main themes were identified describing the attributes of a compassionate leader: (1) Virtuous support, (2) Communication, (3) Personal virtues of the manager, (4) Participatory communication, (5) Growth/flourishing/ nurturing and (6) Team cohesion. The first three themes mentioned above collectively accounted for 63% of the responses, and can therefore be considered to be the most important characteristics of compassionate management behaviour. Conclusion The key indicators of compassionate management in nursing and midwifery which were identified emphasise approachability, active and sensitive listening, sympathetic responses to staff members’ difficulties (especially concerning child and other caring responsibilities), active support of and advocacy for the staff team and active problem solving and conflict resolution. While there were differences between the countries’ views on compassionate healthcare management, some themes were widely represented among different countries’ responses, which suggest key indicators of compassionate management that apply across culture

Management and outcomes of patients with left atrial appendage thrombus prior to percutaneous closure

Altres ajuts: Fundación Interhospitalaria para la Investigación Cardiovascular (FIC Foundation); Abbott.Objective: Left atrial appendage (LAA) thrombus has heretofore been considered a contraindication to percutaneous LAA closure (LAAC). Data regarding its management are very limited. The aim of this study was to analyse the medical and invasive treatment of patients referred for LAAC in the presence of LAA thrombus. Methods: This multicentre observational registry included 126 consecutive patients referred for LAAC with LAA thrombus on preprocedural imaging. Treatment strategies included intensification of antithrombotic therapy (IAT) or direct LAAC. The primary and secondary endpoints were a composite of bleeding, stroke and death at 18 months, and procedural success, respectively. Results: IAT was the preferred strategy in 57.9% of patients, with total thrombus resolution observed in 60.3% and 75.3% after initial and subsequent IAT, respectively. Bleeding complications and stroke during IAT occurred in 9.6% and 2.9%, respectively, compared with 3.8% bleeding and no embolic events in the direct LAAC group before the procedure. Procedural success was 90.5% (96.2% vs 86.3% in direct LAAC and IAT group, respectively, p=0.072), without cases of in-hospital thromboembolic complications. The primary endpoint occurred in 29.3% and device-related thrombosis was found in 12.8%, without significant difference according to treatment strategy. Bleeding complications at 18 months occurred in 22.5% vs 10.5% in the IAT and direct LAAC group, respectively (p=0.102). Conclusion: In the presence of LAA thrombus, IAT was the initial management strategy in half of our cohort, with initial thrombus resolution in 60% of these, but with a relatively high bleeding rate (∼10%). Direct LAAC was feasible, with high procedural success and absence of periprocedural embolic complications. However, a high rate of device-related thrombosis was detected during follow-up

Phylogenetic diversity of Amazonian tree communities

This is the peer reviewed version of the following article: Honorio Coronado, E. N., Dexter, K. G., Pennington, R. T., Chave, J., Lewis, S. L., Alexiades, M. N., Alvarez, E., Alves de Oliveira, A., Amaral, I. L., Araujo-Murakami, A., Arets, E. J. M. M., Aymard, G. A., Baraloto, C., Bonal, D., Brienen, R., Cerón, C., Cornejo Valverde, F., Di Fiore, A., Farfan-Rios, W., Feldpausch, T. R., Higuchi, N., Huamantupa-Chuquimaco, I., Laurance, S. G., Laurance, W. F., López-Gonzalez, G., Marimon, B. S., Marimon-Junior, B. H., Monteagudo Mendoza, A., Neill, D., Palacios Cuenca, W., Peñuela Mora, M. C., Pitman, N. C. A., Prieto, A., Quesada, C. A., Ramirez Angulo, H., Rudas, A., Ruschel, A. R., Salinas Revilla, N., Salomão, R. P., Segalin de Andrade, A., Silman, M. R., Spironello, W., ter Steege, H., Terborgh, J., Toledo, M., Valenzuela Gamarra, L., Vieira, I. C. G., Vilanova Torre, E., Vos, V., Phillips, O. L. (2015), Phylogenetic diversity of Amazonian tree communities. Diversity and Distributions, 21: 1295–1307. doi: 10.1111/ddi.12357, which has been published in final form at 10.1111/ddi.12357Aim: To examine variation in the phylogenetic diversity (PD) of tree communities across geographical and environmental gradients in Amazonia. Location: Two hundred and eighty-three c. 1 ha forest inventory plots from across Amazonia. Methods: We evaluated PD as the total phylogenetic branch length across species in each plot (PDss), the mean pairwise phylogenetic distance between species (MPD), the mean nearest taxon distance (MNTD) and their equivalents standardized for species richness (ses.PDss, ses.MPD, ses.MNTD). We compared PD of tree communities growing (1) on substrates of varying geological age; and (2) in environments with varying ecophysiological barriers to growth and survival. Results: PDss is strongly positively correlated with species richness (SR), whereas MNTD has a negative correlation. Communities on geologically young- and intermediate-aged substrates (western and central Amazonia respectively) have the highest SR, and therefore the highest PDss and the lowest MNTD. We find that the youngest and oldest substrates (the latter on the Brazilian and Guiana Shields) have the highest ses.PDss and ses.MNTD. MPD and ses.MPD are strongly correlated with how evenly taxa are distributed among the three principal angiosperm clades and are both highest in western Amazonia. Meanwhile, seasonally dry tropical forest (SDTF) and forests on white sands have low PD, as evaluated by any metric. Main conclusions: High ses.PDss and ses.MNTD reflect greater lineage diversity in communities. We suggest that high ses.PDss and ses.MNTD in western Amazonia results from its favourable, easy-to-colonize environment, whereas high values in the Brazilian and Guianan Shields may be due to accumulation of lineages over a longer period of time. White-sand forests and SDTF are dominated by close relatives from fewer lineages, perhaps reflecting ecophysiological barriers that are difficult to surmount evolutionarily. Because MPD and ses.MPD do not reflect lineage diversity per se, we suggest that PDss, ses.PDss and ses.MNTD may be the most useful diversity metrics for setting large-scale conservation priorities.FINCyT - PhD studentshipSchool of Geography of the University of LeedsRoyal Botanic Garden EdinburghNatural Environment Research Council (NERC)Gordon and Betty Moore FoundationEuropean Union's Seventh Framework ProgrammeERCCNPq/PELDNSF - Fellowshi

Impact of common cardio-metabolic risk factors on fatal and non-fatal cardiovascular disease in Latin America and the Caribbean: an individual-level pooled analysis of 31 cohort studies

Background: Estimates of the burden of cardio-metabolic risk factors in Latin America and the Caribbean (LAC) rely on relative risks (RRs) from non-LAC countries. Whether these RRs apply to LAC remains un- known. Methods: We pooled LAC cohorts. We estimated RRs per unit of exposure to body mass index (BMI), systolic blood pressure (SBP), fasting plasma glucose (FPG), total cholesterol (TC) and non-HDL cholesterol on fatal (31 cohorts, n = 168,287) and non-fatal (13 cohorts, n = 27,554) cardiovascular diseases, adjusting for regression dilution bias. We used these RRs and national data on mean risk factor levels to estimate the number of cardiovascular deaths attributable to non-optimal levels of each risk factor. Results: Our RRs for SBP, FPG and TC were like those observed in cohorts conducted in high-income countries; however, for BMI, our RRs were consistently smaller in people below 75 years of age. Across risk factors, we observed smaller RRs among older ages. Non-optimal SBP was responsible for the largest number of attributable cardiovascular deaths ranging from 38 per 10 0,0 0 0 women and 54 men in Peru, to 261 (Dominica, women) and 282 (Guyana, men). For non-HDL cholesterol, the lowest attributable rate was for women in Peru (21) and men in Guatemala (25), and the largest in men (158) and women (142) from Guyana. Interpretation: RRs for BMI from studies conducted in high-income countries may overestimate disease burden metrics in LAC; conversely, RRs for SBP, FPG and TC from LAC cohorts are similar to those esti- mated from cohorts in high-income countries

Towards precision medicine: defining and characterizing adipose tissue dysfunction to identify early immunometabolic risk in symptom-free adults from the GEMM family study

Interactions between macrophages and adipocytes are early molecular factors influencing adipose tissue (AT) dysfunction, resulting in high leptin, low adiponectin circulating levels and low-grade metaflammation, leading to insulin resistance (IR) with increased cardiovascular risk. We report the characterization of AT dysfunction through measurements of the adiponectin/leptin ratio (ALR), the adipo-insulin resistance index (Adipo-IRi), fasting/postprandial (F/P) immunometabolic phenotyping and direct F/P differential gene expression in AT biopsies obtained from symptom-free adults from the GEMM family study. AT dysfunction was evaluated through associations of the ALR with F/P insulin-glucose axis, lipid-lipoprotein metabolism, and inflammatory markers. A relevant pattern of negative associations between decreased ALR and markers of systemic low-grade metaflammation, HOMA, and postprandial cardiovascular risk hyperinsulinemic, triglyceride and GLP-1 curves was found. We also analysed their plasma non-coding microRNAs and shotgun lipidomics profiles finding trends that may reflect a pattern of adipose tissue dysfunction in the fed and fasted state. Direct gene differential expression data showed initial patterns of AT molecular signatures of key immunometabolic genes involved in AT expansion, angiogenic remodelling and immune cell migration. These data reinforce the central, early role of AT dysfunction at the molecular and systemic level in the pathogenesis of IR and immunometabolic disorders

Mycobacterium tuberculosis Lineage Influences Innate Immune Response and Virulence and Is Associated with Distinct Cell Envelope Lipid Profiles

The six major genetic lineages of Mycobacterium tuberculosis are strongly associated with specific geographical regions, but their relevance to bacterial virulence and the clinical consequences of infection are unclear. Previously, we found that in Vietnam, East Asian/Beijing and Indo-Oceanic strains were significantly more likely to cause disseminated tuberculosis with meningitis than those from the Euro-American lineage. To investigate this observation we characterised 7 East Asian/Beijing, 5 Indo-Oceanic and 6 Euro-American Vietnamese strains in bone-marrow-derived macrophages, dendritic cells and mice. East Asian/Beijing and Indo-Oceanic strains induced significantly more TNF-α and IL-1β from macrophages than the Euro-American strains, and East Asian/Beijing strains were detectable earlier in the blood of infected mice and grew faster in the lungs. We hypothesised that these differences were induced by lineage-specific variation in cell envelope lipids. Whole lipid extracts from East Asian/Beijing and Indo-Oceanic strains induced higher concentrations of TNF-α from macrophages than Euro-American lipids. The lipid extracts were fractionated and compared by thin layer chromatography to reveal a distinct pattern of lineage-associated profiles. A phthiotriol dimycocerosate was exclusively produced by East Asian/Beijing strains, but not the phenolic glycolipid previously associated with the hyper-virulent phenotype of some isolates of this lineage. All Indo-Oceanic strains produced a unique unidentified lipid, shown to be a phenolphthiocerol dimycocerosate dependent upon an intact pks15/1 for its production. This was described by Goren as the ‘attenuation indictor lipid’ more than 40 years ago, due to its association with less virulent strains from southern India. Mutation of pks15/1 in a representative Indo-Oceanic strain prevented phenolphthiocerol dimycocerosate synthesis, but did not alter macrophage cytokine induction. Our findings suggest that the early interactions between M. tuberculosis and host are determined by the lineage of the infecting strain; but we were unable to show these differences are driven by lineage-specific cell-surface expressed lipids