Eugenia uniflora (pitanga) juice as a new alternative vehicle for Limosilactobacillus fermentum ATCC 23271: evaluation of antioxidant and anti-infective effects.

Probiotic-containing foods are among the most appreciated functional foods; however, probiotic-based dairy products cannot be consumed by people who are lactose intolerant, allergic to milk, or vegetarian or vegan individuals. Thus, new non-dairy matrices have been tested for probiotics delivery. This study evaluated the growth and viability of Limosilactobacillus fermentum ATCC 23271 and Lacticaseibacillus rhamnosus ATCC 9595 in Pitanga juice (Eugenia uniflora L.). The effects of the fermentation on the antioxidant and anti-infective properties of the juice were also analyzed. The E. uniflora juice allowed lactobacilli growth without supplementation, reaching rates around 8.4 Log CFU/mL and producing organic acids (pH values < 4) after 72 h of fermentation. The strain remained viable after 35 days of refrigerated storage. Fermentation by these bacteria increases the antioxidant capacity of the juice. The central composite rotational design was employed to evaluate the effects of bacterial inoculum and pulp concentration on growth and organic acids production by L. fermentum ATCC 23271. The strain was viable and produced organic acids in all tested combinations. L. fermentum-fermented juice and its cell-free supernatant significantly increased the survival of Tenebrio molitor larvae infected by enteroaggregative Escherichia coli 042. The results obtained in this study provide more insights into the potential of Pitanga juice to develop a functional non-dairy probiotic beverage with antioxidant and anti-infective properties.

The essential oil of Melaleuca alternifolia incorporated into hydrogel induces antimicrobial and anti-inflammatory effects on infected wounds by Staphylococcus aureus.

Staphylococcus aureus is one of the most common bacterial isolates found in wounds. Thus, innovative dressings, such as hydrogels, are interesting vehicles for incorporating bioactive compounds like those from Melaleuca alternifolia essential oil (MaEO). In this study, we evaluated the antimicrobial and anti-inflammatory potential of MaEO incorporated into an alginate and chitosan hydrogel for treating wounds infected by S. aureus. The hydrogel incorporated with MaEO 1% (HMa 1%) was homogeneous with a bright pale-yellow color and the characteristic smell of Melaleuca. The incorporation of MaEO 1% does not affect the stability of the hydrogel, which was stable up to 90 days of storage. The Scanning electron microscopy analysis revealed that hydrogels showed irregular surfaces and interconnected porous structures with accumulations of oil crystals distributed throughout the formulation. HMa 1% has a high moisture content (95.1%) and can absorb simulated wound fluid. Regarding the antimicrobial effects, HMa 1% reduced the growth of S. aureus ATCC 6538 in both in vitro conditions and in an ex vivo model of wounds using porcine skin. In addition, the dairy topical treatment of murine skin lesions with HMa 1% induced a significant reduction of the wound area, inflammation score, and bacterial load, as well as tissue re-epithelialization and modulation of inflammatory mediators. Therefore, hydrogel incorporated with MaEO 1% has excellent potential to be used in the pharmacotherapy of infected wounds.

The Potential of Lactiplantibacillus plantarum ATCC 14917 in the Development of Alginate-Based Gel Formulations with Anti-Staphylococcus aureus Properties.

This study aimed to evaluate the potential of lactic acid bacteria (LAB) in developing alginate-based gel formulations to inhibit Staphylococcus aureus. Initially, the antagonistic actions of three lactic acid bacteria (LAB) (Lacticaseibacillus rhamnosus ATCC 10863, Lactiplantibacillus plantarum ATCC 14917, Limosilactobacillus fermentum ATCC 23271) were evaluated against S. aureus ATCC 25923. All tested LAB inhibited S. aureus, but the highest activity was observed for L. plantarum ATCC 14917 (p < 0.05). The antimicrobial effects of L. plantarum ATCC 14917 cell suspensions, sonicate cells extract, and cell-free supernatants (pH 5 or 7) were analyzed using a broth-based assay. The cell suspensions inhibited S. aureus at concentrations ≥ 10%, and these effects were confirmed by a time-kill assay. Alginate-based gels were formulated with cell suspensions, sonicate cells extract, and cell-free supernatant (pH 5). These formulations inhibited S. aureus growth. Based on the results, the alginate gel with cell suspensions at 10% was selected for further characterization. L. plantarum ATCC 14917 survived in the alginate-based gel, especially when stored at 5 °C. At this temperature, the L. plantarum-containing alginate gel was stable, and it was in compliance with microbiological standards. These findings suggest it can be a promising agent for the topical treatment of infections induced by S. aureus.

Immunomodulatory Effects of Cinnamaldehyde in Staphylococcus aureus-Infected Wounds.

Cinnamaldehyde (CNM) is an essential-oil component with reported anti-infective, anti-inflammatory, and healing effects, making it an interesting compound for the treatment of wound infection. Herein, we evaluated the effects of topical administration of CNM in experimental wounds infected by Staphylococcus aureus. Swiss mice (n = 12/group) were randomly allocated into three groups (CON: animals with uninfected lesions; Sa: animals with untreated infected lesions; Sa + CNM: animals with infected wounds and treated with CNM). Excisional lesions (64 mm2) were induced at the dorsal area followed by the addition of S. aureus (80 µL of a 1.5 × 108 CFU/mL bacterial suspension). The wounds were treated with CNM (200 µg/wound/day) or vehicle (2% DMSO) for 10 days. Skin samples were taken on the 3rd or 10th treatment day for quantification of inflammatory mediators, bacterial load, immunophenotyping, and histological analysis. The treatment with CNM improved the healing process and attenuated the severity of skin lesions infected by S. aureus. These effects were associated with significant decreases in bacterial loads in CNM-treated wounds. The levels of neutrophils, TNF-α, IL-6, NO, and VEGF were decreased in the lesions treated with CNM. Taken together, these data provide further evidence of the effectiveness of CNM for the treatment of skin infections.

Short-Term Intake of Theobroma grandiflorum Juice Fermented with Lacticaseibacillus rhamnosus ATCC 9595 Amended the Outcome of Endotoxemia Induced by Lipopolysaccharide.

Endotoxemia is a condition caused by increasing levels of lipopolysaccharide (LPS) characterized by an impaired systemic response that causes multiple organ dysfunction. Lacticaseibacillus rhamnosus ATCC 9595 is a strain with probiotic potential which shows immunomodulatory properties. The incorporation of this bacterium in food rich in bioactive compounds, such as cupuaçu juice (Theobroma grandiflorum), could result in a product with interesting health properties. This work evaluated the effects of the oral administration of cupuaçu juice fermented with L. rhamnosus on the outcome of LPS-induced endotoxemia in mice. C57BL/6 mice (12/group) received oral doses (100 µL) of saline solution and unfermented or fermented cupuaçu juice (108 CFU/mL). After 5 days, the endotoxemia was induced by an intraperitoneal injection of LPS (10 mg/kg). The endotoxemia severity was evaluated daily using a score based on grooming behavior, mobility, presence of piloerection, and weeping eyes. After 6 h and 120 h, the mice (6/group) were euthanized for analysis of cell counts (in peritoneal lavage and serum) and organ weight. L. rhamnosus grew in cupuaçu juice and produced organic acids without the need for supplementation. The bacteria counts were stable in the juice during storage at 4 °C for 28 days. The fermentation with L. rhamnosus ATCC 9595 changed the metabolites profile of cupuaçu juice due to the biotransformation and enhancement of some compounds. In general, the administration of L. rhamnosus-fermented juice allowed a significant improvement in several characteristics of endotoxemic status (weight loss, hypothermia, severity index, cell migration). In addition, treatment with fermented juice significantly reduced the weight of the spleen, liver, intestine, and kidneys compared to the saline-treated endotoxemic group. Taken together, our data show that short-term intake therapy of cupuaçu juice fermented with L. rhamnosus ATCC 9595 can reduce systemic inflammation in an experimental model of LPS-induced endotoxemia in mice.

Schinus terebinthifolius Leaf Lectin (SteLL) Reduces the Bacterial and Inflammatory Burden of Wounds Infected by Staphylococcus aureus Promoting Skin Repair.

Staphylococcus aureus is commonly found in wound infections where this pathogen impairs skin repair. The lectin isolated from leaves of Schinus terebinthifolius (named SteLL) has antimicrobial and antivirulence action against S. aureus. This study evaluated the effects of topical administration of SteLL on mice wounds infected by S. aureus. Seventy-two C57/BL6 mice (6−8 weeks old) were allocated into four groups: (i) uninfected wounds; (ii) infected wounds, (iii) infected wounds treated with 32 µg/mL SteLL solution; (iv) infected wounds treated with 64 µg/mL SteLL solution. The excisional wounds (64 mm2) were induced on the dorsum and infected by S. aureus 432170 (4.0 × 106 CFU/wound). The daily treatment started 1-day post-infection (dpi). The topical application of both SteLL concentrations significantly accelerated the healing of S. aureus-infected wounds until the 7th dpi, when compared to untreated infected lesions (reductions of 1.95−4.55-fold and 1.79−2.90-fold for SteLL at 32 µg/mL and 64 µg/mL, respectively). The SteLL-based treatment also amended the severity of wound infection and reduced the bacterial load (12-fold to 72-fold for 32 µg/mL, and 14-fold to 282-fold for 64 µg/mL). SteLL-treated wounds show higher collagen deposition and restoration of skin structure than other groups. The bacterial load and the levels of inflammatory markers (IL-6, MCP-1, TNF-α, and VEGF) were also reduced by both SteLL concentrations. These results corroborate the reported anti-infective properties of SteLL, making this lectin a lead candidate for developing alternative agents for the treatment of S. aureus-infected skin lesions.

Antimicrobial and anti-inflammatory effects of Eugenia brejoensis essential oil in mice wounds infected by Staphylococcus aureus.

Eugenia brejoensis Mazine (Myrtaceae) is source of an essential oil (EbEO) with anti-infective activities against Staphylococcus aureus. This study evaluated the antimicrobial and anti-inflammatory potentials of EbEO in S. aureus-infected skin wounds. The excisional lesions (64 mm2) were induced on Swiss mice back (6 to 8-week-old) that were allocated into 3 groups (n = 12): 1) non-infected wounds (CON); 2) wounds infected with S. aureus ATCC 6538 (Sa); 3) S. aureus-infected wounds and treated with EbEO (Sa + EbEO). The infected groups received approximately 104 CFU/wound. The animals were treated with EbEO (10 µg/wound/day) or vehicle from the 1-day post-infection (dpi) until the 10th dpi. The clinical parameters (wound area, presence of exudate, edema intensity, etc.) were daily analyzed. The levels of inflammatory mediators (cytokines, nitric oxide, VEGF) and bacterial load were measured at the cutaneous tissue at 4th dpi and 10th dpi. Topical application of EbEO accelerated wound contraction with an average contraction of 83.48 ± 11.27 % of the lesion area until 6th dpi. In this period, the rates of lesion contraction were 54.28 ± 5.57% and 34.5 ± 2.67% for CON and Sa groups, respectively. The positive effects of EbEO on wound contraction were associated with significantly (p < 0.05) reduction on bacterial load and the release of inflammatory mediators (IL-6, IL-17A, TNF-α, NO and VEGF). Taken together, these data confirm the antimicrobial potential of EbEO and provide insights into its anti-inflammatory effects, making this essential oil an interesting candidate for the development of new therapeutic alternatives for infected cutaneous wounds.

Immunomodulatory and anti-infective effects of Cratylia mollis lectin (Cramoll) in a model of wound infection induced by Staphylococcus aureus.

This work evaluated the immunomodulatory and anti-infective effects of Cratylia mollis lectin (Cramoll) in a model of wound infection induced by S. aureus. Swiss mice were divided into 3 groups (n = 12/group): non-inoculated (Control group); inoculated with S. aureus (Sa group); inoculated with S. aureus and treated with Cramoll (Sa + Cramoll group). In each animal, one lesion (64 mm2) was induced on the back and contaminated with S. aureus (~4.0 × 106 CFU/wound). The treatment with Cramoll (5 µg/animal/day) started 1-day post-infection (dpi) and extended for 10 days. Clinical parameters (wound size, inflammatory aspects, etc.) were daily recorded; while cytokines levels, bacterial load and histological aspects were determined in the cutaneous tissue at 4th dpi or 11th dpi. The mice infected with S. aureus exhibited a delay in wound contraction and the highest inflammatory scores. These effects were impaired by the treatment with Cramoll which reduced the release of key inflammatory mediators (TNF-α, NO, VEGF) and the bacterial load at wound tissue. Histological evaluations showed a restauration of skin structures in the animals treated with Cramoll. Taken together, these results provide more insights about the healing and immunomodulatory properties of Cramoll and suggest this lectin as a lead compound for treatment of wound infection.

Interplay between ESKAPE Pathogens and Immunity in Skin Infections: An Overview of the Major Determinants of Virulence and Antibiotic Resistance.

The skin is the largest organ in the human body, acting as a physical and immunological barrier against pathogenic microorganisms. The cutaneous lesions constitute a gateway for microbial contamination that can lead to chronic wounds and other invasive infections. Chronic wounds are considered as serious public health problems due the related social, psychological and economic consequences. The group of bacteria known as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter sp.) are among the most prevalent bacteria in cutaneous infections. These pathogens have a high level of incidence in hospital environments and several strains present phenotypes of multidrug resistance. In this review, we discuss some important aspects of skin immunology and the involvement of ESKAPE in wound infections. First, we introduce some fundamental aspects of skin physiology and immunology related to cutaneous infections. Following this, the major virulence factors involved in colonization and tissue damage are highlighted, as well as the most frequently detected antimicrobial resistance genes. ESKAPE pathogens express several virulence determinants that overcome the skin's physical and immunological barriers, enabling them to cause severe wound infections. The high ability these bacteria to acquire resistance is alarming, particularly in the hospital settings where immunocompromised individuals are exposed to these pathogens. Knowledge about the virulence and resistance markers of these species is important in order to develop new strategies to detect and treat their associated infections.

Polysaccharide-Based Formulations for Healing of Skin-Related Wound Infections: Lessons from Animal Models and Clinical Trials.

Skin injuries constitute a gateway for pathogenic bacteria that can be either part of tissue microbiota or acquired from the environmental. These microorganisms (such as Acinetobacter baumannii, Enterococcus faecalis,Pseudomonas aeruginosa, and Staphylococcus aureus) produce virulence factors that impair tissue integrity and sustain the inflammatory phase leading for establishment of chronic wounds. The high levels of antimicrobial resistance have limited the therapeutic arsenal for combatting skin infections. Thus, the treatment of non-healing chronic wounds is a huge challenge for health services worldwide, imposing great socio-economic damage to the affected individuals. This scenario has encouraged the use of natural polymers, such as polysaccharide, in order to develop new formulations (membranes, nanoparticles, hydrogels, scaffolds) to be applied in the treatment of skin infections. In this non-exhaustive review, we discuss the applications of polysaccharide-based formulations in the healing of infected wounds in animal models and clinical trials. The formulations discussed in this review were prepared using alginate, cellulose, chitosan, and hyaluronic acid. In addition to have healing actions per se, these polysaccharide formulations can act as transdermal drug delivery systems, controlling the release of active ingredients (such as antimicrobial and healing agents). The papers show that these polysaccharides-based formulations are efficient in controlling infection and improve the healing, even in chronic infected wounds. These data should positively impact the design of new dressings to treat skin infections.