Insights into chitosan antibiofilm activity against methicillin-resistant Staphylococcus aureus.

AIMS: Chitosan is a natural compound that has been validated as a viable antimicrobial agent against Staphylococcus aureus. With this work we sought to evaluate the planktonic and sessile sensitivity of methicillin-resistant S. aureus to chitosan's activity and evaluate if methicillin-resistant S. aureus (MRSA) would be more or less sensitive to chitosan's activity than methicillin-sensitive S. aureus (MSSA). METHODS AND RESULTS: A group comprised of reference strains and clinical multiresistant isolates of MSSA and MRSA were used. Methicilin resistance effect upon chitosan activity was assessed in planktonic setting and in different phases of sessile colonization, namely adhesion, biofilm formation and mature biofilm through biomass and metabolism inhibition. The results obtained showed that S. aureus methicillin resistance mechanism did not impair chitosan's activity as the highest bacterial susceptibility was registered for MRSA. Chitosan was highly effective in inhibiting MSSA and MRSA strains in both planktonic and sessile settings with biofilm inhibition percentages reaching as high as 90% for MRSA. CONCLUSIONS: Staphylococcus aureus methicillin resistance did not impair chitosan's antimicrobial and antibiofilm activities and MRSA and MSSA were inhibited both in planktonic and sessile settings at low concentrations with great efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the obtained results chitosan shows potential as an alternative for the control of biofilm-related recalcitrant MRSA infections.

Topical application of probiotics in skin: adhesion, antimicrobial and antibiofilm in vitro assays.

AIMS: When skin dysbiosis occurs as a result of skin disorders, probiotics can act as modulators, restoring microbial balance. Several properties of selected probiotics were evaluated so that their topical application could be considered. METHODS AND RESULTS: Adhesion, antimicrobial, quorum sensing and antibiofilm assays were carried out with several probiotic strains and tested against selected skin pathogens. All tested strains displayed significant adhesion to keratin. All lactobacilli with the exception of Lactobacillus delbrueckii, showed antimicrobial activity against skin pathogens, mainly due to organic acid production. Most of them also prevented biofilm formation, but only Propioniferax innocua was able to break down mature biofilms. CONCLUSIONS: This study demonstrates that although all tested probiotics adhered to human keratin, they showed limited ability to prevent adhesion of some potential skin pathogens. Most of the tested probiotics successfully prevented biofilm formation, suggesting that they may be successfully used in the future as a complement to conventional therapies in the treatment of a range of skin disorders. SIGNIFICANCE AND IMPACT OF STUDY: The topically used probiotics may be a natural, targeted treatment approach to several skin disorders and a complement to conventional therapies which present many undesirable side effects.

A diversity profile from the staphylococcal community on atopic dermatitis skin: a molecular approach.

AIMS: The aim of this study was to determine the biodiversity of the skin staphylococcal community from patients with atopic dermatitis (AD) and superantigen (SAg) detection from Staphylococcus aureus isolates. METHODS AND RESULTS: In this study, we developed a novel multiplex PCR that allows the identification and discrimination of bacteria belonging to the Staphylococcus genus both Staph. aureus and coagulase-negative Staphylococcus - Staph. capitis, Staph. epidermidis, Staph. haemolyticus and Staph. hominis isolated from the skin of patients with AD. In addition, a multiplex PCR assay that allows the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs), SE-like toxins and toxic shock syndrome toxin-1 was also performed and applied in Staph. aureus isolates. The microflora of the skin of patients with AD was dominated by Staph. aureus (69 isolates, 35·6%) followed by Staph. epidermidis (59 isolates, 30·4%) species. The SElM and SElN genes were the most frequently detected in our study (15 isolates, 71·4%), followed by SEG and SElO (14 isolates, 66·7%). CONCLUSIONS: Our molecular-based approach successfully identified the staphylococcal microflora that was relatively specific to patients with AD. Considering skin colonization and expression of virulence factors, the Staph. aureus may play a relevant role in AD pathophysiology. SIGNIFICANCE AND IMPACT OF THE STUDY: This ability to classify disease-related microbial species provides new insights into the relevance of those microbes in human disorders.

Study of the effects of chitosan upon Streptococcus mutans adherence and biofilm formation.

The main aim of this work was to access the potential use of high and low molecular weight chitosans as potential oral antimicrobials, particularly as antibiofilm agents. Chitosan's interference with Streptococcus mutans capability to adhere and form biofilms was assessed. Additionally the effect upon mature and polymicrobial biofilms was also evaluated. The results obtained showed that chitosan was capable of interfering with S. mutans adhesion and primary biofilm formation. This action was observed up to a week with little to none decrease in efficiency. In addition chitosan was capable of inhibiting biofilms formed by two microorganisms and was capable of acting upon mature biofilms leading to significant reductions (94%) in biofilm survival. However clear statistical differences (p < 0.05) were registered in all assays with, in most assays, HMw chitosan presenting higher efficiency than LMw chitosan. Considering this results chitosan's potential as a valid alternative to traditional antimicrobials in oral health it's evident.

A systematic review of natural products for skin applications: Targeting inflammation, wound healing, and photo-aging.

BACKGROUND: Every day the skin is constantly exposed to several harmful factors that induce oxidative stress. When the cells are incapable to maintain the balance between antioxidant defenses and reactive oxygen species, the skin no longer can keep its integrity and homeostasis. Chronic inflammation, premature skin aging, tissue damage, and immunosuppression are possible consequences induced by sustained exposure to environmental and endogenous reactive oxygen species. Skin immune and non-immune cells together with the microbiome are essential to efficiently trigger skin immune responses to stress. For this reason, an ever-increasing demand for novel molecules capable of modulating immune functions in the skin has risen the level of their development, particularly in the field of natural product-derived molecules. PURPOSE: In this review, we explore different classes of molecules that showed evidence in modulate skin immune responses, as well as their target receptors and signaling pathways. Moreover, we describe the role of polyphenols, polysaccharides, fatty acids, peptides, and probiotics as possible treatments for skin conditions, including wound healing, infection, inflammation, allergies, and premature skin aging. METHODS: Literature was searched, analyzed, and collected using databases, including PubMed, Science Direct, and Google Scholar. The search terms used included "Skin", "wound healing", "natural products", "skin microbiome", "immunomodulation", "anti-inflammatory", "antioxidant", "infection", "UV radiation", "polyphenols", "polysaccharides", "fatty acids", "plant oils", "peptides", "antimicrobial peptides", "probiotics", "atopic dermatitis", "psoriasis", "auto-immunity", "dry skin", "aging", etc., and several combinations of these keywords. RESULTS: Natural products offer different solutions as possible treatments for several skin conditions. Significant antioxidant and anti-inflammatory activities were reported, followed by the ability to modulate immune functions in the skin. Several membrane-bound immune receptors in the skin recognize diverse types of natural-derived molecules, promoting different immune responses that can improve skin conditions. CONCLUSION: Despite the increasing progress in drug discovery, several limiting factors need future clarification. Understanding the safety, biological activities, and precise mechanisms of action is a priority as well as the characterization of the active compounds responsible for that. This review provides directions for future studies in the development of new molecules with important pharmaceutical and cosmeceutical value.

Chitosan: antimicrobial action upon staphylococci after impregnation onto cotton fabric.

BACKGROUND: High levels of viable Staphylococcus aureus, which are often found on inflamed skin surfaces, are usually associated with atopic dermatitis. Textiles, owing to their high specific surface area and intrinsic hydrophilicity, retain moisture while also providing excellent environmental conditions for microbial growth and proliferation. Recently, a number of chemicals have been added to textiles, so as to confer antimicrobial activity. AIMS: To evaluate the antimicrobial action of chitosan upon selected skin staphylococci. METHODS AND RESULTS: We isolated staphylococci from normal skin of 24 volunteers and studied their survival upon contact with chitosan-impregnated cotton fabric. Low and high molecular weight chitosans were used at two concentrations; all four did effectively reduce the growth of some staphylococci (namely Staph. aureus), by up to 5 log cycles, thus unfolding a potential towards control and even prevention of related skin disorders. CONCLUSION: Our data suggest an effective, but selective antibacterial action of chitosans towards skin bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The possibility to use a natural biopolymer incorporated in a textile to alleviate and even treat some of the symptoms associated with this skin condition may raise an alternative to existing medical treatments. The selectivity observed prevents full elimination of bacteria from the skin surface, which is an advantage.

Evaluation and insights into chitosan antimicrobial activity against anaerobic oral pathogens.

The objective of this study was to assess the antimicrobial capability of non-chemically altered chitosan as an alternative to traditional antimicrobials used in the treatment of oral infections. The action mechanism of chitosan was also ascertained. High and low molecular weight chitosan showed antimicrobial activity at low concentrations for all tested bacteria with the MICs varying between 1 and 7 mg/ml with a drop of efficacy relatively to the action of LMW chitosan. In addition chitosan showed also to be an effective bactericidal presenting bactericidal effect within 8 h at the latest. Additionally the evaluation of chitosan's action mechanism showed that both MWs acted upon the bacterial cell wall and were not capable of interacting with the intracellular substances, as showed by the inefficacy obtained in the flocculation assay.

Probiotic growth in skin-like conditions.

Although probiotics' main known effects are in the digestive system, over the last years several benefits that come from their topical use, have been investigated. Several studies have reported beneficial effects on different skin disorders, such as atopic dermatitis, acne, eczema, psoriasis, wound healing, skin aging and reactive skin. Their main action is assigned to the inhibition of skin colonization by pathogens. In this work, the growths of three probiotic strains were evaluated in the presence of abiotic factors similar to those found in skin, namely, UV radiation, temperature, pH, NaCl and fatty acids. Lactobacillus rhamnosus showed increased growth under the pH of 6, but no differences in its growth were found for the various NaCl concentrations tested. Lactobacillus delbrueckii increased the number of bacterial cells in 88.8% when grown in 10 mM NaCl concentration, while Propioniferax innocua showed increased growth at 45 °C. All tested probiotic bacteria were able to grow under skin-like conditions. However, L. rhamnosus was the probiotic that showed the best results. The results obtained in this study indicate that the used probiotics may be beneficial in the treatment of skin diseases, since they are able to successfully thrive in skin-like conditions.

Chitosan as an effective inhibitor of multidrug resistant Acinetobacter baumannii.

Over the last two decades worldwide levels of antibiotic resistance have risen leading to the appearance of multidrug resistant microorganisms. Acinetobacter baumannii is a known skin pathogen which has emerged as a major cause of nosocomial outbreaks due to its capacity to colonize indwelling medical devices and natural antibiotic resistance. With chitosan being an effective antimicrobial agent against antibiotic resistant microorganisms, the aim of this work was to access its potential as an alternative to traditional antimicrobials in the management of A. baumannii growth. What the results showed was that both chitosan MW's tested were active upon A. baumannii's planktonic and sessile growth. For planktonic growth MICs and MBCs were obtained at relatively low concentrations (0.5-2mg/mL) while for sessile growth chitosan proved to be an effective inhibitor of A. baumannii's adhesion and biofilm formation. Considering these results chitosan shows a high potential for control of A. baumannii infections.

Chitosan nanoparticles as alternative anti-staphylococci agents: Bactericidal, antibiofilm and antiadhesive effects.

Chitosan is a biocompatible, bioactive, non-toxic polymer that due to these characteristics has been widely used as a carrier for targeted delivery of bioactive molecules. In recent years, and considering that chitosan has a strong antimicrobial potential, the scientific community's focus has shifted onto the possible antimicrobial activity of chitosan nanoparticles. With this in mind, the aim of this work was to produce low molecular weight chitosan nanoparticles, through the ionic gelation method and characterize their potential biological activity against three staphylococci (MSSA, MRSA and MRSE) in planktonic and sessile environments. The chitosan nanoparticles produced had an average size of 244±12nm, an average charge of 17.3±1.4mV and had a MIC of 1.25mg/mL for all tested microorganisms. Bactericidal activity was only registered for MSSA and MRSA with the time-inhibition curves showing bactericidal activity within 1h. Assays regarding chitosan nanoparticles' impact upon sessile populations showed that they were effective in preventing MRSE adhesion and highly effective in reducing MRSA and MSSA biofilm formation.

Microbiological characterization of Serra da Estrela cheese throughout its Appellation d'Origine Protégée region.

The purpose of this study was to assess the typical microbiological quality of the most famous Portuguese traditional cheese, Serra da Estrela, and to assess its ripening time and geographical dependence. Ninety-six experimental cheeses manufactured from sixteen batches of milk on eight dairy farms scattered over the Appellation d' Origine Protégée (AOP) region were qualitatively and quantitatively evaluated microbiologically at various ripening times. Viable counts were performed after inoculation on appropriate selective media for aerobic mesophiles and proteolytic and lipolytic microflora, as well as lactococci, lactobacilli, species of Enterobacteriaceae, lactic streptococci, staphylococci, and yeasts. Members of the Enterobacteriaceae and lactic acid bacteria were the predominant microbial groups on all dairy farms throughout maturation; the latter are probably the microbial group responsible for most proteolytic and lipolytic breakdown in Serra da Estrela cheese. The microbial groups whose numbers were most affected by dairy-to-dairy variation were species of Enterobacteriaceae staphylococci, and enterococci, which are the most critical groups in terms of health hazards. It is therefore suggested that tighter control should be implemented at the level of choice of raw materials, in milk-handling practices, and in general throughout the manufacturing process in attempts to standardize production and consistently reduce microbiological risks (even though the distinctiveness of a few final organoleptic characteristics may somehow be reduced.

A comprehensive study into the impact of a chitosan mouthwash upon oral microorganism's biofilm formation in vitro.

Modern dentistry emphasizes the importance of dental plaque control to improve oral health. To that end the development of oral care formulations has been geared toward the incorporation of antiplaque agents that may play a crucial role in oral health maintenance. In later years the research into antiplaque agents has led to the discovery of compounds with significant capability to affect biofilm formation. Among these compounds was chitosan, a polysaccharide which showed great ability to interfere with Streptococcus mutans biofilm formation. As such the aim of this work was to incorporate chitosan into a mouthwash matrix and assess its effect upon biofilm formation of oral microorganisms. This assessment was performed via study of the impact the mouthwash upon microbial adherence, biofilm formation and mature biofilms. Additionally, the action of the chitosan mouthwash was compared with two commercially available mouthwashes. The results here obtained show that only the chitosan containing mouthwash was capable of interfering with all microorganisms' adherence, biofilm formation and mature biofilms while at the same time showing vastly superior activity than both commercial mouthwashes assayed. As such a chitosan mouthwash shows great potential as a natural and efficient alternative to traditional mouthwashes.

Chitosan mouthwash: toxicity and in vivo validation.

A previous study showed that a chitosan mouthwash would be a valid alternative to current mouthwashes as it demonstrated, in vitro, significantly higher antibiofilm activity than two commercial mouthwashes. As such, the aim of this work was to verify the safety of the developed product and to validate, in vivo, the biological activity ascertained in vitro. Chitosan mouthwash safety was evaluated through Ames, MTT and V79 chromosomal aberration assay while antimicrobial activity was evaluated through in vivo assays. The results showed that the chitosan mouthwash was safe, presenting lower cytotoxicity than a commercial mouthwash, and that it effectively reduced viable counts of Streptococcus spp. and Enterococcus spp. by ca. 5.5 log of CFU. Furthermore, in direct comparison with a commercial mouthwash the chitosan mouthwash possessed significantly higher antimicrobial activity. The conjunction of these results proves that the chitosan mouthwash is a safe, effective, natural alternative to the existent chemical mouthwashes.

A novel direct contact method for the assessment of the antimicrobial activity of dental cements.

Dental cements are a crucial part of the odontological treatment, however, due to the hazardous nature and reduced biological efficiency of some of the used materials, newer and safer alternatives are needed, particularly so those possessing higher antimicrobial activity than their traditional counterparts. The evaluation of the antimicrobial properties of solid and semi-solid antimicrobials, such as dental cements and gels, is challenging, particularly due to the low sensitivity of the current methods. Thus, the main aim of this study was the evaluation of the antimicrobial activity of a novel chitosan containing dental cement while simultaneous assessing/validating a new, more efficient, method for the evaluation of the antimicrobial activity of solid and gel like materials. The results obtained showed that the proposed method exhibited a higher sensitivity than the standard 96 well microtiter assay and allowed the determination of bactericidal activity. Additionally, it is interesting to note that the chitosan containing cement, which presented higher antimicrobial activity than the traditional zinc oxide/eugenol mix, was capable of inducing a viable count reduction above 5 log of CFU for all of the studied microorganisms.

Volatile free fatty acids as ripening indicators for Serra da Estrela cheese.

Serra da Estrela cheese is an artisanal cheese manufactured from raw ewe's milk coagulated with Cynara cardunculus; it is highly appreciated for its unique flavor and bouquet. This research effort focused on a search for the molecules responsible for those organoleptic characteristics. Eighty cheeses manufactured in 4 dairies located in the Appelation d'Origine Protegee region of Serra da Estrela, Portugal, were thus assayed for volatiles, in an attempt to characterize their odorous profile. Cheeses were analyzed from the time of manufacture up to 180 d of ripening. The volatile fraction was assayed by solid phase microextraction-gas chromatography/mass spectroscopy, and several compounds belonging to different chemical groups (e.g., fatty acids, esters, carbonyl compounds, pyrazines, and sulfur compounds) were detected in that traditional cheese. Among these, free fatty acids (FFA) were quantitatively the dominant family present. Furthermore, sensory descriptors for the typical aroma of this cheese included acidic, sweaty, and sheepy-like. Acetic, isobutyric, and isovaleric acids increased in concentration during the ripening process up to 90 d, and remained constant thereafter. Preliminary sensory analysis was performed by addition of the major FFA to an unripened cheese matrix; results showed that they could successfully be used as ripening indicators for this cheese. Such key molecules may thus be used to monitor ripening, and hence find the optimal consumption time for this gourmet dairy product.

Amino acid catabolism and generation of volatiles by lactic acid bacteria.

Twelve isolates of lactic acid bacteria, belonging to the Lactobacillus, Lactococcus, Leuconostoc, and Enterococcus genera, were previously isolated from 180-d-old Serra da Estrela cheese, a traditional Portuguese cheese manufactured from raw milk and coagulated with a plant rennet. These isolates were subsequently tested for their ability to catabolize free amino acids, when incubated independently with each amino acid in free form or with a mixture thereof. Attempts were made in both situations to correlate the rates of free amino acid uptake with the numbers of viable cells. When incubated individually, leucine, valine, glycine, aspartic acid, serine, threonine, lysine, glutamic acid, and alanine were degraded by all strains considered; arginine tended to build up, probably because of transamination of other amino acids. When incubated together, the degradation of free amino acids by each strain was dependent on pH (with an optimum pH around 6.0). The volatiles detected in ripened Serra da Estrela cheese originated mainly from leucine, phenylalanine, alanine, and valine, whereas in vitro they originated mainly from valine, phenylalanine, serine, leucine, alanine, and threonine. The wild strains tested offer a great potential for flavor generation, which might justify their inclusion in a tentative starter/nonstarter culture for that and similar cheeses.