Deletion of codY impairs Staphylococcus epidermidis biofilm formation, generation of viable but non-culturable cells and stimulates cytokine production in human macrophages.

Introduction. Staphylococcus epidermidis biofilms are one of the major causes of bloodstream infections related to the use of medical devices. The diagnosis of these infections is challenging, delaying their treatment and resulting in increased morbidity and mortality rates. As such, it is urgent to characterize the mechanisms employed by this bacterium to endure antibiotic treatments and the response of the host immune system, to develop more effective therapeutic strategies. In several bacterial species, the gene codY was shown to encode a protein that regulates the expression of genes involved in biofilm formation and immune evasion. Additionally, in a previous study, our group generated evidence indicating that codY is involved in the emergence of viable but non-culturable (VBNC) cells in S. epidermidis.Gap statement/Hypothesis. As such, we hypothesized that the gene codY has have an important role in this bacterium virulence.Aim. This study aimed to assess, for the first time, the impact of the deletion of the gene codY in S. epidermidis virulence, namely, in antibiotic susceptibility, biofilm formation, VBNC state emergence and in vitro host immune system response.Methodology. Using an allelic replacement strategy, we constructed and then characterized an S. epidermidis strain lacking codY, in regards to biofilm and VBNC cell formation, susceptibility to antibiotics as well as their role in the interaction with human blood and plasma. Additionally, we investigate whether the codY gene can impact the activation of innate immune cells by evaluating the production of both pro- and anti-inflammatory cytokines by THP-1 macrophages.Results. We demonstrated that the deletion of the gene codY resulted in biofilms with less c.f.u. counts and fewer VBNC cells. Furthermore, we show that although WT and mutant cells were similarly internalized in vitro by human macrophages, a stronger cytokine response was elicited by the mutant in a toll-like receptor 4-dependent manner.Conclusion. Our results indicate that codY contributes to S. epidermidis virulence, which in turn may have an impact on our ability to manage the biofilm-associated infections caused by this bacterium.

Nanostructured Antimicrobials for Quality and Safety Improvement in Dairy Products.

In the food sector, one of the most important economic activities is the dairy industry, which has been facing many challenges in order to meet the increasing demand by consumers for natural and minimally processed products with high quality. In this sense, the application of innovative and emerging technologies can be an interesting alternative, for example, the use of nanotechnology in packaging and as delivery systems. This technology has the potential to improve the quality and safety of dairy products, representing an interesting approach for delivering food preservatives and improving the mechanical, barrier and functional properties of packaging. Several applications and promising results of nanostructures for dairy product preservation can be found throughout this review, including the use of metallic and polymeric nanoparticles, lipid-based nanostructures, nanofibers, nanofilms and nanocoatings. In addition, some relevant examples of the direct application of nanostructured natural antimicrobials in milk and cheese are presented and discussed, as well as the use of milk agar as a model for a preliminary test. Despite their high cost and the difficulties for scale-up, interesting results of these technologies in dairy foods and packaging materials have promoted a growing interest of the dairy industry.

codY and pdhA Expression Is Induced in Staphylococcus epidermidis Biofilm and Planktonic Populations With Higher Proportions of Viable but Non-Culturable Cells.

Staphylococcus epidermidis biofilm cells can enter a physiological state known as viable but non-culturable (VBNC), where, despite being alive, they do not grow in conventional laboratory media. As such, the presence of VBNC cells impacts the diagnosis of S. epidermidis biofilm-associated infections. Previous transcriptomics analysis of S. epidermidis strain 9142 biofilms with higher proportions of VBNC cells suggested that the genes pdhA, codY and mazEF could be involved in the induction of the VBNC state. However, it was previously demonstrated that VBNC induction is strain-dependent. To properly assess the role of these genes in VBNC induction, the construction of mutant strains is necessary. Thus, herein, we assessed if VBNC cells could be induced in strain 1457, a strain amenable to genetic manipulation, and if the previously identified genes were involved in the modulation of the VBNC state in this strain. Furthermore, we evaluated the formation of VBNC cells on planktonic cultures. Our results showed that despite being commonly associated with biofilms, the proportion of VBNC cells can be modulated in both biofilm and planktonic cultures and that the expression of codY and pdhA was upregulated under VBNC inducing conditions in both phenotypes. Overall, our study revealed that the formation of VBNC cells in S. epidermidis is independent of the mode of growth and that the genes codY and pdhA seem to be relevant for the regulation of this physiological condition.

Lipid-Based Nanostructures for the Delivery of Natural Antimicrobials.

Encapsulation can be a suitable strategy to protect natural antimicrobial substances against some harsh conditions of processing and storage and to provide efficient formulations for antimicrobial delivery. Lipid-based nanostructures, including liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid nanocarriers (NLCs), are valuable systems for the delivery and controlled release of natural antimicrobial substances. These nanostructures have been used as carriers for bacteriocins and other antimicrobial peptides, antimicrobial enzymes, essential oils, and antimicrobial phytochemicals. Most studies are conducted with liposomes, although the potential of SLNs and NLCs as antimicrobial nanocarriers is not yet fully established. Some studies reveal that lipid-based formulations can be used for co-encapsulation of natural antimicrobials, improving their potential to control microbial pathogens.

Virulence Factors in Coagulase-Negative Staphylococci.

Coagulase-negative staphylococci (CoNS) have emerged as major pathogens in healthcare-associated facilities, being S. epidermidis, S. haemolyticus and, more recently, S. lugdunensis, the most clinically relevant species. Despite being less virulent than the well-studied pathogen S. aureus, the number of CoNS strains sequenced is constantly increasing and, with that, the number of virulence factors identified in those strains. In this regard, biofilm formation is considered the most important. Besides virulence factors, the presence of several antibiotic-resistance genes identified in CoNS is worrisome and makes treatment very challenging. In this review, we analyzed the different aspects involved in CoNS virulence and their impact on health and food.

Modulation of volatile release and antimicrobial properties of pink pepper essential oil by microencapsulation in single- and double-layer structured matrices.

The bioactivity of essential oils applied in foods to act as natural preservatives can be reduced due to interactions with other components of the food matrix. Microencapsulation can help to increase the functionality of these compounds. In addition, the electrostatic interaction between proteins and polysaccharides can result in double-layered encapsulating structures, ensuring greater protection to essential oils than using only protein as surface active agent. In this work, pink pepper essential oil was microencapsulated by spray drying of single-layer emulsions, stabilized by soy protein isolate (SPI), and of double-layer emulsions, stabilized by soy protein isolate/high methoxyl pectin (SPI/HMP). Pink pepper essential oil showed predominance of α-pinene, ß-pinene, ß-mircene, δ-3-carene, d-limonene, and germacrene D. Compared to SPI microcapsules, SPI/HMP microcapsules better preserved the total volatile content identified in pure oil, showed less water adsorption during storage at relative humidity ≥75% and improved antimicrobial properties. When stored for 20 days (25 °C/RH = 52.8%), both microcapsules allowed more gradual release of volatiles compared with non-encapsulated oil. Microencapsulation by spray drying did not have negative effects on the antioxidant activity of the encapsulated oil, as the microcapsules showed similar results to the non-encapsulated oil, around 11 µg Trolox/mg of oil. After storage, however, the non-encapsulated oil showed greater losses of its antioxidant activity due to higher rates of volatile release. In the in vitro antimicrobial activity assay, both microcapsules inhibited growth of Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes and Listeria innocua, although no inhibition was observed against Gram-negative bacteria. When added in milk, both microcapsules reduced bacterial growth, whereas non-encapsulated oil showed no satisfactory inhibition. Faster reduction of microbial growth in milk was observed for SPI/HMP microcapsules. Inhibition results were better for skim milk than for whole milk, suggesting that the interaction of essential oil with other lipids present in milk decreased its bioactivity. Microencapsulation positively affected the functionality of pink pepper essential oil, highlighting its potential for application as a natural preservative in food products.

Comparative analysis between biofilm formation and gene expression in Staphylococcus epidermidis isolates.

AIM: To understand the relationship between ica, aap and bhp gene expression and the implications in biofilm formation in selected clinical and commensal Staphylococcus epidermidis isolates. MATERIAL & METHODS: Isolates were analyzed regarding their biofilm-forming capacity, biochemical matrix composition, biofilm spatial organization and expression of biofilm-related genes. RESULTS: On polysaccharide intercellular adhesin-dependent biofilms, aap and bhp contributions for the biofilm growth were negligible, despite very high levels of expression. In contrast, smaller increases in icaA expression contributed significantly to biofilm growth. Interestingly, no biological differences were observed between clinical and commensal strains. CONCLUSION: These results reinforce the concept that S. epidermidis is an 'accidental pathogen,' and that the ica operon is the main mechanism of biofilm formation in clinical and commensal isolates.

Nanostructures for delivery of natural antimicrobials in food.

Natural antimicrobial compounds are a topic of utmost interest in food science due to the increased demand for safe and high-quality foods with minimal processing. The use of nanostructures is an interesting alternative to protect and delivery antimicrobials in food, also providing controlled release of natural compounds such as bacteriocins and antimicrobial proteins, and also for delivery of plant derived antimicrobials. A diversity of nanostructures are capable of trapping natural antimicrobials maintaining the stability of substances that are frequently sensitive to food processing and storage conditions. This article provides an overview on natural antimicrobials incorporated in nanostructures, showing an effective antimicrobial activity on a diversity of food spoilage and pathogenic microorganisms.

Different cell disruption methods for obtaining carotenoids by Sporodiobolus pararoseus and Rhodothorula mucilaginosa.

Since carotenoids are synthesized inside the cell, it is desirable to find an efficient method to extract carotegenic pigments. This study aimed at comparing the effectiveness of different chemical and mechanical techniques to disrupt the cell wall of Sporidiobolus pararoseus and Rhodotorula mucilaginosa yeasts isolated from environmental samples. Among the techniques under study, the ultrasonic bath and the abrasion with glass beads yielded the most promising results for S. pararoseus (84.8 ± 2.3 and 76.9 ± 2.1 µg/g, respectively). The ultrasonic bath yielded the highest specific concentration of carotenoids for R. mucilaginosa (193.5 ± 25.8 µg/g), while the biomass freezing process improved neither the extractability nor the specific concentration of carotenoids. Lyophilization increased the specific concentrations of carotenoids from S. pararoseus and R. mucilaginosa by 20 and 13.7%, respectively, while the freezing process did not significantly affect (p > 0.05) the recovery of carotenoids from both yeasts; thus, it may be eliminated from the process.

Mycological analysis of ambrosia and creamy dulce de leche / Análise micológica de ambrosia e doce de leite pastoso

Creamy dulce de leche is a product made from concentrated milk by heating, and adding sucrose. Ambrosia is a variety of dulce de leche prepared by adding eggs and sugar in to milk. Aiming at identifying the genera of filamentous fungi isolated from creamy dulce de leche and ambrosia, 40 ambrosia and 20 creamy dulce deleche samples were analyzed in the present study. Filamentous fungi and yeast counts and the identification of genera of the isolated filamentous fungi were performed. Thirty-nine samples (65 per cent) showed filamentous fungi and yeast counts that exceeded the accep table limits. Six fungus genera were identified, and amongthem the Penicillium was the most frequent. These findings are a warning of the requirement for strict control on good manufacturing practices as well as on the inspection and monitoring of ambrosia andcreamy dulce de leche trading.