Physical Activity Plays an Important Role in Body Weight Regulation

Emerging literature highlights the need to incorporate physical activity into every strategy intended to prevent weight gain as well as to maintain weight loss over time. Furthermore, physical activity should be part of any plan to lose weight. The stimulus of exercise provides valuable metabolic adaptations that improve energy and macronutrient balance regulation. A tight coupling between energy intake and energy expenditure has been documented at high levels of physical exercise, suggesting that exercise may improve appetite control. The regular practice of physical activity has also been reported to reduce the risk of stress-induced weight gain. A more personalized approach is recommended when planning exercise programs in a clinical weight loss setting in order to limit the compensatory changes associated to exercise-induced weight loss. With modern environment promoting overeating and sedentary behavior, there is an urgent need for a concerted action including legislative measures to promote healthy active living in order to curb the current epidemic of chronic diseases

Protein from meat or vegetable sources in meals matched for fiber content has similar effects on subjective appetite sensations and energy intake - A randomized acute cross-over meal test study

Higher-protein meals decrease hunger and increase satiety compared to lower-protein meals. However, no consensus exists about the different effects of animal and vegetable proteins on appetite. We investigated how a meal based on vegetable protein (fava beans/split peas) affected ad libitum energy intake and appetite sensations, compared to macronutrient-balanced, iso-caloric meals based on animal protein (veal/pork or eggs). Thirty-five healthy men were enrolled in this acute cross-over study. On each test day, participants were presented with one of four test meals (~3550 kilojoules (kJ) 19% of energy from protein), based on fava beans/split peas (28.5 g fiber), pork/veal or eggs supplemented with pea fiber to control for fiber content (28.5 g fiber), or eggs without supplementation of fiber (6.0 g fiber). Subjective appetite sensations were recorded at baseline and every half hour until the ad libitum meal three hours later. There were no differences in ad libitum energy intake across test meals (p > 0.05). Further, no differences were found across meals for hunger, satiety, fullness, prospective food consumption, or composite appetite score (all p > 0.05). Iso-caloric, macronutrient-balanced, fiber-matched meals based on vegetable protein (fava beans/split peas) or animal protein (veal/pork or eggs) had similar effects on ad libitum energy intake and appetite sensations

The murine lung microbiome in relation to the intestinal and vaginal bacterial communities

BACKGROUND: This work provides the first description of the bacterial population of the lung microbiota in mice. The aim of this study was to examine the lung microbiome in mice, the most used animal model for inflammatory lung diseases such as COPD, cystic fibrosis and asthma. Bacterial communities from broncho-alveolar lavage fluids and lung tissue were compared to samples taken from fecal matter (caecum) and vaginal lavage fluid from female BALB/cJ mice. RESULTS: Using a customized 16S rRNA sequencing protocol amplifying the V3-V4 region our study shows that the mice have a lung microbiome that cluster separately from mouse intestinal microbiome (caecum). The mouse lung microbiome is dominated by Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria overlapping the vaginal microbiome. We also show that removal of host tissue or cells from lung fluid during the DNA extraction step has an impact on the resulting bacterial community profile. Sample preparation needs to be considered when choosing an extraction method and interpreting data. CONCLUSIONS: We have consistently amplified bacterial DNA from mouse lungs that is distinct from the intestinal microbiome in these mice. The gut microbiome has been extensively studied for its links to development of disease. Here we suggest that also the lung microbiome could be important in relation to inflammatory lung diseases. Further research is needed to understand the contribution of the lung microbiome and the gut-lung axis to the development of lung diseases such as COPD and asthma

Efficacy of a synthetic antimicrobial peptidomimetic versus vancomycin in a Staphylococcus epidermidis device-related murine peritonitis model

Objectives: Biofilm-forming Staphylococcus epidermidis is a prevalent cause of peritonitis during peritoneal dialysis. We compared the efficacy of a synthetic antimicrobial peptidomimetic (Ltx21) versus vancomycin in a murine model mimicking a device-related peritonitis. Methods: Silicone implants, pre-colonized with an S. epidermidis biofilm, were inserted into the peritoneal cavity of BALB/c mice. Three groups (36 mice in each) with pre-colonized implants received intraperitoneal treatment with Ltx21, vancomycin or placebo. Mice were euthanized on day 3 (n¼ 12), day 6 (n¼ 12) or day 8 (n¼ 12) post-implantation. Controls were mice with sterile implants (n¼ 18) and mice without surgery (n ¼6). Bacterial reductions in cfu were analysed from implants and peritoneal fluid (PF). Inflammatory responses in serum and PF were measured. Results: Vancomycin resulted in a stronger reduction in cfu counts, both on pre-colonized implants and in PF, compared with Ltx21 and placebo. Complete bacterial clearance of the implants was not achieved in any of the groups. The implants pre-colonized with S. epidermidis 1457 resulted in a low-grade peritonitis. We observed, only on day 6, a significant increase in the PF leucocyte count in the group with pre-colonized implants compared with the group with sterile implants (P ¼ 0.0364). Conclusions: Treatment with vancomycin or Ltx21 was not sufficient to achieve complete bacterial clearance of implants, underlining the difficulties of treating such infections. The low-grade infection may attenuate the inflammatory response and contribute to impaired bacterial clearance. Keywords: biofilms, device-related peritonitis, mouse model Introduction Gram-positive bacteria, in particular coagulase-negative staphylococci, are a prevalent cause of peritoneal dialysis catheter-related peritonitis. 1 This is a serious complication that may lead to catheter removal, peritoneal membrane dysfunction and transfer to haemodialysis. 2 Peritonitis is believed to occur by bacterial entrance to the peritoneal cavity through catheter colonization. 2 Staphylococcal biofilm formation on catheters results in a reduced ability to combat such infections, both by the host immune system and by conventional treatment with antibiotics. Synthetic antimicrobial peptidomimetics (SAMPs) are novel antimicrobial agents derived from cationic antimicrobial peptides that are widespread in nature. 5 Their modes of action are not completely resolved. However, a central mechanism is bacterial membrane disruption, affecting both dormant and dividing bacteria. 7 This study aimed to investigate the efficacy of a SAMP (Ltx21) versus vancomycin in a murine model mimicking devicerelated Staphylococcus epidermidis biofilm-associated peritonitis. We assessed bacterial clearance and the host innate immune response to understand the pathophysiological mechanisms involved. Methods Bacterial isolates and MICs S. epidermidis 1457, used in this experiment, was originally isolated from a central venous catheter infection. S. epidermidis 1457 forms a thick biofilm under the in vitro growth conditions used in this study. 7 All three SAMPs have the same tripeptide sequence, with two arginine moieties providing their cationic properties and a modified tryptophan providing the lipophilic bulk. The SAMPs differ by C-terminal modifications, of which Ltx21 has an additional phenylalanine attached compared with Ltx9 and Ltx5. For Ltx21 the MIC was 6 mg/L (determined by the microbroth dilution method) and the minimal biofilm inhibitory concentration was 60 mg/L (determined by the Alamar blue method), comparable to those values previously reported for Ltx5 and Ltx9. 7 Species confirmation of small colony variants (SCVs) was performed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Animals and animal ethics One hundred and thirty-two female BALB/c mice (Taconic M&B A/S Ry, Denmark), aged 7 -8 weeks, were used. Information on the experimental treatment of the animals and the duration of experiments (days) is provided in Device-related S. epidermidis biofilm-associated peritonitis model Silicone implants (5 mm×5 mm×2 mm; Ole Dich, Denmark) were inserted into the murine intraperitoneal cavity in order to mimic a device-related peritonitis. Briefly, the implants were prepared by incubation for 120 h in an S. epidermidis 1457 culture. The inoculum was adjusted to an optical density at 600 nm that was equivalent to that of a 2 McFarland standard in 0.9% NaCl and further suspended in tryptic soy broth (TSB) with 1% glucose to induce biofilm formation. Every 24 h the implants were rinsed in PBS and transferred to a sterile flask containing fresh medium (TSB with 1% glucose). Mice were anaethetized by subcutaneous injections (0.15 mL) in the groin area with a mixture of 0.0375 mL of 0.315 mg/mL fentanyl/10 mg/mL fluanisone (VetaPharma Ltd, UK) and 0.0375 mL of 5 mg/L midazolam (Hameln Pharmaceuticals, Germany) in 0.075 mL of sterile water. Insertion of implants and intraperitoneal treatment were performed as previously described. 9,10 Vancomycin (Sandoz, Australia) and Ltx21 (Lytix Biopharma AS, Tromsø, Norway) were both dissolved in 0.9% NaCl to a final concentration of 1 mg/mL and 0.5 mg/mL, respectively. All animals received intraperitoneal injections (400 mL) every 24 h for up to 7 days. Treatment was initiated 2 h post-implantation. The vancomycin dose was 20 mg/kg, based on previous studies. 11,12 The Ltx21 dose was 10 mg/kg, based on previous toxicology studies, a pilot treatment study and in vitro MIC studies. On the days of implant removal, mice were anaethetized by subcutaneous injection of 0.1 mL of pentobarbital (200 mg/mL) (KVL, Denmark). After general anaesthesia, blood was drawn by cardiac puncture and transferred to tubes containing heparin for fluorescence-activated cell sorting (FACS) (n¼6 animals) or 50 mg/L lepirudin (Refludan, Hoechst, Germany) for complement analysis (n¼6 animals). Peritoneal lavage was performed by injecting 5 mL of PBS into the peritoneal cavity, followed by gently massaging the abdomen before withdrawing the peritoneal fluid (PF). Implants were removed from the peritoneal cavity and transferred to tubes containing 1 mL of NaCl and 20 glass beads (Lenz, Laborglasinstrumente, Germany). Mice were euthanized by removal of the heart under general anaesthesia. Bacteriology Implants removed from the animals were vortexed for 30 s followed by 5 min of sonication at 40 kHz in an ultrasound bath (Bransonic 3510, Branso Ultrasonic Corporation, USA). One hundred microlitres of both the implant-derived suspension and PF was serial diluted and plated on blood agar plates (SSI, Denmark) for bacterial enumeration. The cfu counts were determined after incubation at 378C overnight. Prolonged incubation was necessary to detect SCVs. Cytokines, chemokines and complement Quantification of tumour necrosis factor-a (TNF-a), interleukin-1b (IL-1b), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 2 (MIP-2) and monocyte chemotactic protein-1 (MCP-1/CCL2) was performed on plasma and/or PF using the Fluorokine MAP system (R&D Systems, UK) in combination with a dual-laser, flow-based sorting and detection analyser (Luminex Corporation, USA), according to the manufacturer's description. Complement factors C3a and C5a from plasma and PF were quantified using ELISA kits (USCN Life Science Inc., Wuhan, China) according to the manufacturer's instructions. Haematological parameters and flow cytometry The total leucocyte concentration and the fractions of granulocytes and macrophages were estimated in PF and blood as previously described. 13 Briefly, the fixed samples were analysed using FACS Canto (Becton Dickinson, USA). Light scatter and logarithmically amplified fluorescence parameters from at least 10 000 events were recorded in list mode after gating on forward light scatter to avoid debris, cell aggregates and bacteria. Statistics The data were analysed using GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA) or IMB SPSS Statistics 19. We used the two-way analysis of variance (ANOVA) test with Bonferroni corrections for multiple comparisons. The non-parametric Mann-Whitney U-test was Results Clinical observation Independent of treatment groups the mice exhibited signs of illness such as ruffled fur and reduced activity levels during the first 2 days. The mortality rate for mice with infected implants was 6/108 (vancomycin¼2, Ltx21¼2 and placebo¼2). The death of these mice was not anticipated, and compared with surviving mice they did not exhibit signs of illness or distress prior to death. Bacteriology Untreated pre-colonized implants had cfu counts of 3×10 8 cfu/ implant. Treatment in vivo with vancomycin and Ltx21 resulted in moderate reductions in the bacterial counts on the implants Cellular response We found no indication of an elevated systemic cellular response, either in mice with pre-colonized implants or in mice with sterile implants (data not shown). A local cellular response was observed in PF. Higher levels of leucocytes in PF from mice with pre-colonized implants receiving Ltx21, vancomycin or placebo were observed on day 6 (P ¼ 0.0364) when compared with animals with sterile implants Cytokine response Levels of TNF-a, IL-1b, GM-CSF, MIP-2 and MCP-1/CCL2 were measured in plasma and in PF on days 3, 6 and 8 post-implantation (data not shown). No significant differences were observed between the groups on any days regarding levels of IL-1b, MIP-2 and TNF-a. Mice with sterile implants showed significantly higher levels of MCP-1 in PF (P ¼ 0.005) on day 6 compared with mice with pre-colonized implants treated with placebo. GM-CSF was measured in blood only. Significantly increased values of GM-CSF were detected on days 3, 6 and 8 in mice with pre-colonized implants (P ¼0.0028) receiving Ltx21, vancomycin or placebo compared with mice with sterile implants. Significantly increased values of GM-CSF were also found on day 3 (P ¼0.02) and day 6 (P ¼ 0.04) in animals with sterile implants compared with animals without surgery. Complement activation The activation products of the complement system, C3a and C5a, were measured in plasma and PF. No significant activation of complement was observed within the groups with pre-colonized implants compared with the control groups without surgery. Discussion This murine model mimicking a device-related S. epidermidis biofilm-associated peritonitis enabled us to study the effects of two different treatment regimens and the host innate immune response. Our aim was to investigate whether Ltx21 could eradicate pre-formed S. epidermidis biofilms on peritoneal implants. Vancomycin 7 SAMPs have been found to have high levels of serum albumin binding. 14 Although in vitro time -kill kinetic studies have demonstrated rapid killing of bacteria, 6 protein binding might occur instantaneously upon administration of such peptides. 14 This reduces the amount of available peptide and might explain the reduced efficacy of Ltx21 in vivo compared with the good efficacy observed in vitro. 7,14 Vancomycin resulted in better bacterial clearance than Ltx21. However, complete biofilm clearance was not achieved by any of the two study drugs, despite high intraperitoneal dosing. On days 6 and 8 post-implantation we observed SCVs associated with implants in both treatment groups and in the placebo group. SCVs have previously been associated with persistent, subclinical and resistant infections associated with implanted medical devices. The overall low levels of granulocytes and macrophages in both blood and PF on days 3, 6 and 8 indicated a low-grade infection. Induction of macrophage apoptosis and mechanisms interfering with phagocytosis and macrophage activation has been observed for both S. epidermidis and Staphylococcus haemolyticus. 18,19 Schommer et al. 19 demonstrated that biofilm production by S. epidermidis 1457 resulted in reduced phagocytosis and macrophage activation, yielding low activation of the transcription factor NF-kB, leading to a significantly reduced IL-1b synthesis in mouse macrophage-like cells. Furthermore, in a mouse model, an S. aureus biofilm induced macrophage death and a significant reduction in IL-1b, TNF-a and MCP-1 production. 20 These observations are in line with findings from our study. In general, we found no consistent increase in cytokine production in the infected groups compared with the sterile groups. In S. epidermidis biofilm infections, a recent study reported that granulocytes are recruited and activated, but are not capable of engulfing bacteria embedded in the biofilm. No complement activation was observed in this S. epidermidis biofilm peritonitis model. In contrast, a previous study by our group demonstrated that S. epidermidis 1457 biofilm induced a strong complement activation in an ex vivo full blood model. There are limitations with this study. The model used is a suitable peritonitis model allowing simultaneous sampling of several parameters in response to treatment of an implant-associated biofilm infection. However, the current study could have benefitted from inclusion of additional animals, allowing for prolonged observation of persistence. One could argue that the use of pre-colonized implants is clinically irrelevant. However, in order to establish a biofilm infection to study the efficacy of the two different treatment regimens, we found that pre-colonization was necessary in order to obtain an infection in immunocompetent mice. Conclusions Our observations demonstrate failure of the novel SAMP Ltx21 and vancomycin in efficiently eradicating the S. epidermidis implantassociated biofilm infection. The reduced efficacy of the SAMP in vivo compared with previous in vitro results reflects the importance of performing animal studies. The presence of a persistent implant infection, which is not cleared by the innate immune system, is demonstrated. We demonstrated that this model allows for study of the complex interplay between the host immune system and the effects of antimicrobial treatment

Less is more: Antibiotics at the beginning of life.

Antibiotic exposure at the beginning of life can lead to increased antimicrobial resistance and perturbations of the developing microbiome. Early-life microbiome disruption increases the risks of developing chronic diseases later in life. Fear of missing evolving neonatal sepsis is the key driver for antibiotic overtreatment early in life. Bias (a systemic deviation towards overtreatment) and noise (a random scatter) affect the decision-making process. In this perspective, we advocate for a factual approach quantifying the burden of treatment in relation to the burden of disease balancing antimicrobial stewardship and effective sepsis management

Analysis of Antibiotic Exposure and Early-Onset Neonatal Sepsis in Europe, North America, and Australia.

IMPORTANCE Appropriate use of antibiotics is life-saving in neonatal early-onset sepsis (EOS), but overuse of antibiotics is associated with antimicrobial resistance and long-term adverse outcomes. Large international studies quantifying early-life antibiotic exposure along with EOS incidence are needed to provide a basis for future interventions aimed at safely reducing neonatal antibiotic exposure. OBJECTIVE To compare early postnatal exposure to antibiotics, incidence of EOS, and mortality among different networks in high-income countries. DESIGN, SETTING, AND PARTICIPANTS This is a retrospective, cross-sectional study of late-preterm and full-term neonates born between January 1, 2014, and December 31, 2018, in 13 hospital-based or population-based networks from 11 countries in Europe and North America and Australia. The study included all infants born alive at a gestational age greater than or equal to 34 weeks in the participating networks. Data were analyzed from October 2021 to March 2022. EXPOSURES Exposure to antibiotics started in the first postnatal week. MAIN OUTCOMES AND MEASURES The main outcomes were the proportion of late-preterm and full-term neonates receiving intravenous antibiotics, the duration of antibiotic treatment, the incidence of culture-proven EOS, and all-cause and EOS-associated mortality. RESULTS A total of 757 979 late-preterm and full-term neonates were born in the participating networks during the study period; 21 703 neonates (2.86%; 95% CI, 2.83%-2.90%), including 12 886 boys (59.4%) with a median (IQR) gestational age of 39 (36-40) weeks and median (IQR) birth weight of 3250 (2750-3750) g, received intravenous antibiotics during the first postnatal week. The proportion of neonates started on antibiotics ranged from 1.18% to 12.45% among networks. The median (IQR) duration of treatment was 9 (7-14) days for neonates with EOS and 4 (3-6) days for those without EOS. This led to an antibiotic exposure of 135 days per 1000 live births (range across networks, 54-491 days per 1000 live births). The incidence of EOS was 0.49 cases per 1000 live births (range, 0.18-1.45 cases per 1000 live births). EOS-associated mortality was 3.20% (12 of 375 neonates; range, 0.00%-12.00%). For each case of EOS, 58 neonates were started on antibiotics and 273 antibiotic days were administered. CONCLUSIONS AND RELEVANCE The findings of this study suggest that antibiotic exposure during the first postnatal week is disproportionate compared with the burden of EOS and that there are wide (up to 9-fold) variations internationally. This study defined a set of indicators reporting on both dimensions to facilitate benchmarking and future interventions aimed at safely reducing antibiotic exposure in early life