Microbiota en teléfonos móviles de médicos oftalmólogos / Microbiota in mobile phones of medical ophthalmologist

Objetivo: Determinar la microbiota en teléfonos móviles utilizados durante la consulta oftalmológica por parte del personal médico, de los pacientes y de los familiares. Métodos: Se analizaron los teléfonos celulares del personal médico y de los pacientes y/o los familiares en el área de consulta de la especialidad. Se realizó una encuesta para evaluar el patrón de uso y la desinfección de los teléfonos móviles. Se tomó una muestra de raspado de los celulares. Las muestras obtenidas fueron inoculadas en medios de cultivo y se incubaron a 37 °C durante 24 h. Se identificó género y especie en los cultivos positivos y se analizaron los resultados obtenidos utilizando estadística descriptiva. Resultados: Se analizaron 71 teléfonos celulares del personal médico y 52 de los pacientes y/o los familiares. Los microorganismos aislados en los teléfonos celulares de los médicos oftalmólogos fueron: estafilococos coagulasa negativa (ECN) 50%, Staphylococcus aureus 32,4%, enterobacterias 4,2%, actinomicetos 4,2 y 9,8% resultaron negativos. Por otro lado, en los teléfonos celulares de los pacientes y los familiares, los microorganismos aislados fueron Staphylococcus aureus 75%, estafilococos coagulasa negativa (ECN) 24% y enterobacterias 1%. Conclusiones: Los resultados obtenidos muestran que los teléfonos celulares, tanto del personal médico como de los pacientes y sus familiares, contienen bacterias consideradas patógenas que podrían establecer una infección. Es relevante establecer una práctica rutinaria de limpieza del teléfono celular y concienciar a la población de los hábitos de higiene, puesto que en ellos queda el cuidado de sus ojos después de la consulta

Objective: To determine the microbiota of mobile phones used during the ophthalmological consultation by medical personnel, patients, and family members. Methods: An analysis was made on the mobile phones of the medical staff and of patients and/or family members in the area of clinical specialty. A survey was conducted to evaluate the pattern of use and disinfection of mobile phones. A smear sample was taken from the mobile phones. The specimens obtained were inoculated in culture media and incubated at 37 °C for 24 hours. Genus and species were identified in the positive cultures and the results obtained were analysed using descriptive statistics. Results: An analysis was made on 71 mobile phones of medical personnel and 52 from patients and/or family members. The microorganisms isolated in the mobile phones of the ophthalmologists were: coagulase-negative staphylococci 50%, Staphylococcus aureus 32.4%, enterobacteria 4.2%, Actinomycetes 4.2%, and 9.8% were negative. On the other hand, in the phones of patients and relatives, the isolated microorganisms were Staphylococcus aureus 75%, coagulase-negative staphylococci 24%, and enterobacteria 1%. Conclusions: The results obtained show that mobile phones, both of the medical staff and of the patients and their relatives, contain bacteria considered pathogenic that could cause an infection. It is important to establish a routine practice of cleaning mobile phones and to make the population aware of hygiene habits, since they are responsible for the care of their eyes after consultation

Perfil bacteriológico de superficies y equipos en un servicio de Fisioterapia / Bacteriological profile of surfaces and equipment in a Physiotherapy Unit

Objetivo: Determinar el perfil bacteriológico en superficies de trabajo, aditamentos y equipos del área de Fisioterapia de una institución prestadora de salud de nivel 1 de complejidad en salud de la ciudad de Popayán, Cauca, Colombia, durante el mes de diciembre del 2015. Métodos: Se realizó un estudio descriptivo a partir de la toma de 13 muestras elegidas al azar entre superficies de trabajo, aditamentos y equipos del área de fisioterapia, el aislamiento se realizó a partir de medios de cultivos no selectivos y la identificación bacteriana por técnicas manuales. Resultados: De 13 muestras obtenidas, el 38,5 por mil fueron negativas y el 61,5 por mil fueron positiva, en las cuales en el 53,9 por mil se encontraron estafilococos coagulasa negativa y en el 7,6 por mil se aísla Micrococcus sp. y Bacillus sp. Conclusiones: La desinfección de las superficies de trabajo, aditamentos y equipos debe realizarse con un agente de mayor eficacia y potencia contra bacterias grampositivas, a fin de reducir contaminación de material inertes y posibles infecciones cruzadas

Objective: To determine the bacteriological profile in work areas, fittings, and equipment of the Physiotherapy area of Popayán, Cauca, Colombia. Methods: A descriptive study was performed based on 13 randomly selected samples from work surfaces, fittings, and equipment in the physiotherapy area. The isolation was performed using non-selective culture media, and bacterial identification was by using manual techniques. Results: Of the 13 samples obtained, 38.5 per-mille were negative and 61.5 per-mille were positive, in which 53.9 per-mille were coagulase negative staphylococcus, and 7.6 per-mille isolated Micrococcus sp. and Bacillus sp. Conclusions: Disinfection of work surfaces, fixtures and equipment should be performed with a more effective and potent agent against gram-positive bacteria, in order to reduce contamination of inert material and possible cross-infection

Recuperación de episodios de infección por Clostridium difficile tras la aplicación de un documento de consenso / Recovery from episodes of Clostridium difficile infection following the implementation of a consensus document

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Enrichment and Recovery of Mammalian Cells from Contaminated Cultures Using Aqueous Two-Phase Systems.

This Article describes a density-based method for removing contaminants, including microorganisms and nonviable cells, from mammalian cell cultures using an aqueous two-phase system (ATPS). The properties of a 7% w/w polyethylene glycol (PEG)-11% w/w Ficoll ATPS can be tuned to prepare a biocompatible system that removes contaminants with little to no adverse effects on the viability or growth of the cultured cells after treatment. This system can be used to enrich cell culture populations for viable cells and to reduce the number of microorganism contaminants in a culture, which increases the chances of subsequent antibiotic treatments being successful. We test the effectiveness of our method in model contaminated cultures of both adherent (HeLa) and suspension (HL-60 II) mammalian cells contaminated with bacteria (E. coli) and yeast (S. cerevisiae). An average of 70.2 ± 4.6% of HeLa cells added to the system are subsequently recovered, and 55.9 ± 2.1% of HL-60 II cells are recovered. After sedimenting to the interface of the ATPS, these cells have an average viability of 98.0 ± 0.2% and 95.3 ± 2.2%, respectively. By removing unwanted cells, desired cell populations can be recovered, and cultures that would otherwise need to be discarded can continue to be used.

Actualización en técnicas para diagnóstico microbiológico de infecciones fúngicas / Microbiological diagnosis of fungal infections: an update

Durante los últimos años se ha producido un incremento constante de las infecciones fúngicas, especialmente en pacientes inmunocomprometidos y sometidos a terapias invasivas, en los que un diagnóstico precoz es importante para su tratamiento y evolución debido a la alta mortalidad ocasionada por estas infecciones. Dadas las limitaciones del diagnóstico clásico, basado en la recuperación, cultivo y estudio de las características macroscópicas y microscópicas del hongo, se están desarrollando y aplicando nuevas técnicas basadas en la detección de diferentes metabolitos, antígenos y ácidos nucleicos fúngicos, así como anticuerpos producidos en respuesta a la infección, que facilitan el diagnóstico rápido de estas infecciones, en especial cuando varias de estas técnicas se utilizan combinadas. Algunos de estos nuevos métodos, en general dotados de buena sensibilidad y fiabilidad, requieren metodologías complejas, personal experimentado y están disponibles en muy pocos laboratorios. El presente trabajo muestra una revisión de las técnicas disponibles y otras en desarrollo para el diagnóstico de las infecciones fúngicas, su utilidad clínica y su posibilidad de utilización de forma rutinaria por los laboratorios clínicos y/o la necesidad de contar con laboratorios de referencia (AU)

Recent years have seen an increase in fungal infections, especially in immunocompromised patients undergoing invasive therapy. Conventional diagnosis methods based on culture are labour-intensive, time-consuming, have variable sensitivity, and in some cases are infeasible due to clinical factors. New assays such as detection of different metabolites, antigens, fungal nucleic acids and antibodies play an important role in the diagnosis and monitoring of response to therapy, especially when several of these techniques are used in combination. Some of these new methods with good sensitivity and reliability require complex methodologies, experienced personnel and are available in very few clinical laboratories. We present a review of the available techniques and other investigational platforms that have recently emerged as promising tools for the diagnosis of fungal infections, their clinical utility and their potential routine use in clinical laboratories and/or the need to rely on reference laboratorios (AU)

Controles interlaboratorio: algo más que un examen de conocimientos / Interlaboratory controls: more than just a knowledge test

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ADN de Chlamydia trachomatis en leucocitos de sangre periférica de neonatos / Chlamydia trachomaatis DNA in leukocytes of peripheral blood from neonates

INTRODUCCIÓN: El diagnóstico de infección por Chlamydia trachomatis es difícil en recién nacidos; sin embargo, este se realiza mediante el cultivo celular o por la detección de anticuerpos IgM anti-C. trachomatis (anti-CT). La detección de ADN de C. trachomatis en leucocitos de sangre mediante reacción en cadena de la polimerasa (PCR) podría ser una mejor herramienta para el diagnóstico de infección por este patógeno. MATERIAL Y MÉTODOS: Se analizaron 44 recién nacidos, todos ellos prematuros y con peso menor de 2.500 g. De cada paciente se obtuvieron muestras de sangre y de lavado nasofaríngeo. El ADN de los leucocitos fue obtenido mediante la técnica de fenol-cloroformo. La detección de C. trachomatis fue llevada a cabo mediante la amplificación del gen ompA utilizando el PCR de punto final. Además, se realiza- ron las pruebas de cultivo celular y la detección de anticuerpos IgM anti-CT mediante la técnica de microinmunofluorescencia. RESULTADOS: Veinte pacientes fueron PCR-positivo (45,5%), y la prueba se asoció significativamente con la presencia de neumonía (RR = 2,28; IC 95%: 1,01-5,17; p = 0,035). El cultivo celular de lavado nasofaríngeo solo fue positivo en 7 muestras y no hubo asociación significativa con algún dato clínico o de laboratorio. El título de anticuerpos anti-CT asociado al PCR-positivo fue 1:32 (RR = 2,74; IC 95%: 1,21-6,23; p = 0,008); sin embargo, este título no se asoció a la presencia de neumonía. CONCLUSIÓN: La detección de ADN en leucocitos de sangre periférica podría ser útil para el diagnóstico de infección por C. trachomatis

INTRODUCTION: Diagnosis of Chlamydia trachomatis infection in newborns is difficult; however, this diagnosis is performed by cell culture or by detection of IgM antibodies against C. trachomatis. Detection of C. trachomatis DNA in peripheral blood leukocytes using polymer chain reaction (PCR) may be a better tool for the diagnosis of infection by this pathogen. MATERIAL AND METHODS: A total of 44 premature newborns, all weighing less than 2500 g, were included in the study. A blood sample and nasopharyngeal lavages were obtained from each newborn. Leukocyte DNA was obtained by phenol-chloroform extraction technique. Detection of C. trachomatis was performed by amplifying the ompA gene using the PCR endpoint. Cell culture tests and the detection of IgM antibodies against C. trachomatis by microimmunofluorescence assay were also performed. RESULTS: Twenty newborns were PCR-positive (45.5%), with this test being significantly associated with the presence of pneumonia (RR = 2.28; 95% CI: 1.01 to 5.17; P = .035). The cell culture of nasopharyngeal lavage was positive in only 7 samples and no significant association was observed with any clinical or laboratory data. The titer of IgM antibodies against C. trachomatis associated with PCR-positive was 1:32 (RR = 2.74; 95% CI: 1.21 to 6.23; P = .008), however this titer was not associated with the presence of pneumonia. CONCLUSION: DNA detection in peripheral blood leukocytes could be useful for diagnosis of C. trachomatis infection

Removal of transmissible spongiform encephalopathy prion from large volumes of cell culture media supplemented with fetal bovine serum by using hollow fiber anion-exchange membrane chromatography.

Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco's modified Eagle's medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth media supplemented with FBS.

Recogida de urocultivo por sondaje vesical en niños: una observación sistemática / Coleta de urocultura por sonda vesical em criança: uma observação sistemática / Collect of uroculture by urinary catheter in child: a systematic observation

La infección urinaria se caracteriza por la invasión de microorganismos en el tracto urinario, siendo una de las patologías más frecuentes en todos los grupos de edad, especialmente en niños. El urocultivo se considera el método estándar-oro para el diagnóstico de laboratorio, ofreciendo un alto valor predictivo positivo, si se garantiza una técnica aséptica durante la recolección. El estudio de tipo evaluativo, prospectivo y cuantitativo, tiene por objeto comparar los resultados de los urocultivos por sondaje vesical recogidos por enfermeras del Ambulatorio y Enfermería de un Hospital Universitario Pediátrico teniendo en cuenta el material y la técnica utilizada en el procedimiento.La población de estudio consistió en 12 enfermeros, 4 residentes y los resultados de 300 muestras de cultivos de orina en el período junio-agosto de 2012. Los datos fueron recolectados a través de la observación sistemática y la documentación técnica. Los resultados mostraron una mayor contaminación del procedimiento en la enfermería (13,3%) que en el Ambulatorio (1,3%), donde se utilizó kit específico de urocultivo (AU)

A infecção urinária se caracteriza pela invasão de microrganismos no trato urinário, sendo uma das patologias mais frequentes em todas as faixas etárias, principalmente, em crianças. A urocultura é considerada o método padrão-ouro de diagnóstico laboratorial por oferecer alto valor preditivo positivo, se garantida uma técnica asséptica durante a coleta da urina. O estudo do tipo avaliativo, prospectivo e quantitativo objetivou comparar os resultados das uroculturas por sonda vesical coletada por enfermeiros do Ambulatório e Enfermaria de um Hospital Universitário Pediátrico, considerando o material e a técnica utilizada no procedimento. A população estudada foi composta por 12 enfermeiros, 4 residentes de enfermagem e pelos resultados das 300 amostras das uroculturas no período de junho a agosto de 2012. Os dados foram coletados mediante observação sistemática e técnica documental. Os resultados apontaram maior contaminação do procedimento na Enfermaria (13,3%) do que no Ambulatório (1,3%), onde se utilizou kit específico de urocultura

Urinary tract infection is characterized by invasion of microorganisms in the urinary tract, one of the most frequent pathologies in all age groups, especially in children. The uroculture is considered the gold standard method of diagnosis for offering high positive predictive value, if guaranteed aseptic technique during the urine collected. The assessment study, prospective and quantitative aimed to compare the results of urine cultures for catheter collected by nurses in the clinic and infirmary of a University Hospital Pediatric, considering the material and the technique used in the procedure. The study population consisted of 12 nurses and 4 nursing home residents and the results of 300 samples of uroculture in the period June-August 2012. Data were collected through systematic observation and technical documentation. Results showed higher contamination procedure Infirmary (13.3%) than in the Clinic (1.3%), which we used urine specific kit (AU)

Optimización de la metodología de cultivo de condrocitos de discos procedentes de hernias discales y comprobación de la presencia del Toll-like receptor 4 (TLR 4) / Optimization of methods of chondrocyte cell culture from human disc hernia subjects and testing the presence of Toll-like receptor 4 (TLR 4)

Objetivo: Optimizar la técnica de cultivo de células del disco intervertebral en humanos y confirmar la expresión del Toll-like receptor 4 (TLR4 ). Material y método: Se cultivaron muestras de núcleo pulposo obtenidas durante la cirugía de hernia discal. Las células cultivadas de los nueve pacientes (todos con degeneración del disco moderada-grave en la escala de Pfirrmann) fueron observadas para determinar su capacidad de crecimiento. Las células cultivadas obtenidas se estudiaron con el fin de determinar su fenotipo mediante inmunotinción y rtPCR. La presencia de TLR4 fue comprobada por los mismos métodos. Resultados: Las células aisladas se sembraron a diferentes concentraciones. La concentración ideal se obtuvo para las condiciones óptimas del cultivo. Se confirmó que el fenotipo de las células fue condrocitario y se confirmó la presencia del receptor TLR4 en las células cultivadas. Conclusión: Se confirma la presencia tanto de ARNm como de la proteína del receptor TLR4 en los condrocitos del disco intervertebral. Este hallazgo allana el camino para la caracterización de las funciones de este receptor en los procesos inflamatorios de la hernia de disco (AU)

Objective: To optimize the technique of culturing human intervertebral disc cells, and to confirm the expression of toll-like receptor 4 (TLR4) in these cells. Material and method: Samples of nucleus pulposus obtained during disc hernia surgery were cultured. Cells from nine patients (all with moderate-severe disc degeneration scores, based on the Pfirmann scale) were followed up to determine their growing capacity. The obtained cultured cells were tested for the chondrocyte phenotype by immunostaining and rt-PCR and the presence of TLR4 was tested by the same methods. Results: The cells were isolated and seeded at different concentrations. The ideal concentration was obtained for optimal culture conditions. The cells were confirmed to have the chondrocyte phenotype and TLR4 was confirmed to be present in the cultured cells. Conclusion: This study confirms the presence of both mRNA and TLR4 protein in intervertebral disc chondrocytes. This paves the way for elucidating of the roles of this receptor in the inflammatory processes of disc hernia (AU)

Single step, rapid identification of pathogenic microorganisms in a culture bottle.

Efforts to treat bloodstream infections, which have a relatively high mortality rate, are delayed by the lengthy multi-step process required to identify the causative bacteria. Due to this delay, broad spectrum antibiotics are prescribed on a presumptive basis, leading to the rise of antibiotic resistant microorganisms. Here, as proof of principle, we describe a colourimetric sensor that rapidly identifies opportunistic pathogenic bacteria in a single step in TSB media. The device is composed of a reaction chamber and an array of chemoresponsive dyes deposited on a substrate in a prearranged pattern. This single step, disposable, automated system can detect and identify of eight strains of bacteria, starting with clinically relevant concentrations bacteria in twenty four hours in TSB media. Thus, this technology may be used to streamline the current blood culture process by combining detection and identification in a single step.

Invasion of solid culture media: a widespread phenotypic feature of clinical bacterial isolates

Objetivos. El crecimiento en profundidad en medios de cultivo sólidos es un fenómeno habitual en hongos filamentosos y levaduras. Sin embargo, son muy escasas las especies bacterianas en las que se ha documentado este fenómeno. El objetivo de este trabajo fue comprobar la capacidad de invasión del agar de un amplio abanico de especies bacterianas grampositivas y gramnegativas de interés clínico. Material y métodos. Se cultivaron tres cepas de cada una de once especies bacterianas sobre agar Columbia hasta un máximo de 15 días. Colonias aisladas fueron procesadas e incluidas en resina epoxi por métodos histológicos y secciones transversales semifinas teñidas con azul de toluidina fueron visualizadas por microscopía óptica. Resultados. Se observó crecimiento en el interior del agar en al menos una de las cepas de nueve de las especies estudiadas. En bacilos gramnegativos, las invasiones eran escasas en número, pequeñas y con morfología redondeada o triangular. En cocos grampositivos las invasiones eran de gran tamaño, numerosas y de morfología variable (lentiforme, globular, irregular, en forma de punta de flecha, etc.) según la especie. Conclusiones. En nuestra opinión, el crecimiento en el interior del agar puede significar una estrategia de supervivencia común a muchas especies bacterianas y hasta ahora prácticamente no descrita. Esta estrategia podría ser el reflejo de un tropismo a favor de gradiente de nutrientes, o bien un fenómeno de diseminación y colonización de nuevos nichos ecológicos, con posibles implicaciones en la patogenia(AU)

Objectives. The in-depth growth in solid culture media is a common feature in filamentous fungi and yeasts. However, there are very few bacterial species in which this phenomenon has been documented. The aim of this work was to assess the agar invasiveness of a wide range of Gram-positive and Gram-negative bacterial species of clinical interest. Material and methods. Three different clinical isolates for each of eleven bacterial species were plated onto Columbia blood agar and let grow up to 15 days. Isolated colonies were processed by histological methods, embedded in epoxy resin, and then, semithin sections were stained with toluidine blue and visualized by light microscopy. Results. Growth within the agar was observed in at least one strain in 9 of the 11 studied species. Invasions of Gramnegative rods were small, not plentiful, and round or triangleshaped. In Gram-positive cocci, invasions were of big size, abundant and of variable shape (lentiform, globular, irregular, arrowhead) depending on the species. Conclusions: We propose that the growth within the agar can indicate a survival strategy common to many bacterial species, and so far, not previously reported. This strategy could be either a nutrient gradient tropism or the spread and colonization of new ecological niches, with potential implications in pathogeny(AU)

Optimization of flask culture medium and conditions for hyaluronic acid production by a streptococcus equisimilis mutant nc2168

A mutant designated NC2168, which was selected from wild-type Streptococcus equisimilis CVCC55116by ultraviolet ray combined with60Co-γ ray treatment and does not produce streptolysin, was employed to produce hyaluronic acid (HA). In order to increase the output of HA in a flask, the culture medium and conditions for NC2168 were optimized in this study. The influence of culture medium ingredients including carbon sources, nitrogen sources and metal ions on HA production was evaluated using factional factorial design. The mathematical model, which represented the effect of each medium component and their interaction on the yield of HA, was established by the quadratic rotary combination design and response surface method. The model estimated that, a maximal yield of HA could be obtained when the concentrations of yeast extract, peptone, glucose, and MgSO4 were set at 3 g/100 mL, 2 g/100 mL, 0.5 g/100 mL and 0.15 g/100 mL, respectively. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a remarkable increase in the output of HA and the maximum of the predicted HA production was 174.76 mg/L. The model developed was accurate and reliable for predicting the production of HA by NC2168.Cultivation conditions were optimized by an orthogonal experimental design and the optimal conditions were as follows: temperature 33ºC, pH 7.8, agitation speed 200 rpm, medium volume 20 mL.

Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment

A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO) were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 10(8) pfu/mL) without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL), after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino / Potential of neural transdifferentiation of mesenchymal stem cells from equine bone marrow

Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1. Com relação a análise imunofenotípica foi observado uma maior (P<0,001) expressão dos marcadores GFAP e β3 tubulina ao término do P2 quando comparado ao P1. A habilidade das CTMs em gerar tipos celulares relacionados a linhagem neural é complexa e multifatorial, dependendo não só dos agentes indutores, mas também do ambiente no qual estas células são cultivadas. Desta forma um maior número de estudos é necessário para o melhor entendimento do processo de transdiferenciação neural a partir de CTMs de equinos.

The first studies showing the potential of neural transdifferentiation of mesenchymal stem cells (MSCs) from bone marrow (BM) were conducted in camundogos and humans in the early 2000s. After this period, the number of research and publications with the same purpose increased, but with rare or scarce studies in horses. The aim of this study was to evaluate in vitro neuronal transdifferentiation potential of MSCs from equine BM using two protocols: P1 (forksolin and retinoic acid) and P2 (2-βmecarptoetanol). After confirming the mesenchymal lineages, by positivity for the marker CD90 (X=97.94%), negative for the marker CD34 and positive response for osteogenic differentiation, MSCs were subjected to neural transdifferentiation (P1 and P2) for morphological analysis and expression of neural markers GFAP and β3 tubulin by flow cytometry. The results revealed morphological changes in varying degrees between the tested protocols. In protocol 1, twenty four hours after incubation with the media of neural differentiation, a large proportion of cells (>80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension. It was also evidenced with the use of this protocol, lower cellular adhesion after exposure to media when compared to P1. Regarding the immunophenotyping analysis it was observed a higher (P<0.001) expression of the markers GFAP and β3 tubulin at the end of P2 compared to P1. The ability of MSCs to generate cell types related to neural lineage is complex and multifactorial, depending not only of inducing agents, but also the environment in which these cells will be cultivated. Thus a greater number of studies are necessary to better understand the process of neural transdifferentiation of MSCs from equine.

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos / In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.

The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12.Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Aislamientos por Staphylococcus lugdunensis. ¿Patógeno o contaminante en pacientes oncohematológicos? / Isolation by Staphylococcus lugdunensis. Pathogens or contaminants in haematology-oncology patients?

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Crecimiento fúngico en medios de cultivo que simulan un ambiente extremo / Fungal growth in culture media simulating an extreme environment

Antecedentes. Hay un creciente interés por el estudio de los microorganismos que habitan ambientes extremos por razones que van desde incrementar el conocimiento sobre el origen de la vida hasta la búsqueda de nuevas aplicaciones biotecnológicas. Objetivos. En el presente trabajo se aborda el estudio de la tolerancia de hongos aislados del Arroyo de Aguas Agrias (AAS; Tharsis, Huelva, España), un ambiente ácido y rico en metales, frente a medios de cultivo preparados con agua procedente de este ecosistema extremo (medio AASW). También se investigó la posibilidad de crecimiento en estas condiciones de cepas de colección de hongos y levaduras. Métodos. Para los hongos miceliares se calculó un índice de tolerancia, definido como el cociente entre el diámetro de crecimiento de las colonias sobre AASW y el que se produce en un medio control. Para las levaduras se determinó la concentración mínima inhibitoria de AASW. Resultados. En general, los hongos aislados del AAS manifestaron diferencias en su capacidad para germinar y crecer sobre el medio AASW. Las cepas de colección del género Aspergillus fueron capaces de crecer sobre el medio AASW, pero mostraron diferencias en su tolerancia al mismo en comparación con los aislamientos ambientales. Conclusiones. Los hongos extremotolerantes pueden manifestar diferencias en su tolerancia a medios de cultivo que simulan las condiciones de su hábitat natural. Los resultados de nuestro trabajo sugieren que la capacidad de los hongos para crecer en ambientes ácidos, ricos en metales, puede ser más común de lo que pudiera pensarse, y pone de manifiesto la importancia de determinar los factores específicos que son responsables de la tolerancia a esos ambientes extremos(AU)

Background. There is an increasing interest in the study of microorganisms that inhabit extreme environments for reasons that vary from gaining insight into the origin of life to the searching of new biotechnological applications. Aims. In this work, we studied the tolerance of fungi isolated from the Aguas Agrias Stream (AAS; Tharsis, Huelva, Spain), an acidic metal-rich environment, to a culture medium prepared with water from this extreme ecosystem (AASW medium). The ability of some culture collection strains of moulds and yeasts to grow on AASW medium was also assessed. Methods. For moulds, a tolerance index was calculated by dividing the growth diameter of colonies on AASW medium by the diameter in the control medium, and their germinative potential was recorded. For yeasts and yeast-like fungi, the minimum inhibitory concentration of AASW was determined. Results. In general, the fungi isolated from the AAS showed differences in their ability to germinate and grow on AASW medium. Collection strains of the genus Aspergillus could grow on AASW medium, but showed some differences in tolerance when compared to environmental isolates. Conclusions. Extremotolerant fungi can manifest differences in their tolerance to culture media that simulate the conditions of their natural habitat. The results of this work suggest that the ability of fungi to grow in acidic, metal-rich environments might be more widespread than previously thought, and highlight the importance of determining the factors that are responsible for tolerance to these extreme environments(AU)

Efecto de distintos antimicrobianos sobre el crecimiento de Listeria monocytogenes / Effect of different antimicrobial agents on the growth of Listeria monocytogenes

En este estudio se ha evaluado la eficacia de dos ácidos orgánicos contemplados en la lista positiva de aditivos alimentarios, el lactato sódico (E-325) y el diacetato sódico (E-262), sobre el crecimiento de Listeria monocytogenes. Estos aditivos se adicionaron en diferentes concentraciones a un medio de cultivo líquido, determinando el incremento de densidad óptica del medio a 600 nm durante 24 horas a 37 ºC, con respecto al medio sin inocular que se tomó como blanco, realizando la medida cada hora. El incremento de absorbancia se midió con respecto al tiempo, evaluando el crecimiento de la bacteria a través de la interpretación de la tasa máxima de incremento de absorbancia (micro) y el tiempo mínimo requerido para detectar un incremento en la densidad óptica del medio (epsilon). Este último parámetro se puede equiparar al tiempo de latencia o tiempo de adaptación al medio. Así, para el lactato sódico, se observó que ejerce un efecto negativo dosis dependiente sobre el crecimiento de L. monocytogenes, prolongando el tiempo que necesitó la bacteria para adaptarse al medio de cultivo (epsilon), sin afectar a la tasa de crecimiento (micro) una vez que esta comenzó a crecer. El diacetato sódico mostró ser más efectivo que el lactato sódico frente al crecimiento de la bacteria, incrementando el tiempo de adaptación al medio, así como disminuyendo la tasa de crecimiento. Además, el diacetato sódico consiguió inhibir de forma completa el crecimiento de la bacteria a concentraciones iguales o superiores a 0.2%(AU)

The effectiveness of two organic acids included in the positive list for additives, sodium lactate (E-325) and sodium diacetate (E-262), was evaluated against Listeria monocytogenes growth. Different concentrations of these additives were added to the liquid culture medium. The optical density increments at 600 nm was measured for a 24 hours period under 37 0C, using non-inoculated medium as blank. The measurements were taken every hour in sterile 96 wells plates each. After this analysis, a graphical representation of absorbance increment against time was done, extrapolating the maximum absorbance increment rate (micro) and the minimum time required to detect an absorbance increment (epsilon) from the graphic. These two parameters made possible to evaluate the bacterial growth. After the analysis of epsilon and micro for lactate concentrations, a negative effect in bacterial growth was observed, extending epsilon value. Nevertheless, once the bacterial growth started, any effect on micro value was detected. A higher inhibitory effect was observed after the analysis of these parameters for diacetate concentrations, an extension on epsilon value as well as a micro value descent was found. In this way, a total inhibition of growth occurred when diacetate concentration was 0,2% or higher(AU)

Formulation of economical microbial feed using degraded chicken feathers by a novel Streptomyces sp: mitigation of environmental pollution

A new Streptomyces sp. IF 5 was isolated from the feather dumped soil and found to have a tremendous keratinase activity. The strain enabled the degradation of the chicken feathers very effectively in 60 h. The 16S rRNA sequence of 1474 bp long was submitted to the National centre for Biotechnological information. The keratinolytic activity in the culture medium was 1181 U/ml. The release and analyses of sulphydryl groups in the culture medium evident the degradation activity by the Streptomyces sp. IF 5. The idea of the present study was to use the degraded chicken feathers as the substrate for the growth and cultivation of microorganisms. We have designed a very economical culture medium that includes the usage of some basal salts alone and degraded chicken feathers (10 g/l). The results of the specific growth rate of the tested microbes confirm the usage of the new designed medium for microbial culturing.