Aislamiento de cocaína y benzoilecgonina en muestras de orina por extracciones líquido-líquido y en fase sólida y confirmación por Cromatografía líquida de alta resolución (HPLC)

El consumo ilícito de cocaína se ha incrementado extraordinariamente en los últimos años, por lo que resulta indispensable el desarrollo de metodologías seguras, rápidas y eficientes para su detección. En este trabajo se desarrollo una técnica de HPLC de fase reversa con detector de UV para identificar y cuantificar a la cocaína y la benzoilecgonina, con resultados satisfactorios en los parámetros del control de calidad.Se realizó un estudio de recobrado para el aislamiento de la cocaína y la benzoilecgonina en orina con extracciones liquido liquido y en fase sólida con tres tipos de columnas comerciales (Bond Elut Certify, Extrelut 3 y Supelclean LC-18). Las fracciones obtenidas con la extracción líquido-líquido resultaron muy contaminados, con porcentajes de recobrados bajos (45% y 28% para la cocaína y la benzoilecgonina, respectivamente. En las extracciones en fase sólida para la cocaína resultaron muy eficientes las columnas Supelclean LC-18 (87-102 %) y Extrelut 3 (70-102 %), mientras que para el aislamiento de la benzoilecgonina resultaron más eficiente las columnas Extrelut 3 (86-101 %) y Supelclean LC-18 (73-89%). Las columnas Bond Elut Certify resultaron poco eficientes para el aislamiento de la cocaína (53-79%) y con un recobrado aún mas bajo para su metabolito (2-21% %)

Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus

Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus

α,α-disubstituted β-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal

Bacterial biofilms account for up to 80% of all infections and complicate successful therapies due to their intrinsic tolerance to antibiotics. Biofilms also cause serious problems in the industrial sectors, for instance due to the deterioration of metals or microbial contamination of products. Efforts are put in finding novel strategies in both avoiding and fighting biofilms. Biofilm control is achieved by killing and/or removing biofilm or preventing transition to the biofilm lifestyle. Previous research reported on the anti-biofilm potency of α,α-disubstituted β-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Dalton. In the current study it was investigated if these derivatives cause a fast disintegration of biofilm bacteria and removal of Staphylococcus aureus biofilms. One hour incubation of biofilms with all three derivatives resulted in reduced metabolic activity and membrane permeabilization in S. aureus (ATCC 25923) biofilms. Bactericidal properties of these derivatives were attributed to a direct effect on membranes of biofilm bacteria. The green fluorescence protein expressing Staphylococcus aureus strain AH2547 was cultivated in a CDC biofilm reactor and utilized for disinfectant efficacy testing of A3, following the single-tube method (American Society for Testing and Materials designation number E2871). A3 at a concentration of 90 μM acted as fast as 100 μM chlorhexidine and was equally effective. Confocal laser scanning microscopy studies showed that chlorhexidine treatment lead to fluorescence fading indicating membrane permeabilization but did not cause biomass removal. In contrast, A3 treatment caused a simultaneous biofilm fluorescence loss and biomass removal. These dual anti-biofilm properties make α,α-disubstituted β-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms

Growth Mode and Carbon Source Impact the Surfaceome Dynamics of Lactobacillus rhamnosus GG

Bacterial biofilms have clear implications in disease and in food applications involving probiotics. Here, we show that switching the carbohydrate source from glucose to fructose increased the biofilm formation and the total surface-antigenicity of a well-known probiotic, Lactobacillus rhamnosus GG. Surfaceomes (all cell surface-associated proteins) of GG cells grown with glucose and fructose in planktonic and biofilm cultures were identified and compared, which indicated carbohydrate source-dependent variations, especially during biofilm growth. The most distinctive differences under these conditions were detected with several surface adhesins (e.g., MBF, SpaC pilus protein and penicillin-binding proteins), enzymes (glycoside hydrolases, PrsA, PrtP, PrtR, and HtrA) and moonlighting proteins (glycolytic, transcription/translation and stress-associated proteins, r-proteins, tRNA synthetases, Clp family proteins, PepC, PepN, and PepA). The abundance of several known adhesins and candidate moonlighters, including enzymes acting on casein-derived peptides (ClpP, PepC, and PepN), increased in the biofilm cells grown on fructose, from which the surface-associated aminopeptidase activity mediated by PepC and PepN was further confirmed by an enzymatic assay. The mucus binding factor (MBF) was found most abundant in fructose grown biofilm cells whereas SpaC adhesin was identified specifically from planktonic cells growing on fructose. An additional indirect ELISA indicated both growth mode- and carbohydrate-dependent differences in abundance of SpaC, whereas the overall adherence of GG assessed with porcine mucus indicated that the carbon source and the growth mode affected mucus adhesion. The adherence of GG cells to mucus was almost completely inhibited by anti-SpaC antibodies regardless of growth mode and/or carbohydrate source, indicating the key role of the SpaCBA pilus in adherence under the tested conditions. Altogether, our results suggest that carbon source and growth mode coordinate mechanisms shaping the proteinaceous composition of GG cell surface, which potentially contributes to resistance, nutrient acquisition and cell-cell interactions under different conditions. In conclusion, the present study shows that different growth regimes and conditions can have a profound impact on the adherent and antigenic features of GG, thereby providing new information on how to gain additional benefits from this probiotic.Peer reviewe

Structural and Functional Dynamics of Staphylococcus aureus Biofilms and Biofilm Matrix Proteins on Different Clinical Materials

Medical device-associated staphylococcal infections are a common and challenging problem. However, detailed knowledge of staphylococcal biofilm dynamics on clinically relevant surfaces is still limited. In the present study, biofilm formation of the Staphylococcus aureus ATCC 25923 strain was studied on clinically relevant materials—borosilicate glass, plexiglass, hydroxyapatite, titanium and polystyrene—at 18, 42 and 66 h. Materials with the highest surface roughness and porosity (hydroxyapatite and plexiglass) did not promote biofilm formation as efficiently as some other selected materials. Matrix-associated poly-N-acetyl-β-(1-6)-glucosamine (PNAG) was considered important in young (18 h) biofilms, whereas proteins appeared to play a more important role at later stages of biofilm development. A total of 460 proteins were identified from biofilm matrices formed on the indicated materials and time points—from which, 66 proteins were proposed to form the core surfaceome. At 18 h, the appearance of several r-proteins and glycolytic adhesive moonlighters, possibly via an autolysin (AtlA)-mediated release, was demonstrated in all materials, whereas classical surface adhesins, resistance- and virulence-associated proteins displayed greater variation in their abundances depending on the used material. Hydroxyapatite-associated biofilms were more susceptible to antibiotics than biofilms formed on titanium, but no clear correlation between the tolerance and biofilm age was observed. Thus, other factors, possibly the adhesive moonlighters, could have contributed to the observed chemotolerant phenotype. In addition, a protein-dependent matrix network was observed to be already well-established at the 18 h time point. To the best of our knowledge, this is among the first studies shedding light into matrix-associated surfaceomes of S. aureus biofilms grown on different clinically relevant materials and at different time points

Combined Effect of Naturally-Derived Biofilm Inhibitors and Differentiated HL-60 Cells in the Prevention of Staphylococcus aureus Biofilm Formation

Nosocomial diseases represent a huge health and economic burden. A significant portion is associated with the use of medical devices, with 80% of these infections being caused by a bacterial biofilm. The insertion of a foreign material usually elicits inflammation, which can result in hampered antimicrobial capacity of the host immunity due to the effort of immune cells being directed to degrade the material. The ineffective clearance by immune cells is a perfect opportunity for bacteria to attach and form a biofilm. In this study, we analyzed the antibiofilm capacity of three naturally derived biofilm inhibitors when combined with immune cells in order to assess their applicability in implantable titanium devices and low-density polyethylene (LDPE) endotracheal tubes. To this end, we used a system based on the coculture of HL-60 cells differentiated into polymorphonuclear leukocytes (PMNs) and Staphylococcus aureus (laboratory and clinical strains) on titanium, as well as LDPE surfaces. Out of the three inhibitors, the one coded DHA1 showed the highest potential to be incorporated into implantable devices, as it displayed a combined activity with the immune cells, preventing bacterial attachment on the titanium and LDPE. The other two inhibitors seemed to also be good candidates for incorporation into LDPE endotracheal tubes

Combined Effect of Naturally-Derived Biofilm Inhibitors and Differentiated HL-60 Cells in the Prevention of Staphylococcus aureus Biofilm Formation

Nosocomial diseases represent a huge health and economic burden. A significant portion is associated with the use of medical devices, with 80% of these infections being caused by a bacterial biofilm. The insertion of a foreign material usually elicits inflammation, which can result in hampered antimicrobial capacity of the host immunity due to the effort of immune cells being directed to degrade the material. The ineffective clearance by immune cells is a perfect opportunity for bacteria to attach and form a biofilm. In this study, we analyzed the antibiofilm capacity of three naturally derived biofilm inhibitors when combined with immune cells in order to assess their applicability in implantable titanium devices and low-density polyethylene (LDPE) endotracheal tubes. To this end, we used a system based on the coculture of HL-60 cells differentiated into polymorphonuclear leukocytes (PMNs) and Staphylococcus aureus (laboratory and clinical strains) on titanium, as well as LDPE surfaces. Out of the three inhibitors, the one coded DHA1 showed the highest potential to be incorporated into implantable devices, as it displayed a combined activity with the immune cells, preventing bacterial attachment on the titanium and LDPE. The other two inhibitors seemed to also be good candidates for incorporation into LDPE endotracheal tubes

Evaluaciones toxicológicas de un extracto acuoso del alga marina Bryothamnion triquetrum (Gmelin) M.A.Howe en estudios in vitro y modelos animales

Objetivo: El objetivo de este trabajo fue evaluar la toxicidad de un extracto acuoso del alga marina Bryothamnion triquetrum. Métodos: El ensayo de Ames se desarrolló con las cepas de S. typhimurium TA 1535, TA 1537 y TA 1538 con y sin activación metabólica. El estudio de citotoxicidad se realizó con células intestinales Caco-2 durante 24 y 48 horas de exposición al extracto y la viabilidad fue evaluada con la técnica de yoduro de propidio. El Estudio de Toxicidad Aguda se realizó con ratones Balc/c machos por vía oral e intraperitoneal y el Ensayo de Toxicidad por Dosis Repetidas se desarrolló con ratas Wistar de ambos sexos, durante 3 meses por vía oral con dosis de 8 y 32 mg/kg. Resultados: En el estudio de citotoxicidad con células Caco-2 se obtuvieron CL50 de 9,3 y 4,5 mg/mL con exposiciones de 24 y 48 horas respectivamente. El ensayo de Ames evidencia que no es mutágeno directo ni promutágeno hasta 1000 μg. La DL50 del extracto por vía intraperitoneal fue de 1205 mg/kg y por vía oral no se observó mortalidad en dosis de 2000 mg/kg. En el estudio de Toxicidad por Dosis Repetidas no se observó toxicidad. Conclusiones: A partir de estos resultados se puede postular que el extracto acuoso del alga marina B. triquetrum es inocuo, consideración necesaria, entre otras, para su posible uso como nutracéutico y/o fitofármaco.Aim: The aim of this work was to evaluate the toxicity of an aqueous extract from seaweed Bryothamnion triquetrum. Materials and Methods: Ames assay was developed with S. typhimurium TA 1535, TA 1537 and TA 1538 with and without metabolic activation. Citotoxicity study was carried out with intestinal cells Caco-2 during 24 and 48 hours of exhibition to the extract and the viability was evaluated with the technique of Propidium iodide. Acute Toxicity was carried out with mice Balc/c males for via oral and intraperitoneal and the Toxicity for Repeated Dose was developed with rats Wistar of both sexes, during 3 months for via oral with dose of 8 and 32 mg/kg. Results: Results of Ames assays showed that this extract is not direct mutagen or promutagen in quantity until 1000 μg. The cytotoxic effect (LC50) of Caco-2 cells after 24 and 48 h of exposition were 9,3 and 4,5 mg/mL respectively. The LD50 of the extract, with intraperitoneal administration was 1205 mg/kg and by oral via not produce mortality in doses until 2000 mg/kg. At the doses of 8 and 32 mg/kg of extract, the repeated oral administration produced no toxic effects. Conclusions: In summary, this paper adds convincing evidences in support of innocuous of the aqueous extract of B.triquetrum. Altogether; these results represent another step towards the use of this natural product as phytotherapeutical agent.CNPq (Brasil), número de proyecto PV 400001/2009 8

Minimum information guideline for spectrophotometric and fluorometric methods to assess biofilm formation in microplates

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioflm.2019.100010.The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the necessary information that a manuscript should include for the experiments in a study to be clearly interpreted and independently reproduced. As a result of several discussions between international groups working in the area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm formation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set of recommendations. The intention of the minimum information guideline is to improve the quality of scientific communication that will augment interlaboratory reproducibility in biofilm microplate assays.This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska – Curie grant agreement No 722467, as part of the Print-Aid consortium. The information and views set out in this article are those of the authors and do not necessarily reflect the official opinion of the European Union. Neither the European Union institutions and bodies nor any person acting on their behalf may be held responsible for the use which may be made of the information contained therein. This work received additional financial support by: project UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE funded by national funds through FCT/MCTES (PIDDAC); Project “LEPABE-2-ECO-INNOVATION” – NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio