Exploring the feasibility of bacteriocins EntK1 and EntEJ97s in treatment of systemic vancomycin resistant enterococci infections in mice.

AIMS: Enterocins K1 and EJ97 have specific antimicrobial activity against Enterococcus faecium and Enterococcus faecalis, respectively. The aim of this study was to investigate the utility of these enterocins for in vivo treatment of systemic enterococcal infections. METHODS AND RESULTS: The antimicrobial effect in blood was analysed and compared against the effect in saline. Colony forming unit counts revealed that the enterocins killed all the bacteria within 1 hour. Additionally, the bactericidal effect against E. faecalis was more rapid in blood, indicating a possible synergy between EntEJ97 and blood. Importantly, no enterocin resistant mutants emerged in these experiments. Injecting the enterocins intraperitoneally in an in vivo mouse model and using fluorescence and minimum inhibitory concentration determination to estimate concentrations of the peptides in plasma, indicate that the enterocins exist in circulation in therapeutic concentrations. Alanine aminotransferase detection, and haemolysis analysis indicates that there is no detectable liver damage or haemolytic effect after injection. CONCLUSIONS: The study revealed that EntK1 and EntEJ97 are able to kill all bacteria ex vivo in the presence of blood. In vivo experiments determine that the enterocins exist in circulation in therapeutic concentrations without causing liver damage or haemolysis. Future experiments should test these peptides for treatment of infection in a relevant in vivo model.

Comparison of mouse models of microbial experience reveals differences in microbial diversity and response to vaccination.

Specific pathogen-free (SPF) laboratory mice dominate preclinical studies for immunology and vaccinology. Unfortunately, SPF mice often fail to accurately model human responses to vaccination and other immunological perturbations. Several groups have taken different approaches to introduce additional microbial experience to SPF mice to better model human immune experience. How these different models compare is unknown. Here, we directly compare three models: housing SPF mice in a microbe-rich barn-like environment (feralizing), adding wild-caught mice to the barn-like environment (fer-cohoused), or cohousing SPF mice with pet store mice in a barrier facility (pet-cohoused); the two latter representing different murine sources of microbial transmission. Pet-cohousing mice resulted in the greatest microbial exposure. Feralizing alone did not result in the transmission of any pathogens tested, while fer-cohousing resulted in the transmission of several picornaviruses. Murine astrovirus 2, the most common pathogen from pet store mice, was absent from the other two model systems. Previously, we had shown that pet-cohousing reduced the antibody response to vaccination compared with SPF mice. This was not recapitulated in either the feralized or fer-cohoused mice. These data indicate that not all dirty mouse models are equivalent in either microbial experience or immune responses to vaccination. These disparities suggest that more cross model comparisons are needed but also represent opportunities to uncover microbe combination-specific phenotypes and develop more refined experimental models. Given the breadth of microbes encountered by humans across the globe, multiple model systems may be needed to accurately recapitulate heterogenous human immune responses.IMPORTANCEAnimal models are an essential tool for evaluating clinical interventions. Unfortunately, they can often fail to accurately predict outcomes when translated into humans. This failure is due in part to a lack of natural infections experienced by most laboratory animals. To improve the mouse model, we and others have exposed laboratory mice to microbes they would experience in the wild. Although these models have been growing in popularity, these different models have not been specifically compared. Here, we directly compare how three different models of microbial experience impact the immune response to influenza vaccination. We find that these models are not the same and that the degree of microbial exposure affects the magnitude of the response to vaccination. These results provide an opportunity for the field to continue comparing and contrasting these systems to determine which models best recapitulate different aspects of the human condition.

Dietary fiber - a scoping review for Nordic Nutrition Recommendations 2023.

Dietary fiber is a term crudely defined as carbohydrates (CHOs) that escape digestion and uptake in the small intestine. Lignin, which is not a CHO, is also a part of the dietary fiber definition. Dietary fibers come in different sizes and forms, with a variety of combinations of monomeric units. Health authorities worldwide have for many years recommended a diet rich in dietary fibers based on consistent findings that dietary fibers are associated with reduced incidences of major non-communicable diseases, including obesity, type 2 diabetes, cardiovascular disease, and colorectal cancer. Most fibers come from common edible foods from the plant kingdom, but fibers are also found in food additives, supplements, and breast milk. The recommended intake in Nordic Nutrition Recommendations 2012 (NNR2012) is 25 g/d for women and 35 g/d for men, whereas the actual intake is significantly lower, ranging from 16 g/d to 22 g/d in women and 18 g/d to 26 g/d in men. New studies since NNR2012 confirm the current view that dietary fiber is beneficial for health, advocating intakes of at least 25 g/day.

Microbial experience through housing in a farmyard-type environment alters intestinal barrier properties in mouse colons.

To close the gap between ultra-hygienic research mouse models and the much more environmentally exposed conditions of humans, we have established a system where laboratory mice are raised under a full set of environmental factors present in a naturalistic, farmyard-type habitat-a process we have called feralization. In previous studies we have shown that feralized (Fer) mice were protected against colorectal cancer when compared to conventionally reared laboratory mice (Lab). However, the protective mechanisms remain to be elucidated. Disruption of the protective intestinal barrier is an acknowledged player in colorectal carcinogenesis, and in the current study we assessed colonic mucosal barrier properties in healthy, feralized C57BL/6JRj male mice. While we found no effect of feralization on mucus layer properties, higher expression of genes encoding the mucus components Fcgbp and Clca1 still suggested mucus enforcement due to feralization. Genes encoding other proteins known to be involved in bacterial defense (Itln1, Ang1, Retnlb) and inflammatory mechanisms (Zbp1, Gsdmc2) were also higher expressed in feralized mice, further suggesting that the Fer mice have an altered intestinal mucosal barrier. These findings demonstrate that microbial experience conferred by housing in a farmyard-type environment alters the intestinal barrier properties in mice possibly leading to a more robust protection against disease. Future studies to unravel regulatory roles of feralization on intestinal barrier should aim to conduct proteomic analyses and in vivo performance of the feralized mice intestinal barrier.

Differential effects of plant and animal fats on obesity-induced dyslipidemia and atherosclerosis in Ldlr-/-.Leiden mice.

Cardiovascular disease (CVD) is closely associated with obesity through risk factors such as dyslipidemia and chronic low-grade inflammation, which may be affected by diet. Dietary fats have been extensively studied in relation to CVD risk, however these studies have not always yielded consistent results, most likely due to lack in control of experimental conditions and confounding factors. Here we studied the effects of different plant and animal fats on dyslipidemia, inflammation, and atherosclerosis. Ldlr-/-.Leiden mice were fed isocaloric energy-dense diets with translational macronutrient composition for 28 weeks. The diets were identical apart from the type of fat they contained: either (1) a mixture of olive and rapeseed oil, (2) sunflower oil, (3) pork fat, (4) beef fat, or (5) milk fat. The fatty acid composition of the diets was determined and effects on circulating lipid and inflammatory risk factors and atherosclerosis were examined, complemented by adipose tissue histology and liver transcriptomics. While visceral fat mass, adipocyte size, and adipose tissue inflammation were not differentially affected by the diets, atherosclerotic lesion load and severity was more pronounced with increasing dietary saturated fatty acid content and decreasing monounsaturated and polyunsaturated fatty acid content, and hence most pronounced with beef and milk fat. These differential effects were accompanied by increases in pro-atherogenic plasma lipids/lipoproteins (e.g., triglycerides, apolipoprotein B), activation of pro-atherogenic cytokine/chemokine signaling pathways in liver, and with circulating pro-atherogenic mediators of inflammation altogether providing a rationale for the differential effects of plant and animal fats.

Lugol's solution and Gentian violet eradicate methicillin-resistant Staphylococcus aureus biofilm in skin wound infections.

The study aimed to evaluate the antibacterial efficacy of Lugol's solution 5% and Gentian violet 1% against methicillin-resistant Staphylococcus aureus (MRSA) biofilm in vivo. The bactericidal efficacy for treatment of MRSA-biofilm skin wound infection was tested in a murine model. Luciferase-tagged S. aureus Xen31, a MRSA-strain derived from S. aureus ATCC-3359130, was used for infection. Wounds were made in the skin of mice and infected with MRSA. The mice were treated with Lugol's solution and Gentian violet. Application of the antimicrobial agents started 24 hours post infection and was repeated daily for five-days. The antimicrobial effect on the biofilm bacteria was evaluated by measuring bioluminescence from MRSA daily for seven-days. Lugol's solution and Gentian violet showed a significant reduction in luminescent signals from the first assessment day to all subsequent days (P < .001). Lugol's solution and Gentian violet effectively eradicated MRSA in biofilm in vivo and could be alternatives or in addition to topical antibiotics when MRSA-biofilm wound infection is suspected.

A transgenic reporter mouse model for in vivo assessment of retinoic acid receptor transcriptional activation.

Background: Vitamin A is essential for a wide range of life processes throughout embryogenesis to adult life. With the aim of developing an in vivo model to monitor retinoic acid receptor (RAR) transactivation real-time in intact animals, we generated transgenic mice carrying a luciferase (luc) reporter gene under the control of retinoic acid response elements (RAREs) consisting of three copies of a direct repeat with five spacing nucleotides (DR5). Methods: Transgenic mice carrying a RARE dependent luciferase reporter flanked with insulator sequence were generated by pronuclear injection. RARE dependent luciferase activity was detected by in vivo imaging or in tissue extracts following manipulations with RAR/retinoid X receptor (RXR) agonists, RAR antagonists or in vitamin A deficient mice. Results: We found a strong induction of luciferase activity in a time and dose dependent manner by retinoic acid as well as RAR agonists, but not by the RXR agonist (using n=4-6 per group; 94 mice). In addition, luciferase activity was strongly reduced in vitamin A-deficient mice (n=6-9; 30 mice). These observations confirm that luciferase activity was controlled by RAR activation in the RARE-luc mouse. Luciferase activity was detectable in various organs, with high activity especially in brain and testis, indicating strong retinoid signalling in these tissues. Conclusion: The RARE-luc transgenic mice, which enabled real-time in vivo assessment of RAR activation, will be useful in understanding the normal physiology of vitamin A, the role of retinoid signalling in pathologies as well as to evaluate pharmacological ligands for RARs.

State-of-the-art methodological investigation of carotenoid activity and metabolism - from organic synthesis via metabolism to biological activity - exemplified by a novel retinoid signalling pathway.

Carotenoids are the most abundant lipophilic secondary plant metabolites and their dietary intake has been related to a large number of potential health benefits relevant for humans, including even reduced total mortality. An important feature is their potential to impact oxidative stress and inflammatory pathways, by interacting with transcription factors. For example, they may act as precursors of bioactive derivatives activating nuclear hormone receptor mediated signalling. These bioactive derivatives, originating e.g. from ß-carotene, i.e. retinoids / vitamin A, can activate the nuclear hormone receptors RARs (retinoic acid receptors). Due to new analytical insights, various novel metabolic pathways were recently outlined to be mediated via distinct nuclear hormone receptor activating pathways that were predicted and further confirmed. In this article, we describe old and novel metabolic pathways from various carotenoids towards novel ligands of alternative nuclear hormone receptors. However, to fully elucidate these pathways, a larger array of techniques and tools, starting from organic synthesis, lipidomics, reporter models, classical in vitro and in vivo models and further omics-approaches and their statistical evaluation are needed to comprehensively and conclusively study this topic. Thus, we further describe state-of-the-art techniques from A to Ω elucidating carotenoid biological mediated activities and describe in detail required materials and methods needed - in practical protocol form - for the various steps of carotenoid investigations.

Dietary intake of micronized avian eggshell membrane in aged mice reduces circulating inflammatory markers, increases microbiota diversity, and attenuates skeletal muscle aging.

Introduction: Avian eggshell membrane (ESM) is a complex extracellular matrix comprising collagens, glycoproteins, proteoglycans, and hyaluronic acid. We have previously demonstrated that ESM possesses anti-inflammatory properties in vitro and regulates wound healing processes in vivo. The present study aimed to investigate if oral intake of micronized ESM could attenuate skeletal muscle aging associated with beneficial alterations in gut microbiota profile and reduced inflammation. Methods: Elderly male C57BL/6 mice were fed an AIN93G diet supplemented with 0, 0.1, 1, or 8% ESM. Young mice were used as reference. The digestibility of ESM was investigated using the static in vitro digestion model INFOGEST for older people and adults, and the gut microbiota profile was analyzed in mice. In addition, we performed a small-scale pre-clinical human study with healthy home-dwelling elderly (>70 years) who received capsules with a placebo or 500 mg ESM every day for 4 weeks and studied the effect on circulating inflammatory markers. Results and discussion: Intake of ESM in elderly mice impacted and attenuated several well-known hallmarks of aging, such as a reduction in the number of skeletal muscle fibers, the appearance of centronucleated fibers, a decrease in type IIa/IIx fiber type proportion, reduced gene expression of satellite cell markers Sdc3 and Pax7 and increased gene expression of the muscle atrophy marker Fbxo32. Similarly, a transition toward the phenotypic characteristics of young mice was observed for several proteins involved in cellular processes and metabolism. The digestibility of ESM was poor, especially for the elderly condition. Furthermore, our experiments showed that mice fed with 8% ESM had increased gut microbiota diversity and altered microbiota composition compared with the other groups. ESM in the diet also lowered the expression of the inflammation marker TNFA in mice and in vitro in THP-1 macrophages. In the human study, intake of ESM capsules significantly reduced the inflammatory marker CRP. Altogether, our results suggest that ESM, a natural extracellular biomaterial, may be attractive as a nutraceutical candidate with a possible effect on skeletal muscle aging possibly through its immunomodulating effect or gut microbiota.

Bacteriocins Revitalize Non-Effective Penicillin G to Overcome Methicillin-Resistant Staphylococcus pseudintermedius.

The rise of antibiotic-resistant bacteria is among the biggest challenges in human and veterinary medicine. One of the major factors that contributes to resistance is use of frontline clinical antibiotics in veterinary practices. To avoid this problem, searching for antimicrobials aimed at veterinary applications is becoming especially important. Thiopeptide micrococcin P1 and leaderless peptide EntEJ97s are two different bacteriocins that are very active against many gram-positive bacteria; however, sensitive bacteria can rapidly develop resistance towards those bacteriocins. To overcome this problem, we searched for synergy between those bacteriocins and conventional antibiotics against methicillin-resistant Staphylococcus pseudintermedius (MRSP): a common pathogen in animal skin infections. The two bacteriocins acted synergistically with each other and with penicillin G against MRSP clinical isolates in both planktonic and biofilm assays; they also prevented resistance development. The therapeutic potential was further validated in a murine skin infection model that showed that a combination of micrococcin P1, EntEJ97s and penicillin G reduced cell-forming units of MRSP by 2-log10 CFU/g. Taken together, our data show that a combination of bacteriocins with conventional antibiotics can not only prevent resistance development but also pave the way to revitalize some old, less useful antibiotics, such as penicillin, which by itself has no effect on methicillin-resistant pathogens.

Intestinal effect of faba bean fractions in WD-fed mice treated with low dose of DSS.

Rodent studies have shown that legumes can reduce chemical induced colonic inflammation, but the role of faba bean fractions for colon health has not been described. We have investigated the role of protein and fiber fractions of faba beans for colonic health and microbiota composition in a low-grade inflammation mice-model when incorporated in a Western diet (WD). The diet of sixty C57BL/6JRj male mice was standardized to a WD (41% fat, 43% carbohydrates) before were randomly assigned to four groups (n = 12) receiving either 1) WD with 30% of the protein replaced with faba-bean proteins, 2) WD with 7% of the fiber replaced with faba-bean fibers, 3) WD with protein and fiber fractions or 4) plain WD (n = 24). Low-grade inflammation was induced by 1% dextran sodium sulfate (DSS) given to mice for the last six days of the trial. Half (n = 12) in group 4) were given only water (controls). Prior to DSS, body weight, energy intake, glucose and insulin tolerance assays were performed. Inflammatory status in the colon was assessed by biomarkers of inflammation and qRT-PCR analyses of inflammatory related genes. Fecal microbiota composition was assessed by 16S rRNA gene sequencing. 1% DSS treatment increased levels in fecal lipocalin-2 and induced disease activity index score, but the presence of faba bean fractions in WD did not influence these indicators nor the expression level of inflammatory associated genes. However, the mice that had faba-bean proteins had a lower amount of Proteobacteria compared the group on plain WD. The Actinobacteria abundance was also lower in the group that had fiber fraction from faba-beans. Overall, outcomes indicated that in a low-grade inflammation model, replacement of protein and or fiber in a WD with faba bean fractions had marginal effects on inflammatory parameters and colonic microbiota.

The ROS-generating enzyme NADPH oxidase 1 modulates the colonic microbiota but offers minor protection against dextran sulfate sodium-induced low-grade colon inflammation in mice.

The enzyme NADPH oxidase 1 (NOX1) is a major producer of superoxide which together with other reactive oxygen and nitrogen species (ROS/RNS) are implicated in maintaining a healthy epithelial barrier in the gut. While previous studies have indicated NOX1's involvement in microbial modulation in the small intestine, less is known about the effects of NOX1-dependent ROS/RNS formation in the colon. We investigated the role of NOX1 in the colon of NOX1 knockout (KO) and wild type (WT) mice, under mild and subclinical low-grade colon inflammation induced by 1% dextran sulfate sodium (DSS). Ex vivo imaging of ROS/RNS in the colon revealed that absence of NOX1 strongly decreased ROS/RNS production, particularly during DSS treatment. Furthermore, while absence of NOX1 did not affect disease activity, some markers of inflammation (mRNA: Tnfa, Il6, Ptgs2; protein: lipocalin 2) in the colonic mucosa tended to be higher in NOX1 KO than in WT mice following DSS treatment. Lack of NOX1 also extensively modulated the bacterial community in the colon (16S rRNA gene sequencing), where NOX1 KO mice were characterized mainly by lower α-diversity (richness and evenness), higher abundance of Firmicutes, Akkermansia, and Oscillibacter, and lower abundance of Bacteroidetes and Alistipes. Together, our data suggest that NOX1 is pivotal for colonic ROS/RNS production in mice both during steady-state (i.e., no DSS treatment) and during 1% DSS-induced low-grade inflammation and for modulation of the colonic microbiota, with potential beneficial consequences for intestinal health.

Comparison of the Immunogenic Properties of Lactiplantibacillus plantarum Carrying the Mycobacterial Ag85B-ESAT-6 Antigen at Various Cellular Localizations.

The bacille Calmette-Guèrin (BCG) vaccine has been used for a century; nonetheless, tuberculosis (TB) remains one of the deadliest diseases in the world. Thus, new approaches to developing a new, more efficient vaccine are desirable. Mucosal vaccines are of particular interest, considering that Mycobacterium tuberculosis first enters the body through the mucosal membranes. We have previously demonstrated the immunogenicity of a recombinant Lactiplantibacillus plantarum delivery vector with TB hybrid antigen Ag85B-ESAT-6 anchored to the cell membrane. The goal of the present study was to analyze the impact of antigen localization in the immune response. Thus, we assessed two novel vaccine candidates, with the TB antigen either non-covalently anchored to the cell wall (LysMAgE6) or located intracellularly (CytAgE6). In addition, we compared two expression systems, using an inducible (LipoAgE6) or a constitutive promoter (cLipoAgE6) for expression of covalently anchored antigen to the cell membrane. Following administration to mice, antigen-specific CD4+ T-cell proliferation and IFN-γ and IL-17A secretion were analyzed for lung cell and splenocyte populations. Generally, the immune response in lung cells was stronger compared to splenocytes. The analyses showed that the type of expression system did not significantly affect the immunogenicity, while various antigen localizations resulted in markedly different responses. The immune response was considerably stronger for the surface-displaying candidate strains compared to the candidate with an intracellular antigen. These findings emphasize the significance of antigen exposure and further support the potential of L. plantarum as a mucosal vaccine delivery vehicle in the fight against TB.

A High-Fat Western Diet Attenuates Intestinal Changes in Mice with DSS-Induced Low-Grade Inflammation.

BACKGROUND: A Western diet (WD) is associated with increased inflammation in the large intestine, which is often ascribed to the high dietary fat content. Intestinal inflammation in rodents can be induced by oral administration of dextran sodium sulfate (DSS). However, most studies investigating effects of WD and DSS have not used appropriate low-fat diets (LFDs) as control. OBJECTIVES: To compare the effects of a WD with those of an LFD on colon health in a DSS-induced low-grade colonic inflammation mouse model. METHODS: Six-week-old male C57BL/6JRj mice were fed an LFD (fat = 10.3% energy, n = 24) or a WD (fat = 41.2% energy, n = 24) for 15 wk [Experiment 1 (Exp.1)]. Half the mice on each diet (n = 12) then received 1% DSS in water for 6 d with the remainder (n = 12 in each diet) administered water. Disease activity, proinflammatory genes, inflammatory biomarkers, and fecal microbiota (16S rRNA) were assessed (Exp.1). Follow-up experiments (Exp.2 and Exp.3) were performed to investigate whether fat source (milk or lard; Exp.2) affected outcomes and whether a shift from LFD to WD 1 d prior to 1% DSS exposure caused an immediate effect on DSS-induced inflammation (Exp.3). RESULTS: In Exp.1, 1% DSS treatment significantly increased disease score in the LFD group compared with the WD group (2.7 compared with 0.8; P < 0.001). Higher concentrations of fecal lipocalin (11-fold; P < 0.001), proinflammatory gene expression (≤82-fold), and Proteobacteria were observed in LFD-fed mice compared with the WD group. The 2 fat sources in WDs (Exp.2) revealed the same low inflammation in WD+DSS mice compared with LFD+DSS mice. Finally, the switch from LFD to WD just before DSS exposure resulted in reduced colonic inflammation (Exp.3). CONCLUSIONS: Herein, WDs (with milk or lard) protected mice against DSS-induced colonic inflammation compared with LFD-fed mice. Whether fat intake induces protective mechanisms against DSS-mediated inflammation or inhibits establishment of the DSS-induced colitis model is unclear.

Naturalizing laboratory mice by housing in a farmyard-type habitat confers protection against colorectal carcinogenesis.

Living in a farm environment in proximity to animals is associated with reduced risk of developing allergies and asthma, and has been suggested to protect against other diseases, such as inflammatory bowel disease and cancer. Despite epidemiological evidence, experimental disease models that recapitulate such environments are needed to understand the underlying mechanisms. In this study, we show that feralizing conventional inbred mice by continuous exposure to a livestock farmyard-type environment conferred protection toward colorectal carcinogenesis. Two independent experimental approaches for colorectal cancer induction were used; spontaneous (Apc Min/+ mice on an A/J background) or chemical (AOM/DSS). In contrast to conventionally reared laboratory mice, the feralized mouse gut microbiota structure remained stable and resistant to mutagen- and colitis-induced neoplasia. Moreover, the feralized mice exhibited signs of a more mature immunophenotype, indicated by increased expression of NK and T-cell maturation markers, and a more potent IFN-γ response to stimuli. In our study, hygienically born and raised mice subsequently feralized post-weaning were protected to a similar level as life-long exposed mice, although the greatest effect was seen upon neonatal exposure. Collectively, we show protective implications of a farmyard-type environment on colorectal cancer development and demonstrate the utility of a novel animal modeling approach that recapitulates realistic disease responses in a naturalized mammal.

Hybrid hydrogels for bacteriocin delivery to infected wounds.

Superficial infections in chronic wounds can prevent the wound healing process by the development of persistent infections and drug-resistant biofilms. Topically applied antimicrobial formulations with stabilized and controlled release offer significant benefits for the effective treatment of wound infections. Bacteriocins are the antimicrobial peptides (AMPs) produced by bacteria that are viable alternatives to antibiotics owing to their natural origin and low propensity for resistance development. Herein, we developed a hybrid hydrogel composed of Pluronic F127 (PF127), ethylenediaminetetraacetic acid (EDTA) loaded liposomes, glutathione (GSH), and the bacteriocin Garvicin KS (GarKS) referred to as "GarKS gel". The GarKS gel exhibited suitable viscosity and rheological properties along with controlled release behavior (up to 9 days) for effective peptide delivery following topical application. Potent in vitro antibacterial and anti-biofilm effects of GarKS gel were evident against the Gram-positive bacterium Staphylococcus aureus. The in vivo treatment of methicillin resistant S. aureus (MRSA) infected mouse wounds suggested potent antibacterial effects of the GarKS gel following multiple applications of once-a-day application for three consecutive days. Altogether, these results provide proof-of-concept for the successful development of AMP loaded topical formulation for effective treatment of wound infections.

Induction of colorectal carcinogenesis in the C57BL/6J and A/J mouse strains with a reduced DSS dose in the AOM/DSS model.

BACKGROUND: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide and thus mouse models of CRC are of significant value to study the pathogenesis. The Azoxymethane/Dextran sulfate sodium (AOM/DSS) model is a widely used, robust initiation-promotion model for chemical induction of colitis-associated CRC in rodents. However, the dosage of chemicals, treatment regimens and outcome measures vary greatly among studies employing this model. Thus, the aim of this study was to examine an AOM/DSS model involving a reduced (1%) dose of DSS for induction of carcinogenesis in A/J and C57BL/6J (B6) mice. RESULTS: We show that colonic preneoplastic lesions can be reliably detected in A/J and B6 mice by use of a AOM/DSS model involving a single injection of 10 mg/kg AOM followed by three 7-day cycles of a low-dose (1%) DSS administration. Supporting existing evidence of A/J mice exhibiting higher susceptibility to AOM than B6 mice, our AOM/DSS-treated A/J mice developed the highest number of large colonic lesions. Clinical symptoms in both strains subjected to the AOM/DSS treatment did not persist in-between treatment cycles, demonstrating that the animals tolerated the treatment well. CONCLUSIONS: Our findings suggest that a reduced dose of DSS in the AOM/DSS model can be considered in future studies of early phase colorectal carcinogenesis in the A/J and B6 mouse strains using preneoplastic lesions as an outcome measure, and that such regimen may reduce the risk of early trial terminations to accommodate human endpoints. Overall, our data emphasize the importance of devoting attention towards choice of protocol, outcome measures and mouse strain in studies of CRC in mice according to the study purpose.

A Strong Synergy Between the Thiopeptide Bacteriocin Micrococcin P1 and Rifampicin Against MRSA in a Murine Skin Infection Model.

Antibiotic-resistant bacterial pathogens have become a serious threat worldwide. One of these pathogens is methicillin-resistant Staphylococcus aureus (MRSA), a major cause of skin and soft tissue infections. In this study we identified a strain of Staphylococcus equorum producing a substance with high antimicrobial activity against many Gram-positive bacteria, including MRSA. By mass spectrometry and whole genome sequencing the antimicrobial substance was identified as the thiopeptide bacteriocin micrococcin P1 (MP1). Based on its properties we developed a one-step purification protocol resulting in high yield (15 mg/L) and high purity (98%) of MP1. For shorter incubation times (5-7 h) MP1 was very potent against MRSA but the inhibitory effect was overshadowed by resistance development during longer incubation time (24h or more). To overcome this problem a synergy study was performed with a number of commercially available antibiotics. Among the antibiotics tested, the combination of MP1 and rifampicin gave the best synergistic effect, with MIC values 25 and 60 times lower than for the individual drugs, respectively. To assess the therapeutic potential of the MP1-rifampicin combination, we used a murine skin infection model based on the use of the multidrug-resistant luciferase-tagged MRSA strain Xen31. As expected, neither of the single antimicrobials (MP1 or rifampicin) could eradicate Xen31 from the wounds. By contrary, the MP1-rifampicin combination was efficient not only to eradicate but also to prevent the recurrence of Xen31 infection. Furthermore, compared to fucidin cream, which is commonly used in skin infection treatments, MP1-rifampicin combination was superior in terms of preventing resistance development. Our results show that combining MP1, and probably other thiopeptides, with antibiotics can be a promising strategy to treat SSTIs caused by MRSA and likely many other Gram-positive bacteria.

Exploring the Therapeutic Potenital of the Leaderless Enterocins K1 and EJ97 in the Treatment of Vancomycin-Resistant Enterococcal Infection.

The membrane-bound protease Eep is an important virulence factor in pathogenic enterococci. The protein is involved in stress response via the RIP pathway which is crucial for pathogenic enterococci to evade host immune attacks during infection. Eep serves also as a receptor for the bacteriocins enterocin K1 and enterocin EJ97. The bacteriocins kill Enterococcus faecium and E. faecalis, respectively, and their antibiotic resistant derivatives including vancomycin resistant enterococci (VRE). This functional duality of Eep makes these two enterocins very promising as options in the prospective treatment of enterococcal infections because wildtype enterococcal cells (with an intact Eep) are sensitive to the bacteriocins while bacteriocin-resistant-mutants (without a functional Eep) become less virulent. As a first step to explore their therapeutic potential in the treatment of systemic enterococcal infections, we investigated the compatibility of the bacteriocins with human blood, and the phenotypic changes of eep-mutants toward different stress conditions. We found that the bacteriocins were compatible with blood, as they did not cause haemolysis and that the bacteriocins retained most of their antibacterial effect when incubated in blood. The bacteriocins were autoclavable which is a crucial criterium for the development of parenteral administration. Eep-mutants, which became resistant to the bacteriocin were, as expected, less capable to withstand stress conditions such as exposure to lysozyme and desiccation. Further, their ability to chain, a trait implicated in niche adaptation as well as being necessary for genetic transfer via conjugation, was also severely affected. Together, these results indicate that the bacteriocins are promising for treatment of VRE infection.