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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

Abstract Background The aim of this study was to estimate the prevalence of fibromyalgia, as well as to assess the major symptoms of this syndrome in an adult, low socioeconomic status population assisted by the primary health care system in a city in Brazil. Methods We cross-sectionally sampled individuals assisted by the public primary health care system (n = 768, 35–60 years old). Participants were interviewed by phone and screened about pain. They were then invited to be clinically assessed (304 accepted). Pain was estimated using a Visual Analogue Scale (VAS). Fibromyalgia was assessed using the Fibromyalgia Impact Questionnaire (FIQ), as well as screening for tender points using dolorimetry. Statistical analyses included Bayesian Statistics and the Kruskal-Wallis Anova test (significance level = 5%). Results From the phone-interview screening, we divided participants (n = 768) in three groups: No Pain (NP) (n = 185); Regional Pain (RP) (n = 388) and Widespread Pain (WP) (n = 106). Among those participating in the clinical assessments, (304 subjects), the prevalence of fibromyalgia was 4.4% (95% confidence interval [2.6%; 6.3%]). Symptoms of pain (VAS and FIQ), feeling well, job ability, fatigue, morning tiredness, stiffness, anxiety and depression were statically different among the groups. In multivariate analyses we found that individuals with FM and WP had significantly higher impairment than those with RP and NP. FM and WP were similarly disabling. Similarly, RP was no significantly different than NP. Conclusion Fibromyalgia is prevalent in the low socioeconomic status population assisted by the public primary health care system. Prevalence was similar to other studies (4.4%) in a more diverse socioeconomic population. Individuals with FM and WP have significant impact in their well being.

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.)

Antibiotic resistance is a major public health challenge, and Gram-negative multidrug-resistant bacteria are particularly dangerous. The threat of running out of active molecules is accelerated by the extensive use of antibiotics in the context of the COVID-19 pandemic, and new antibiotics are urgently needed. Colistin and polymyxin B are natural antibiotics considered as last resort drugs for multi-resistant infections, but their use is limited because of neuro- and nephrotoxicity. We previously reported a series of synthetic analogues inspired in natural polymyxins with a flexible scaffold that allows multiple modifications to improve activity and reduce toxicity. In this work, we focus on modifications in the hydrophobic domains, describing analogues that broaden or narrow the spectrum of activity including both Gram-positive and Gram-negative bacteria, with MICs in the low µM range and low hemolytic activity. Using biophysical methods, we explore the interaction of the new molecules with model membranes that mimic the bacterial inner and outer membranes, finding a selective effect on anionic membranes and a mechanism of action based on the alteration of membrane function. Transmission electron microscopy observation confirms that polymyxin analogues kill microbial cells primarily by damaging membrane integrity. Redistribution of the hydrophobicity within the polymyxin molecule seems a plausible approach for the design and development of safer and more selective antibiotics.

The use of biosurfactants in bioremediation of hydrocarbons and in the removal of heavy metals in crude oils is considered an attractive subject. The vanadium pollution in soil and sediments had attracted research interest in exploring eco-friendly methods of remediation. The present study was conducted to evaluate the potential of a biosurfactant to remove vanadium from artificially contaminated sand. The biosurfactant producer's strain selection process was carried out from 23 strains in two steps. In the primary screening, four preliminary tests were carried out: the emulsification index (24 and 72 h), the surface tension, and the rate of bacterial adhesion to hydrocarbons. In the secondary screening, the surface tension and rhamnolipids concentration were determined, also critical micellar concentration and dilution were calculated. The RNA 16s of selected strain was sequence and the strain was identified as Pseudomonas sp. By chromatographic and spectroscopic assays, the structure of the rhamnolipids was determined. The maximal vanadium removal efficiency (85.5%) was achieved with a rhamnolipids' concentration of 240 mg l -1 . The vanadium concentration was determined by spectroscopic technique. Rhamnolipids produced by this strain can potentially be used in the removal of vanadium.

A newly marine Halomonas pacifica strain Cnaph3 was isolated, as a naphthalene degrader and biosurfactant producer, from contaminated seawater collected in Ataya's fishing harbor, located in Kerkennah Islands, Tunisia. Chromatography flame ionization detector analysis revealed that 98.8% of naphthalene (200 mg/L) was degraded after 7 days of incubation, at 30 g/L NaCl and 37 °C. Strain Cnaph3 showed also a noticeable capacity to grow on a wide range of aliphatic, aromatic, and complex hydrocarbons. Interestingly, strain Cnaph3 showed a significant potential to produce biosurfactants in the presence of all tested substrates, particularly on glycerol (1%, v/v). Electrospray ionization analysis of the biosurfactant, designated Bios-Cnaph3, suggested a lipopeptide composition. The critical micelle concentration of Bios-Cnaph3 was about 500 mg/L. At this concentration, the surface tension of the water was reduced to 27.6 mN/m. Furthermore, Bios-Cnaph3 displayed interesting stabilities over a wide range of temperatures (4-105 °C), salinities (0-100 g/L NaCl), and pH (2.2-12.5). In addition, it showed promising capacities to remove used motor oil from contaminated soils. The biodegradation and biosurfactant-production potential of the Halomonas sp. strain Cnaph3 would present this strain as a favorite agent for bioremediation of hydrocarbon-contaminated sites under saline conditions., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest regarding the publication of this manuscript., (© King Abdulaziz City for Science and Technology 2020.)

After publication of our article [1] the authors have notified us that there are changes in the primary outcome and the statistical analysis plan of the study.

Background: Randomised clinical trials are considered to be the most reliable study design for assessing the efficacy and safety of health interventions., Aims: To analyse worldwide obstetrics research carried out through randomised clinical trials, from 2002 to 2013., Methods: A bibliometric analysis was performed. Publications on obstetrics that were published journals indexed in the MEDLINE database from 2002 to 2013 were analysed. The major medical subject headings used in the search were obstetrics, pregnancy complications and obstetrics surgical procedures. The main study outcome was index of research productivity., Results: Our study search strategy yielded a total of 142,659 articles and 9967 clinical trials. The growth rate of scientific production in obstetrics during this period was 55.43% (n = 5094). The growth rate of production of randomised clinical trials in this specialty, meanwhile, was 97.84% (n = 544). Most of the identified authors (n = 22,622, 71.21%) published only one paper during the study period. Patterns of co-authorship among the 20 most productive authors were identified. After applying Bradford's law, six journals in the nucleus (the most prolific journals) were found. Of all the clinical trials in obstetrics published between 2002 and 2013, 10.3% were published in journals belonging to categories other than Obstetrics and Gynecology. The most common research topic in 2002 and 2013 was the use of analgesia and anesthesia in obstetrics., Conclusions: Total scientific production rate in obstetrics increased from 2002 to 2013, especially randomised clinical trials. However, randomised clinical trials continue to represent a small proportion of total production.

Background: Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management., Methods: The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test., Results: In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups., Conclusions: The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Catanionic surfactant mixtures form a wide variety of organized assemblies and aggregates with improved physicochemical and biological properties. The green catanionic mixture N α N ω -Bis(N α caproylarginine) α,ω-propyldiamide (C 3 (CA) 2 ):Lichenysin (molar ratio 8:2) showed antimicrobial synergies against Yersinia enterocolitica, Bacillus subtilis, Escherichia coli O157:H7, and Candida albicans. Flow cytometry and viability studies indicated that this catanionic mixture increases the probability of Y. enterocolitica (38.2%) and B. subtilis (17.1%) cells entering a viable but nonculturable state. Zeta potential showed that one of the cationic charges of C 3 (CA) 2 is neutralized by Lichenysin. An isotherm study demonstrated the formation of a stable aggregate between the two surfactants. The catanionic aggregate was able to interact with bacterial phospholipids. The lowest hemolysis (22.1 μM) was obtained with the catanionic mixture, although an irritant potential (0.70) was characterized. According to the therapeutic index, the C 3 (CA) 2 :Lichenysin mixture was the formulation least toxic to eukaryotic cells. Partial neutralization of C 3 (CA) 2 by Lichenysin modified the mode of action that enhances the transition of bacterial cells into a viable but nonculturable state (VBNC) and improved the cell selectivity.

Recently it has been demonstrated that catanionic mixtures of oppositely charged surfactants have improved physicochemical-biological properties compared to the individual components. Isotherms of mixtures of an anionic biosurfactant (lichenysin) and a cationic aminoacid surfactant (C 3 (LA) 2 ) indicate a strong interaction suggesting the formation of a new "pseudo-surfactant". The antimicrobial properties of the mixture lichenysin and C 3 (LA) 2 M80:20, indicate a synergistic effect of the components. The mechanism of action on the bacterial envelope was assessed by flow cytometry and Transmission Electron Microscopy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: One promising strategy for reducing human salmonellosis induced by Salmonella Enteritidis is to supplement animal diets with natural feed additives such as mannan oligosaccharides (MOSs)., Objective: We sought to investigate the potential role of Salmosan (S-βGM), an MOS product extremely rich in β-galactomannan, in preventing epithelial barrier function disruption induced by S. Enteritidis colonization in an in vitro model of intestinal Caco-2 cells in culture., Methods: Differentiated Caco-2 cells were incubated for 3 h with S. Enteritidis at a multiplicity of infection of 10 in the absence or presence of 500 μg S-βGM/mL. Paracellular permeability (PP) was assessed by transepithelial electrical resistance (TER), d-mannitol, and fluorescein isothiocyanate-dextran (FD-4) flux. Tight junction proteins and cytoskeletal actin were also localized by confocal microscopy. Reactive oxygen species (ROS) and lipid peroxidation products were evaluated. Scanning and transmission electron microscopy were used to visualize S. Enteritidis adhesion to, and invasion of, the Caco-2 cell cultures., Results: Compared with controls, TER was significantly reduced by 30%, and d-mannitol and FD-4 flux were significantly increased by 374% and 54% in S. Enteritidis-infected cultures, respectively. The presence of S-βGM in infected cultures induced total recoveries of TER and FD-4 flux to values that did not differ from the control and a partial recovery of d-mannitol flux. These effects were confirmed by immunolocalization of actin, zonula occludens protein 1, and occludin. Similar results were obtained for Salmonella Dublin. The protection of S-βGM on PP in infected cultures may be associated with a total recovery of ROS production to values that did not differ from the control. Moreover, S-βGM has the capacity to agglutinate bacteria, leading to a significant reduction of 32% in intracellular S Enteritidis., Conclusion: The results demonstrate that S-βGM contributes to protecting epithelial barrier function in a Caco-2 cell model disrupted by S. Enteritidis., (© 2016 American Society for Nutrition.)

Aims: Apply response surface methodology (RSM) to develop and optimize an economical medium for lichenysin production, which is a surfactant produced by Bacillus licheniformis and evaluate the application of lichenysin in the prevention and disruption of pathogenic micro-organism biofilm that creates health problems in the food industry and hospitals., Results: An economical medium containing molasses was optimized to enhance lichenysin production by RSM. A production of 3·2 g l(-1) of lichenysin was achieved with an optimum medium containing 107·82 g l(-1) of molasses, 6·47 g l(-1) of NaNO3 and 9·7 g l(-1) of K2 HPO4 /KH2 PO4 , in which molasses and phosphate salts had a significant effect on biosurfactant production. Lichenysin was effectively applied in a surface pre-treatment to avoid microbial biofilm development of methicillin-resistant Staphylococcus aureus (MRSA) (68·73%) and Candida albicans (74·35%), with ED50 values of 8·3 and 17·2 μg ml(-1) respectively. It was also very efficient in a surface post-treatment to remove biofilms of MRSA (55·74%) and Yersinia enterocolitica (51·51%), with an ED50 of 2·79 and 4·09 μg ml(-1) respectively., Conclusions: Lichenysin was found to have notable anti-adhesion activity, being able to prevent and eliminate the biofilm formation by pathogenic strains associated with foodborne illness. This new medium resulted in a four-fold increase in production compared with the nonoptimized medium., Significance and Impact of the Study: Molasses can be regarded as a useful resource for biotechnological applications, such as the production of lichenysin. The use of agro-industrial substrates has an important role in the sustainable and competitive development of several industrial sectors, as well as in industrial residues management. Additionally, lichenysin is particularly effective in preventing biofilm formation by strains problematic for the food industry and in the hospital environment. Lichenysin also efficiently disrupts biofilm., (© 2015 The Society for Applied Microbiology.)

Aim: To investigate efficacy and safety of second-line treatment with irinotecan-loaded drug-eluting beads (DEBIRI) and cetuximab (DEBIRITUX) of unresectable colorectal liver metastases., Methods: Patients with the following characteristics were included in the study: unresectable hepatic metastases from colorectal carcinoma (CRC-LM), progression after first line chemotherapy (any type of chemotherapeutic drug and combination was allowed), second line treatment (mandatory), which included for each patient (unregarding the KRas status) two cycles of DEBIRI (using 100-300 μm beads loaded with irinotecan at a total dose 200 mg) followed by 12 cycles of cetuximab that was administered weekly at a first dose of 400 mg/m(2) and then 250 mg/m(2); good performance status (0-2) and liver functionality (alanine aminotransferase and gamma-glutamyl transferase not exceeding three times the upper limit of normal, total bilirubin not exceeding 2.5 mg/mL). Data were collected retrospectively and included: tumor response (evaluated monthly for 6 mo then every 3 mo), overall response rate (ORR), KRas status, type and intensity of adverse events (G according to the Common Terminology Criteria for Adverse Events v3.0, CTCAE), overall survival (OS) and progression free survival (PFS)., Results: Forty consecutive cases of CRC hepatic metastases were included in the study. Median duration of DEBIRITUX was 4.4 mo (range, 4.0-6.5). Sixteen patients (40%) received the planned 2 cycles of DEBIRI and an average of 10 cetuximab cycles. ORR of the whole sample was 50%, in particular 4 patients were complete responders (10%) and 16 (40%) partial responders. The most observed side effects (G2) were: post-embolization syndrome (30%), diarrhea (25%), skin rushes (38%) and asthenia (35%). The retrospective evaluation of KRas status (24 wild type, 16 mutated) showed that the group of patients with wild type KRas had ORR significantly higher than mutant KRas. Median follow-up was 29 mo (8-48 range); median PFS was 9.8 mo and OS was 20.4 mo. Future randomized trials are required in this setting to establish a role for DEBIRITUX compared with systemic chemotherapy., Conclusion: DEBIRITUX seems to be efficacious after first line chemotherapy for the treatment of unresectable CRC-LM.

This work examines the influence of essential oil composition on emulsification with rhamnolipids and their use as therapeutic antimicrobial agents against two opportunistic pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. Rhamnolipids, produced by Pseudomonas aeruginosa, with waste frying oil as the carbon source, were composed of eight rhamnolipid homologues. The rhamnolipid mixture was used to produce emulsions containing essential oils (EOs) of Melaleuca alternifolia, Cinnamomum verum, Origanum compactum and Lavandula angustifolia using the titration method. Ternary phase diagrams were designed to evaluate emulsion stability, which differed depending on the essential oil. The in vitro antimicrobial activity of the EOs alone and the emulsions was evaluated. The antimicrobial activity presented by the essential oils alone increased with emulsification. The surface properties of rhamnolipids contribute to the positive dispersion of EOs and thus increase their availability and antimicrobial activity against C. albicans and S. aureus. Therefore, rhamnolipid-based emulsions represent a promising approach to the development of EO delivery systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Congenital vesicovaginal fistula (CVVF) is a rare, complex female genital malformation that is difficult to diagnose, classify and treat. Symptoms include menouria, cyclical haematuria and urinary incontinence from birth. The aim of this review was to highlight the importance of correct diagnosis and treatment of this congenital anomaly. A comprehensive literature review was conducted to identify articles on CVVF with and without menouria. Forty-one articles were found, of which 31 described 37 original cases of congenital fistula (30 CVVF, six uterovesical fistula and one urethrovaginal fistula): 14 in girls and 23 in adults. The cases were classified according to clinical, diagnostic and therapeutic parameters in order to unify concepts and terms. Menouria occurred in 21 of the 23 adults: 14 cases were CVVF, six cases were congenital uterovesical fistula, and one case was congenital urethrovaginal fistula in a young woman without congenital adrenal hyperplasia. Sixteen (53%) of the 30 patients with CVVF had urinary incontinence or hydrocolpometra, and 14 (47%) had menouria. There were associated anomalies in 91% of cases, 29 (48%) of which involved the urinary tract. Various diagnostic and therapeutic methods were used, and there was confusion surrounding the aetiopathogenesis in most cases. CVVF should be suspected in any girl with urinary incontinence, urinary tract infections from birth, vaginal swelling or hydrocolpometras, as well as in adults with menouria. Diagnosis should be based on physical examination and imaging (cystoscopy during menouria, ultrasound and magnetic resonance imaging). Surgical treatment should be based on correction of the vaginal defects (obstructive problem opening or vaginal reconstruction) and CVVF closure. The embryological origin of CVVF lies in the abnormal persistence of the urogenital sinus due to lack of formation and caudal growth of the urogenital wedge, combined with distinct degrees of agenesis or hypoplasia of the entire urogenital ridge or the mesonephric ducts (which affects development of the Müllerian ducts)., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The taxonomic position of Sphingobacterium antarcticum has been revised by means of 16S rRNA gene sequences, DNA-DNA hybridization, and phenotypic and chemotaxonomic characteristics. All data previously reported, as well as the results of the present phylogenetic analysis, support that Sphingobacterium antarcticum is clearly a member of the genus Pedobacter, also affiliated with the family Sphingobacteriaceae. We propose that Sphingobacterium antarcticum (corrig. Shivaji et al. 1992) should be reclassified as Pedobacter antarcticus comb. nov.

The application of biosurfactants in the biomedical field is growing due to their antimicrobial activity, low cytotoxicity and ability to induce apoptosis in cancer cells. In the light of this therapeutic potential, as well as possible applications in cosmetics or as drug vehicles in pharmaceutical products, a new biosurfactant produced by Sphingobacterium detergens was investigated for its haemolytic activity and cytotoxic and antiproliferative effects in different cell lines. Fraction A showed 100% haemolysis in rabbit erythrocytes, but in Fraction B the rate was only 83%. When comparing cytotoxicity values (IC50) of the two fractions in model fibroblast and keratinocyte cell cultures, Fraction B was less cytotoxic, showing lower values than the reference compound SDS, indicating low skin irritability. Finally, in non-differentiated intestinal Caco-2 cultures, Fractions A and B reduced cell proliferation and induced apoptosis by 44% and 75%, respectively. According to these results, biosurfactants produced by S. detergens have potential application in cosmetic and pharmaceutical formulations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The commercial application of a new biosurfactant such as the one produced by Sphingobacteriumdetergens needs a cost-effective process and knowledge of its properties. In the present study, a specific medium and a downstream process have been developed to enhance biosurfactant production. Optimal concentrations of nutrients in MCA medium were (g/L) the following: KH(2)PO(4), 1; K(2)HPO(4), 2; CO(NH(2))(2) 0.88; CaCl(2) 0.01; FeSO(4)·7H(2)O, 0.01; MgSO(4)·7H(2)O 0.5; KCl, 1.0; trace elements 0.05 mL. Biosurfactant production in the MCA medium required a bacterial co-metabolism of glucose and an n-alkane. A fed-batch culture with supernatant lyophilization prior to organic extraction produced 466 mg/L of organic extract, which represents a 6.9-fold increase in production. The newly obtained biosurfactant was a complex mixture of molecules. The three characterized fractions consisted of the complete fraction and two second-level purification fractions with apolar and polar characteristics. The complete and apolar fractions have been shown to self-aggregate in the form of lamellar liquid crystals at a high concentration and bilayers at lower concentrations. Negatively charged particles were identified, which were neutralized at a low pH with a concomitant increase in size. The pH affected the surface tension of the solutions congruently with phosphate headgroups., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A novel Gram-negative-staining strain, designated 6.2S(T), was isolated from a soil sample and identified as a biosurfactant producer. Its taxonomic position was investigated using a polyphasic approach. The cells were non-motile, non-spore-forming rods. The organism grew optimally at 30-37 °C, with 0-3% (w/v) NaCl, and at pH 7.0. Based on 16S rRNA gene sequence analysis, strain 6.2S(T) was found to be a member of the genus Sphingobacterium and was most closely related to four type species of the genus, showing sequence similarities of 96.8-98.9%. Partial chaperonin 60 (cpn60) gene sequence analysis was useful in resolving the phylogenetic relationships between strain 6.2S(T) and closely related taxa, with similarities ranging from 85.5% (with Sphingobacterium thalpophilum DSM 11723(T)) to 90.3% (with Sphingobacterium canadense CR11(T) and Sphingobacterium multivorum JCM 21156(T)). The results of DNA-DNA hybridization experiments between the novel strain and its closest relatives gave a DNA-DNA relatedness value of less than 70%, and consequently confirmed that this new strain did not belong to a previously described species of the genus Sphingobacterium. The major fatty acids were summed feature 3 (iso-C(15:0) 2 OH and/or C(16:1)ω7c); iso-C(15:0); iso-C(17:0) 3-OH and C(16:0). The G+C content of the genomic DNA was 40.0 mol%. According to its phenotypic and genotypic characteristics and the phylogenetic data, strain 6.2S(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium detergens sp. nov. is proposed. The type strain is 6.2S(T) ( = CECT 7938(T) = LMG 26465(T)).

Strain 6.2S, isolated from soil and identified as a Sphingobacterium sp., is the first strain in this genus to be reported as a biosurfactant producer, being able to reduce the surface tension of its culture supernatant to 32 mN/m. In this work, biosurfactants from the culture supernatant were purified and partially characterized. The crude extract (10 g/L) was very effective in reducing surface tension (22 mN/m). Thin layer chromatography (TLC) indicated that a mixture of various biosurfactants was present in the 6.2S crude extract. After purification, Fraction A, a phospholipid mixture, reduced surface tension to 33 mN/m. Fraction B was a mixture of lipopetides and at least one glycolipid. The surface tension-concentration curve showed two plateaux, the first of which can be attributed to a critical aggregation concentration of the biosurfactant with a protein (2.7 g/L) and the second to the true cmc in water (6.3g/L)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study analyzed the chemical and physical properties of a biosurfactant synthesized by Rhodococcus sp. 51T7. The biosurfactant was a trehalose tetraester (THL) consisting of six components: one major and five minor. The hydrophobic moieties ranged in size from 9 to 11 carbons. The critical micelle concentration (CMC) was 0.037g L(-1) and the interfacial tension against hexadecane was 5mN m(-1). At pH 7.4 the glycolipid CMC/critical aggregation concentration (CAC) was 0.05g L(-1) and at pH 4 it was 0.034g L(-1). A phase diagram revealed effective emulsification with water and paraffin or isopropyl myristate. A composition of 11.3-7.5-81.8 (isopropyl myristate-THL-W) was stable for at least 3 months. The HLB was 11 and the phase behaviour of the glycolipid revealed the formation of lamellar and hexagonal liquid-crystalline textures.

The Ni(2+) accumulation in batch mode from diluted solutions by gel beads of gellan gum (GG), alginate, kappa-carrageenan, agar, agarose, silica gel, polyacrylamide and two mixtures of GG+agar was investigated. All polymeric materials studied accumulated Ni(2+), but gel beads of GG were stable, easily obtainable and showed the highest Ni(2+) accumulation. The pH of the Ni(2+) solution was not critical for Ni(2+) accumulation. Accumulation of metals Cu(2+), Co(2+), Ni(2+), Pb(2+), Cd(2+) and Zn(2+) by GG gel beads reached the equilibrium after 24h. The removal of Pb(2+) and Cu(2+) from the aqueous solution was very efficient, with maximum metal uptake (q(max)) of 0.85 and 0.75 mmol/g dw GG, respectively. The general q(max) sequence was Pb>Cu>Ni approximately Zn=Co>Cd. In an equimolar metal mixture sorption experiment a clear reduction in accumulation was observed, except for Pb(2+) (30%). Heavy metals were desorbed with 100mM sodium citrate.

Pseudomonas sp. EPS-5028 was examined for the ability to accumulate uranium from solutions. The uptake of uranium by this microorganism is very rapid and is affected by pH but not by temperature, metabolic inhibitors, culture time and the presence of various cations and anions. The amount of uranium absorbed by the cells increased as the uranium concentration of the solution increased up to 55 mg uranium/g cell dry weight. Electron microscopy indicated that uranium accumulated intracellularly as needle-like fibrils. Uranium could be removed chemically from the cells, which could then be reused as a biosorbent.

During batch aerobic submerged fermentation, the exopolysaccharide synthesis by Pseudomonas sp. strain EPS-5028 occurred in growth- and non-growth-linked processes. Polysaccharide formation increased when the pH was controlled at 7 during fermentation. Exopolysaccharide production depended on the phosphate content of the medium. The polymer exhibited a pseudoplastic nature, had good thermostability, and was affected neither by pH nor by high concentrations of salt.