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Camila Folle,1 Ana M Marqués,2 Natalia Díaz-Garrido,3– 5 Paulina Carvajal-Vidal,1 Elena Sánchez López,1,6 Joaquim Suñer-Carbó,1,6 Lyda Halbaut,1,6 Mireia Mallandrich,1,6 Marta Espina,1,6 Josefa Badia,3– 5 Laura Baldoma,3– 5 Maria Luisa García,1,6 Ana Cristina Calpena1,6 1Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona, Spain; 2Department of Biology, Healthcare and Environment, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona, Spain; 3Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona, Spain; 4Institute of Biomedicine of the University of Barcelona (IBUB), Barcelona, Spain; 5Research Institute Sant Joan De Déu (IR‑SJD), Barcelona, Spain; 6Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona, Barcelona, SpainCorrespondence: Camila Folle; Elena Sánchez López, Department of Pharmacy and Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona, 08028, Spain, Email camilafolle@ub.edu; esanchezlopez@ub.eduPurpose: Acne vulgaris is one of the most prevalent dermal disorders affecting skin health and appearance. To date, there is no effective cure for this pathology, and the majority of marketed formulations eliminate both healthy and pathological microbiota. Therefore, hereby we propose the encapsulation of an antimicrobial natural compound (thymol) loaded into lipid nanostructured systems to be topically used against acne.Methods: To address this issue, nanostructured lipid carriers (NLC) capable of encapsulating thymol, a natural compound used for the treatment of acne vulgaris, were developed either using ultrasonication probe or high-pressure homogenization and optimized using 22-star factorial design by analyzing the effect of NLC composition on their physicochemical parameters. These NLC were optimized using a design of experiments approach and were characterized using different physicochemical techniques. Moreover, short-term stability and cell viability using HaCat cells were assessed. Antimicrobial efficacy of the developed NLC was assessed in vitro and ex vivo.Results: NLC encapsulating thymol were developed and optimized and demonstrated a prolonged thymol release. The formulation was dispersed in gels and a screening of several gels was carried out by studying their rheological properties and their skin retention abilities. From them, carbomer demonstrated the capacity to be highly retained in skin tissues, specifically in the epidermis and dermis layers. Moreover, antimicrobial assays against healthy and pathological skin pathogens demonstrated the therapeutic efficacy of thymol-loaded NLC gelling systems since NLC are more efficient in slowly reducing C. acnes viability, but they possess lower antimicrobial activity against S. epidermidis, compared to free thymol.Conclusion: Thymol was successfully loaded into NLC and dispersed in gelling systems, demonstrating that it is a suitable candidate for topical administration against acne vulgaris by eradicating pathogenic bacteria while preserving the healthy skin microbiome. Keywords: lipid nanoparticles, thymol, acne vulgaris, NLC, gels, antimicrobial

The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. In this regard, murepavadin is a cyclic antibacterial peptide in development. The synthesis of murepavadin was undertaken in order to optimize the preparative protocol and scale-up, in particular, the use of new activation reagents. In our hands, classical approaches using carbodiimide/hydroxybenzotriazole rendered low yields. The use of novel carbodiimide and reagents based on OxymaPure ® and Oxy-B is discussed together with the proper use of chromatographic conditions for the adequate characterization of peptide crudes. Higher yields and purities were obtained. Finally, the antimicrobial activity of different synthetic batches was tested in three Pseudomonas aeruginosa strains, including highly resistant ones. All murepavadin batches yielded the same highly active MIC values and proved that the chiral integrity of the molecule was preserved throughout the whole synthetic procedure.

Thyme oil (THO) possesses excellent antibacterial and antioxidant properties which are suitable for skin inflammatory disorders such as acne vulgaris. However, THO is insoluble in water and its components are highly volatile. Therefore, these drawbacks may be overcome by its encapsulation in biodegradable PLGA nanoparticles (THO-NPs) that had been functionalized using several strategies. Moreover, cell viability was studied in HaCat cells, confirming their safety. In order to assess therapeutic efficacy against acne, bacterial reduction capacity and antioxidant properties were assessed. Moreover, the anti-inflammatory and wound-healing abilities of THO-NPs were also confirmed. Additionally, ex vivo antioxidant assessment was carried out using pig skin, demonstrating the suitable antioxidant properties of THO-NPs. Moreover, THO and THO-NPs were dispersed in a gelling system, and stability, rheological properties, and extensibility were assessed. Finally, the biomechanical properties of THO-hydrogel and THO-NP-hydrogel were studied in human volunteers, confirming the suitable activity for the treatment of acne. As a conclusion, THO has been encapsulated into PLGA NPs, and in vitro, ex vivo, and in vivo assessments had been carried out, demonstrating excellent properties for the treatment of inflammatory skin disorders.

Acne constitutes one of the most prevalent skin disorder affecting both skin and mental health of patients. However, no cure has been developed so far. In this area, Thymol constitutes a potential candidate since it is able to restore the healthy microbiota of the skin. However, its permeation properties cause its fast elimination and, to avoid this problem, thymol has been loaded into nanostructured lipid carriers (TH-NLCs). Moreover, to increase the suitability of these systems for skin applications, several surface functionalization strategies of TH-NLCs had been assessed. Among the different molecules, phosphatidylcholine-TH-NLCs demonstrated to be safe as well as to provide high antioxidant activity in cellular studies. Therefore, to administer these systems to the skin, functionalized TH-NLCs were dispersed into a carbomer gel developing semi-solid formulations. Rheological properties, porosity and extensibility of TH dispersed in carbomer as well as phosphatidylcholine-TH-NLCs were assessed demonstrating suitable properties for dermal applications. Moreover, both formulations were applied in healthy volunteers demonstrating that gel-phosphatidylcholine-TH-NLCs were able to increase in skin hydration, decrease water loss and reduce skin sebum. Therefore, gel-phosphatidylcholine-TH-NLCs proved to be a suitable system for skin pathologies linked with high sebum generation, loss of hydration and high oxidation, such as acne vulgaris., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Thymol-loaded PLGA nanoparticles (TH-NPs) were incorporated into different semi-solid formulations using variable gelling agents (carbomer, polysaccharide and poloxamer). The formulations were physicochemically characterized in terms of size, polydispersity index and zeta potential. Moreover, stability studies were performed by analyzing the backscattering profile showing that the gels were able to increase the nanoparticles stability at 4 °C. Moreover, rheological properties showed that all gels were able to increase the viscosity of TH-NPs with the carbomer gels showing the highest values. Moreover, the observation of carbomer dispersed TH-NPs under electron microscopical techniques showed 3D nanometric cross-linked filaments with the NPs found embedded in the threads. In addition, cytotoxicity studies showed that keratinocyte cells in contact with the formulations obtained cell viability values higher than 70 %. Furthermore, antimicrobial efficacy was assessed against C. acnes and S. epidermidis showing that the formulations eliminated the pathogenic C. acnes but preserved the resident S. epidermidis which contributes towards a healthy skin microbiota. Finally, biomechanical properties of TH-NPs dispersed in carbomer gels in contact with healthy human skin were studied showing that they did not alter skin properties and were able to reduce sebum which is increased in acne vulgaris. As a conclusion, TH-NPs dispersed in semi-solid formulations and, especially in carbomer gels, may constitute a suitable solution for the treatment of acne vulgaris., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

In the analysis of water samples, the type of filtration membrane material can influence the recovery of Legionella species, although this issue has been poorly investigated. Filtration membranes (0.45 µm) from different materials and manufacturers (numbered as 1, 2, 3, 4, and 5) were compared: mixed cellulose esters (MCEs), nitrocellulose (NC), and polyethersulfone (PES). After membrane filtration of samples, filters were placed directly onto GVPC agar and incubated at 36 ± 2 °C. The highest mean counts of colony-forming units and colony sizes for Legionella pneumophila and Legionella anisa were obtained with PES filters ( p < 0.001). All membranes placed on GVPC agar totally inhibited Escherichia coli and Enterococcus faecalis ATCC 19443 and ATCC 29212, whereas only the PES filter from manufacturer 3 (3-PES) totally inhibited Pseudomonas aeruginosa . PES membrane performance also differed according to the manufacturer, with 3-PES providing the best productivity and selectivity. In real water samples, 3-PES also produced a higher Legionella recovery and better inhibition of interfering microorganisms. These results support the use of PES membranes in methods where the filter is placed directly on the culture media and not only in procedures where membrane filtration is followed by a washing step (according to ISO 11731:2017).

Chocolate agar (CA) is an enriched medium for the isolation and identification of fastidious bacteria. Defibrinated blood is used to manufacture CA, but this expensive product is not always affordable for companies in developing countries. Blood powder (BP) is potentially a cheaper alternative, although its pre-treatment using autoclaving can impair the quality of the media. Therefore, optimization of BP as a substitute for defibrinated blood for CA manufacture deserves further research. CA was manufactured with irradiated BP (dehydrated bovine blood powder) and its physical and microbiological characteristics were compared with those of conventional CA and CA prepared with autoclaved BP. Each medium was seeded with 20-200 CFU of target bacteria using the spiral pouring method. Finally, another medium was prepared using BP pre-treated by grinding and gamma irradiation and its performance assessed. Compared to conventional CA, the medium containing ground and irradiated BP provided a similar CFU count for both fastidious ( Neisseria , Haemophilus , Campylobacter , and Streptococcus ) and non-fastidious ( Moraxella , Staphylococcus , Enterococcus , Klebsiella , and Pseudomonas ) species, unlike the medium prepared with BP subjected only to irradiation, which provided a lower growth of fastidious species. Morphology and characteristics of all bacterial colonies were very similar in conventional CA and the new medium, the number of Pseudomonas CFU being higher in the latter. The medium prepared with ground plus irradiated vs. irradiated BP more closely resembled conventional CA, having a browner background. The new CA medium prepared with ground and gamma irradiation-sterilized BP has comparable productivity properties to conventional CA. Therefore, it could be a more practical and economical methodology to facilitate large-scale CA manufacture.

Although the pour plate method is widely employed in microbiological quality control, it has certain drawbacks, including having to melt the culture medium before seeding. In this study, the preparation of the culture medium was modified by using a lower concentration of agar (10 g/L), which was separated from the nutrients during sterilization. The new protocol was assessed in media frequently used in microbiological quality control of food, cosmetics, and pharmaceutical products, with tryptic soy agar (TSA), Sabouraud 4% dextrose agar (SDA), and violet red bile glucose agar (VRBG). In comparison with the conventionally produced media, the modifications significantly improved the growth of Saccharomyces cerevisiae in SDA, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium, and Candida albicans in TSA and Escherichia coli ATCC 8739 and ATCC 25922 and S. Typhimurium in VRBG. The modified VRBG was also more selective for Pseudomonas aeruginosa. Regarding physicochemical properties, a significantly lower pH was observed in TSA and VRBG and lower strength values in TSA. Sterilizing agar separately from the other components of the medium and reducing the agar concentration to 10 g/L can improve microorganism growth and enhance the selectivity of differential media in the pour plate method. These modifications could facilitate the automation of this culture technique. IMPORTANCE In the era of rapid microbiological methods, there is a need to improve long-established culture techniques. Drawbacks of the pour plate method include having to melt each medium separately before seeding. For this technique, we demonstrate that separating the agar from the other components of commonly used media during sterilization and reducing the agar concentration to 10 g/L can enhance microbial growth. The new protocol could have advantages in routine laboratory practice because less agar is required and the same molten agar suspension can be used to prepare different media. Moreover, these modifications could facilitate the automation of the pour plate method.

The detection of Salmonella in food is based on the use of a selective enrichment broth such as Muller-Kauffman Tetrathionate-Novobiocin (MKTTn), in which tetrathionate plays a key role by providing Salmonella with a growth advantage. As sodium tetrathionate is unstable, it is generated in situ by the addition of iodine (Lugol's solution) before seeding. This step is cumbersome as the solution is easily spilled, compromising the performance of the medium and hindering the work of technicians. The aim of this study was to optimize MKTTn broth by generating tetrathionate ex situ through an external reaction between iodine and thiosulphate followed by lyophilization. Quality control procedures were performed to compare the modified and original media, testing pure productivity (enrichment with 50-120 CFU of Salmonella Thyphimurium ATCC 14028 and Salmonella Enteritidis ATCC 13076 and plating on Xylose Lysine Deoxycholate agar, XLD), mixed productivity (50-120 CFU of Salmonella strains and Pseudomonas aeruginosa and Escherichia coli at ≥10 4  CFU and XLD plating) and selectivity (≥10 4  CFU of P. aeruginosa and Enterococcus faecalis and plating on Tryptone Casein Soy agar, TSA). The modified MKTTn medium (S/L) performed comparably with the original medium in terms of growth of both Salmonella strains (>300 colonies in XLD), alone or with P. aeruginosa and E. coli. Quantitative assays showed no statistically significant differences in the number of colonies grown on XLD after 10 -5 dilution (p = 0.7015 with S. Thyphimurium ATCC 14028 and p = 0.2387 with S. Enteritidis ATCC 13076; ANOVA test). MKTTn medium (S/L) was also selective against E. coli (≤100 colonies) and E. faecalis (<10 colonies). These results suggest that adding tetrathionate as a lyophilisate (S/L) is a feasible alternative to the use of Lugol's solution for the preparation of MKTTn enrichment broth and does not affect the properties of the medium., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Biosurfactants (BS) are amphiphilic molecules that align at the interface reducing the surface tension. BS production is developed as an alternative to synthetic surfactants because they are biodegradable, with low toxicity and high specificity. BS are versatile, and this research proposes using a biosurfactant crude extract (BCE) as part of cleaning products. This paper reported the BCE production from Bacillus subtilis DS03 using a medium with molasses. The BCE product was characterized by different physical and chemical tests under a wide pH range, high temperatures, and emulsifying properties showing successful results. The water surface tension of 72 mN/m was reduced to 34 mN/m with BCE, achieving a critical micelle concentration at 24.66 ppm. BCE was also applied to polystyrene surface as pre-treatment to avoid microbial biofilm development, showing inhibition in more than 90% of Escherichia coli , Staphylococcus aureus , and Listeria monocytogenes above 2000 ppm BCE. The test continued using BCE as post-treatment to remove biofilms, reporting a significant reduction of 50.10% Escherichia coli , 55.77% Staphylococcus aureus , and 59.44% Listeria monocytogenes in a concentration higher than 250 ppm BCE. Finally, a comparison experiment was performed between sodium lauryl ether sulfate (SLES) and BCE (included in commercial formulation), reporting an efficient reduction with the mixtures. The results suggested that BCE is a promising ingredient for cleaning formulations with applications in industrial food applications.

Glycine-vancomycin-polymyxin-cycloheximide agar (GVPC) is a recommended medium for the detection of Legionella spp. in water samples. However, its quality could be improved in terms of recovery of Legionella spp. and selectivity properties. Modifications were introduced in GVPC manufacture: autoclaving conditions (115°C, 15 min) and atmosphere during component-stirring (removal of oxygen and N 2 injection). The use of softer autoclaving conditions (115°C, 15 min) improved the growth of Legionella anisa by the spiral method and Legionella pneumophila after membrane filtration. The medium manufactured with O 2 removal and autoclaving for 15 min at 115°C allowed a faster growth of L. pneumophila (colonies visible at day 2) and a notable increase of L. anisa growth (colonies appearing at day 3, and statistically significant numbers of CFU at day 5). After 3 to 5 days of incubation, the improved media showed higher selectivity properties, particularly for Enterococcus faecalis ATCC 29212 and Pseudomonas aeruginosa ATCC 9027. A further improvement was achieved by the addition of N 2 during ingredient stirring, leading to a statistically significant faster growth of L. pneumophila at days 2 and 3 and L. anisa at day 3. Selectivity properties were also enhanced, resulting in the complete inhibition of both E. faecalis strains and Escherichia coli and complete-partial inhibition of P. aeruginosa. Oxygen removal during GVPC manufacture using a vacuum pump system promotes the growth of L. pneumophila and L. anisa, and markedly inhibits the growth of E. coli, P. aeruginosa, and E. faecalis. IMPORTANCE Currently, GVPC is a recommended medium for the detection of Legionella spp. in water samples. However, recovery of Legionella spp. and selectivity properties can be improved. GVPC medium manufactured without oxygen improved the growth of Legionella pneumophila and Legionella anisa. Oxygen removal during GVPC manufacture also improved selectivity properties. A further improvement was achieved by the addition of N 2 during ingredient stirring, leading to a faster growth of L. pneumophila at days 2 and 3 and L. anisa at day 3 and enhancement of selectivity properties. The introduction of the modified GVPC medium in routine practice can allow a better detection of Legionella spp. in water samples.

Background: There is growing interest in the possible effect of perioperative anesthetic management on the growth and spread of cancer. The impact of perioperative use of opioids on cancer recurrence remains controversial and an assessment cannot yet be established based on current publications. This study aimed to assess the differential expression of opioid receptors between healthy and tumor tissues in patients with stage II and III colorectal cancer undergoing elective surgery by immunohistochemistry (IHC)., Methods: Propensity-score matched case-control study nested in a retrospective cohort of patients with stage II or III colorectal. The primary endpoint was the difference in µ-opioid receptor (MOR) expression measured by IHC between tumor and healthy tissue in subject with or without recurrence. Secondary endpoints were to evaluate the differences in Opioid Growth Factor Receptor (OGFR), cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) in the matched sample and from a from samples of colorectal cancer stored in the Cancer Genome Atlas (TCGA) and Genotype Tissue Expression Project (GTEx)., Results: There was a significant difference in MOR receptor (median 3 [intequartile range IQR: 1-3] and 0 [IQR: 0-2], P<0.001) and OGFR receptor (median 6 [IQR: 5-6] and 2 [IQR: 1-2], P<0.001) in tumor and control tissue respectively. However, there were no significant differences in cAMP nor PKA expression between both types of tissues and in expression in any of the analyzed variables by recurrence status. The MOR and OGFR expression data from TCGA database were similar to our sample size data with lower expression of MOR and higher expression of OGFR in tumoural samples with a skewed distribution for MOR expression in tumor tissue both in patients with and without recurrence., Conclusion: In patients with stage II and III colorectal cancer, overall expression of MOR and OGFR was significantly increased but was not different between previously matched patients with or without recurrence. No differences were found in the analyzed metabolic pathway of cAMP-PKA: These results were confirmed by an in silico analysis of samples from the TCGA-GTEx database., Competing Interests: OD-C: Received payment for educational talks and scientific conferences from MSD (Merck Sharp & Dohme, Inc.). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Belltall, Mazzinari, Garrido-Cano, Giner, Marí, Eroles, Argente-Navarro, Cata and Diaz-Cambronero.)