Corrigendum: Cinnamaldehyde Inhibits Staphylococcus aureus Virulence Factors and Protects against Infection in a Galleria mellonella Model.

[This corrects the article DOI: 10.3389/fmicb.2016.02052.].

A Study of the Disruptive Effect of the Acetate Fraction of Punica granatum Extract on Cryptococcus Biofilms.

Cryptococcosis, caused by yeasts of the genus Cryptococcus, is an infectious disease with a worldwide distribution. Cryptococcus neoformans and Cryptococcus gattii are the species that commonly cause this disease in humans; however, infections caused by Cryptococcus laurentii, especially in immunocompromised patients, are increasingly being reported. Owing to the increase in the resistance of fungi to antifungals, and a lack of treatment options, it is important to seek new therapeutic alternatives such as natural products. Among these are plant species such as Punica granatum, which is used in folk medicine to treat various diseases. This study aimed to evaluate the activity of the acetate fraction of P. granatum leaf extract against environmental and clinical isolates of Cryptococcus. Three environmental isolates of C. laurentii, PMN, PMA, and PJL II, isolated from soils of different municipalities in the state of Maranhão, a clinical isolate, C. gattii, from a patient with neurocryptococcosis, and a standard strain of C. gattii (ATCC 32068) were used. The minimum and fractional inhibitory concentrations (MIC and FIC, respectively) and time-kill curve of the extract and fluconazole were determined to assess the susceptibility profile of the fungal isolates. Larvae of Tenebrio molitor were infected with Cryptococcus strains, and the effects of acetate fraction of P. granatum extract and fluconazole on the survival and fungal burden were determined. The extract activity was tested against pre-formed biofilms. The acetate fraction of P. granatum extract showed promising antifungal activity against all the species of Cryptococcus evaluated in this study, with an MIC value lower than that of fluconazole. The indices obtained in the FIC test indicated that the antimicrobial effect of the combination of the extract and antifungal was indifferent for 80% of the isolates. The P. granatum acetate fraction reduced the pre-formed biofilm of some isolates, showing better activity than fluconazole, which is consistent with results from fluorescence microscopy. This is the first study on the use of P. granatum and its ability to inhibit Cryptococcus biofilms; therefore, further studies and tests are needed to investigate the components and mechanism of action of P. granatum against cryptococcosis agents.

Dioclea violacea lectin modulates the gentamicin activity against multi-resistant strains and induces nefroprotection during antibiotic exposure.

Gentamicin is an aminoglycoside antibiotic used to treat infections of various origins. In the last few decades, the constant use of gentamicin has resulted in increased bacterial resistance and nephrotoxicity in some cases. In this study, we examined the ability of Dioclea violacea lectin (DVL) in modulate the antimicrobial activity of gentamicin and reduce the nephrotoxicity induced by this drug. The minimum inhibitory concentration (MIC) obtained for DVL against all strains studied was not clinically relevant (MIC ≥ 1024 µg/mL). However, when DVL was combined with gentamicin, a significant increase in antibiotic action was observed against Staphylococcus aureus and Escherichia coli. DVL also reduced antibiotic tolerance in S. aureus during 10 days of continuous treatment. In addition, DVL presented a nephroprotective effect, reducing sodium excretion, N-Gal expression and urinary protein, that are important markers of glomerular and tubular injuries. Taken together, studies of inhibition of hemagglutinating activity, fluorescence spectroscopy and molecular docking revealed that gentamicin can interact with DVL via the carbohydrate recognition domain (CRD), suggesting that the results obtained in this study may be directly related to the interaction of DVL-gentamicin and with the ability of the lectin to interact with glycans present in the cells of the peritoneum.

Phylogenetic and Molecular Profile of Staphylococcus aureus Isolated from Bloodstream Infections in Northeast Brazil.

Staphylococcus aureus is a notorious human pathogen associated with serious nosocomial and community-acquired infections, such as pneumonia, meningitis, endocarditis, toxic shock syndrome, and sepsis, among others. The objective of this study was to investigate the molecular profile, antimicrobial resistance, and clonal diversity of S. aureus isolated from the bloodstream. The determination of the minimum inhibitory concentration (MIC) of the antimicrobial was performed by an automated method. The presence of several virulence and resistance genes was evaluated by PCR. In addition, multilocus sequence typing (MLST) was used to analyze the clonal diversity of S. aureus. A high resistance to oxacillin (78%), clindamycin (78%), erythromycin (70%), ciprofloxacin (61%), and gentamicin (52%) was observed among the isolates. In most of them, the following virulence genes were detected: hlb (83%), ebpS (61%), icaA (57%), fnbpA (17%), and clfA (13%). Only one isolate carried the pvl gene. MLST analysis identified five new sequence types (STs): 5429, 5430, 5431, 5432, and 5433, as well as another seven-ST5, ST97, ST398, ST101, ST30, ST461, and ST2779-among the remaining strains. These seven STs and the four new STs are clustered in four clonal complexes: CC1, CC2, CC7, and CC17. Phylogenetic analysis showed the genetic relationship of the five new ST strains with another 18 strains. Altogether, these analyses indicate the horizontal transfer acquisition of virulence factor genes and multidrug resistance.

Biotechnological Production of Statins: Metabolic Aspects and Genetic Approaches.

Statins are drugs used for people with abnormal lipid levels (hyperlipidemia) and are among the best-selling medications in the United States. Thus, the aspects related to the production of these drugs are of extreme importance for the pharmaceutical industry. Herein, we provide a non-exhaustive review of fungal species used to produce statin and highlighted the major factors affecting the efficacy of this process. The current biotechnological approaches and the advances of a metabolic engineer to improve statins production are also emphasized. The biotechnological production of the main statins (lovastatin, pravastatin and simvastatin) uses different species of filamentous fungi, for example Aspergillus terreus. The statins production is influenced by different types of nutrients available in the medium such as the carbon and nitrogen sources, and several researches have focused their efforts to find the optimal cultivation conditions. Enzymes belonging to Lov class, play essential roles in statin production and have been targeted to genetic manipulations in order to improve the efficiency for Lovastatin and Simvastatin production. For instance, Escherichia coli strains expressing the LovD have been successfully used for lovastatin production. Other examples include the use of iRNA targeting LovF of A. terreus. Therefore, fungi are important allies in the fight against hyperlipidemias. Although many studies have been conducted, investigations on bioprocess optimization (using both native or genetic- modified strains) still necessary.

Use of Some Asteraceae Plants for the Treatment of Wounds: From Ethnopharmacological Studies to Scientific Evidences.

Severe wounds result in large lesions and/or loss of function of the affected areas. The treatment of wounds has challenged health professionals due to its complexity, especially in patients with chronic diseases (such as diabetes), and the presence of pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Taking this into consideration, the development of new therapies for wound healing requires immediate attention. Ethnopharmacological studies performed in different countries have shown the use of several plants from the Asteraceae family as wound-healing agents. Evidences gained from the traditional medicine have opened new ways for the development of novel and more efficient therapies based on the pharmacological properties of these plants. In this article, we discuss the literature data on the use of Asteraceae plants for the treatment of wounds, based on the ethnopharmacological relevance of each plant. Special attention was given to studies showing the mechanisms of action of Asteraceae-derived compounds and clinical trials. Ageratina pichinchensis (Kunth) R.M. King and H. Rob. and Calendula officinalis L. preparations/compounds were found to show good efficacy when assessed in clinical trials of complicated wounds, including venous leg ulcers and foot ulcers of diabetic patients. The compounds silibinin [from Silybum marianum (L.) Gaertn.] and jaceosidin (from Artemisia princeps Pamp.) were identified as promising compounds for the treatment of wounds. Overall, we suggest that Asteraceae plants represent important sources of compounds that may act as new and efficient healing products.

Transient Receptor Potential Canonical Channels 4 and 5 Mediate Escherichia coli-Derived Thioredoxin Effects in Lipopolysaccharide-Injected Mice.

Thioredoxin plays an essential role in bacterial antioxidant machinery and virulence; however, its regulatory actions in the host are less well understood. Reduced human Trx activates transient receptor potential canonical 5 (TRPC5) in inflammation, but there is no evidence of whether these receptors mediate bacterial thioredoxin effects in the host. Importantly, TRPC5 can form functional complexes with other subunits such as TRPC4. Herein, E. coli-derived thioredoxin induced mortality in lipopolysaccharide- (LPS-) injected mice, accompanied by reduction of leukocyte accumulation, regulation of cytokine release into the peritoneum, and impairment of peritoneal macrophage-mediated phagocytosis. Dual TRPC4/TRPC5 blockade by ML204 increased mortality and hypothermia in thioredoxin-treated LPS mice but preserved macrophage's ability to phagocytose. TRPC5 deletion did not alter body temperature but promoted additional accumulation of peritoneal leukocytes and inflammatory mediator release in thioredoxin-administered LPS mice. Thioredoxin diminished macrophage-mediated phagocytosis in wild-type but not TRPC5 knockout animals. TRPC5 ablation did not affect LPS-induced responses. However, ML204 caused mortality associated with exacerbated hypothermia and decreased peritoneal leukocyte numbers and cytokines in LPS-injected mice. These results suggest that bacterial thioredoxin effects under LPS stimuli are mediated by TRPC4 and TRPC5, shedding light on the additional mechanisms of bacterial virulence and on the pathophysiological roles of these receptors.

Eugenol Induces Phenotypic Alterations and Increases the Oxidative Burst in Cryptococcus.

Eugenol is a phenolic compound and the main constituent of the essential oil of clove India. Although there are reports of some pharmacological effects of eugenol, this study is the first that proposes to evaluate the antifungal effects of this phenol against both Cryptococcus gattii and C. neoformans cells. The effect of eugenol against yeast cells was analyzed for drug susceptibility, alterations in cell diameter, capsule properties, amounts of ergosterol, oxidative burst, and thermodynamics data. Data demonstrated that there is no interaction between eugenol and fluconazole and amphotericin B. Eugenol reduced the cell diameter and the capsule size, increased cell surface/volume, changed positively the cell surface charge of cryptococcal cells. We also verified increased levels of reactive oxygen species without activation of antioxidant enzymes, leading to increased lipid peroxidation, mitochondrial membrane depolarization and reduction of lysosomal integrity in cryptococcal cells. Additionally, the results showed that there is no significant molecular interaction between eugenol and C. neoformans. Morphological alterations, changes of cellular superficial charges and oxidative stress play an important role in antifungal activity of eugenol against C. gattii and C. neoformans that could be used as an auxiliary treatment to cutaneous cryptococcosis.

Targeting the Immune System with Plant Lectins to Combat Microbial Infections.

The arsenal of drugs available to treat infections caused by eukaryotic and prokaryotic microbes has been declining exponentially due to antimicrobial resistance phenomenon, leading to an urgent need to develop new therapeutic strategies. Host-directed immunotherapy has been reported as an attractive option to treat microbial infections. It consists in the improvement of host defenses by increasing the expression of inflammatory mediators and/or controlling of inflammation-induced tissue injury. Although the in vitro antimicrobial and immunomodulatory activities of lectins have been extensively demonstrated, few studies have evaluated their in vivo effects on experimental models of infections. This review aims to highlight the experimental use of immunomodulatory plant lectins to improve the host immune response against microbial infections. Lectins have been used in vivo both prophylactically and therapeutically resulting in the increased survival of mice under microbial challenge. Other studies successfully demonstrated that lectins could be used in combination with parasite antigens in order to induce a more efficient immunization. Therefore, these plant lectins represent new candidates for management of microbial infections. Furthermore, immunotherapeutic studies have improved our knowledge about the mechanisms involved in host-pathogen interactions, and may also help in the discovery of new drug targets.

Healing activity evaluation of the galactomannan film obtained from Cassia grandis seeds with immobilized Cratylia mollis seed lectin.

Galactomannan films from Cassia grandis seeds, associated or not with Cramoll 1,4, were used on topical wounds of rats for the evaluation of the healing process during 14days. All of the films were evaluated by cytotoxic assay, FTIR and lectin hemagglutinating activity (HA). Forty-five male rats were submitted to aseptic dermal wounds (Ø=0.8cm) and divided in groups (n=15): control, test 1, and test 2, treated respectively with saline, galactomannan film and film with immobilized Cramoll 1,4. Macroscopic evaluations were performed by clinical observations and area measurements, and microscopic analysis by histological criteria. Epithelial cell proliferation and differentiation was immunohistochemically assessed using CK14 and PCNA. The presence of CO peaks in the FTIR spectrum confirmed the immobilization of Cramoll 1,4 in the film, while the residual HA confirmed the stability of the lectin after immobilization with 90.94% of the initial HA. The films presented non-cytotoxicity and cell viability exceeding 80%. All of the animals presented re-epithelization around 10days, furthermore test 2 group showed a diffuse response at the stromal tissue and the basal layer associated with wounds completely closed with 11days of experiment. The results suggest a promising use of the films as topical wound curatives.

Application of Omics Technologies for Evaluation of Antibacterial Mechanisms of Action of Plant-Derived Products.

In the face of increasing bacterial resistance to antibiotics currently in use, the search for new antimicrobial agents has received a boost in recent years, with natural products playing an important role in this field. In fact, several methods have been proposed to investigate the antibacterial activities of natural products. However, given that the ultimate aim is future therapeutic use as novel drugs, it is extremely necessary to elucidate their modes of action, stating the molecular effects in detail, and identifying their targets in the bacterial cell. This review analyzes the application of "omics technologies" to understand the antibacterial mechanisms of bioactive natural products, to stimulate research interest in this area and promote scientific collaborations. Some studies have been specifically highlighted herein by examining their procedures and results (targeted proteins and metabolic pathways). These approaches have the potential to provide new insights into our comprehension of antimicrobial resistance/susceptibility, creating new perspectives for the struggle against bacteria, and leading to the development of novel products in the future.

Encapsulation into Stealth Liposomes Enhances the Antitumor Action of Recombinant Cratylia mollis Lectin Expressed in Escherichia coli.

This study evaluated the in vivo antitumor potential of the recombinant lectin from seeds of Cratylia mollis (rCramoll) expressed in Escherichia coli, free or encapsulated in stealth liposomes, using mice transplanted with sarcoma 180. rCramoll-loaded stealth liposomes (rCramoll-lipo) were formulated by hydration of the lipid film followed by cycles of freezing and thawing, and about 60% of rCramoll was encapsulated. This novel preparation showed particle size, polydispersity index, and pH suitable for the evaluation of antitumor activity in vivo. Tumor growth inhibition rates were 59% for rCramoll and 75% for rCramoll-lipo. Histopathological analysis of the experimental groups showed that both free and encapsulated lectin caused no changes in the kidneys of animals. Hematological analysis revealed that treatment with rCramoll-lipo significantly increased leukocyte concentration when compared with the untreated and rCramoll group. In conclusion, the encapsulation of rCramoll in stealth liposomes improves its antitumor activity without substantial toxicity; this approach was more successful than the previous results reported for pCramoll loaded into conventional liposomes. At this point, a crucial difference between the antitumor action of free and encapsulated rCramoll was found along with their effects on immune cells. Further investigations are required to elucidate the mechanism(s) of the antitumor effect induced by rCramoll.

Cinnamaldehyde Inhibits Staphylococcus aureus Virulence Factors and Protects against Infection in a Galleria mellonella Model.

Bacterial resistance to the available marketed drugs has prompted the search of novel therapies; especially in regards of anti-virulence strategies that aim to make bacteria less pathogenic and/or decrease their probability to become resistant to therapy. Cinnamaldehyde is widely known for its antibacterial properties through mechanisms that include the interaction of this compound with bacterial cell walls. However, only a handful of studies have addressed its effects on bacterial virulence, especially when tested at sub-inhibitory concentrations. Herein, we show for the first time that cinnamaldehyde is bactericidal against Staphylococcus aureus and Enterococcus faecalis multidrug resistant strains and does not promote bacterial tolerance. Cinnamaldehyde actions were stronger on S. aureus as it was able to inhibit its hemolytic activity on human erythrocytes and reduce its adherence to latex. Furthermore, cinnamaldehyde enhanced the serum-dependent lysis of S. aureus. In vivo testing of cinnamaldehyde in Galleria mellonella larvae infected with S. aureus, showed this compound improves larvae survival whilst diminishing bacterial load in their hemolymph. We suggest that cinnamaldehyde may represent an alternative therapy to control S. aureus-induced bacterial infections as it presents the ability to reduce bacterial virulence/survival without promoting an adaptive phenotype.