Effect on the reduction of bacterial load after surgical hand antisepsis with triclosan 0.5% compared to triclosan 0.5% followed by 70% alcoholic solution.

Triclosan 0.5% by scrubbing does not meet the UNE-EN12791 criteria to be used in the surgical hand preparation (SHP). Triclosan 0.5% by scrubbing followed by ethanol 70% hand rubbing is suitable without the additional characteristic of sustained effect. This limited effectiveness implies that triclosan should be avoided in SHP given the restrictions on its use in consumer antiseptic products. The trial was registered at ClinicalTrials.gov (ID: NCT04538365).

Comparing rubbing and scrubbing surgical hand antisepsis with propan-1-ol 60% in accordance with European regulation UNE-EN 12791:2016+A1:2018.

A crossover trial was conducted to compare hand rub and hand scrub-brush methods for reducing bacterial loads when using propan-1-ol-60% according to European regulations. Both methods significantly reduced the bacterial load immediately after antisepsis, but only the hand rub method achieved significant bacterial load reduction 3 hours after the procedure.

Implicación de los alumnos del Grado de Podología en la innovación docente de la Microbiología / Implication of the students of the Degree of Podiatry in the teaching innovation of Microbiology

Se ha planteado como objetivo la mejora de la calidad de la docencia de la Microbiología mediante la actualización de la metodología docente , introduciendo como actividad docente el aprendizaje activo basado en preguntas (inquiry-based learning:IBL) para conseguir mejorar las competencias que deberán adquirir los estudiantes como parte de su formación integral. En este estudio han participado 63 alumnos de Segundo Curso del Grado de Podología de la Universidad Complutense de Madrid (Curso 2017-2018), y se ha calculado el porcentaje de alumnos que participaron en las 4 pruebas (4IBL), 3 pruebas (3 IBL), en 2 (2 IBL), en 1 (1 IBL), y los que no participaron en ninguna y posteriormente se relacionó con las calificaciones obtenidas en la asignatura de Microbiología. Se incluyeron las preguntas IBL que se realizaron en clase en el campus virtual de la asignatura, pero incluyendo la corrección de las mismas.En los alumnos que realizaron alguna prueba IBL se obtuvieron calificaciones mejores que en aquellos que no habían participado en ninguna. Consideramos que el uso del campus virtual unido a la actualización en la metodología docente puede mejorar el rendimiento académico de los estudiantes de Microbiología

Our focus here is to improve teaching methods in University microbiology classes and the development of educational practices that explicit engage students while enhacing how they learn Microbiology. These practices including inquiry-based learning (IBL) in which students are actively engaged in the learning process for improving the skills to be acquired as part of their effective learning . This study included 63 Microbiology students of Second Year Degree in Podiatry at the Complutense University (Academic Year 2017-2018), and calculated the percentage of students who used these resources 4 IBL, 3 IBL , 2IBL , 1IBL , and that did not participate in any and ubsequently related to the student performance in the Microbiology exam . The IBL questions that were done in class ,were included in the virtual campus , including the correction .Students who prepared IBL scored higher en exam questions , findings also indicate that this activity can measure learning and improve exam preparation. We believe that the benefits of active learning methods with virtual campus over lecture instruction are consistent and can increases student performance in Microbiology

Adaptación de la enseñanza de la microbiología en el grado de podología incluyendo docencia práctica / Adaptation of the teaching of microbiology in the degree of podology, including practical teaching

Con la adaptación al EEES (Espacio Europeo de Educación Superior), se ha comenzado a impartir Microbiología dentro de la materia de Biopatología, introduciendo como actividad docente los créditos prácticos de Microbiología para conseguir mejorar las competencias que deberán adquirir los estudiantes como parte de su formación integral. Se compararon los resultados de la evaluación de 34 alumnos del curso 2011-2012, analizando los resultados entre la pregunta de integración teórico-práctica (TP)y la que únicamente se había explicado en la clase teórica (T).Se encontraron diferencias significativas (p<0,001) entre la nota media que presentaron los alumnos que habían superado la asignatura y los que no la habían superado para la prueba T, mientras que estas medias no fueron estadísticamente significativas para la prueba TP .Podemos concluir que el aprendizaje evaluado en la pregunta TP ha sido satisfactorio en los dos grupos, incluso los alumnos que no han superado la asignatura han logrado alcanzar buenos resultados en esta prueba

With the adaptation to the EHEA (European Higher Education Area ), Microbiology has begun to be taught within the field of Biopathology, introducing as a teaching activity the practical credits of Microbiology in order to improve the skills that students should acquire as part of their integral training. The results of the evaluation of 34 students of the 2011-2012 academic year were compared, analyzing the results between the question of theoretical-practical integration (TP) and the one that had only been explained in the theoretical class (T). Significant differences were found ( p <0.001) between the average score of the students who had passed the course and those who had not passed it for the T test, while these means were not statistically significant for the TP test. We can conclude that the learning evaluated in the question TP has been satisfactory in both groups, even students who have not passed the course have achieved good results in this test

Prevalence of Staphylococcus spp. nasal colonization among doctors of podiatric medicine and associated risk factors in Spain.

Background: This study aimed to estimate the prevalence of methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) nasopharyngeal carriage among Doctors of Podiatric Medicine (Podiatrists) and to determine the potential risk factors. Methods: A cross-sectional study was carried out in 2016-2017 among 239 podiatrists in Spain. The presence of MSSA, MRSA, and MRSE was determined by microbiological analysis of nasal exudate and antimicrobial susceptibility was determined. Each podiatrist completed a questionnaire. The questionnaire comprised various parameters such as sex, age, podiatry experience duration, underlying diseases, prior antibiotic treatment, hospitalization during the last year, and use of a protective mask, an aspiration system, or gloves. Results: The prevalence of MSSA, MRSA, and MRSE was 23.0%, 1.3%, and 23.8%, respectively. The MSSA prevalence was higher among podiatrists who did not use an aspiration system (32.3%) compared to those who did (19.3%; p = 0.0305), and among podiatrists with respiratory diseases (36.8%) compared to those without (20.8%; p = 0.0272). The MRSE prevalence was higher among men (33.7%) compared to women (8.6%; p = 0.0089), podiatrists aged ≥50 (38.5%) compared to ≤35 (17.8%; p = 0.0101), and podiatrists with ≥15 (39.3%) compared to ≤5 years of podiatry experience (12.5%; p = 0.0015). Among the S. aureus strains, 84.5% were resistant to penicillin, 22.4% to erythromycin, 20.7% to clindamycin, and 12.7% to mupirocin. The MRSE strains were resistant to penicillin (93.0%), erythromycin (78.9%), and mupirocin (73.7%). Conclusions: The prevalence of S. aureus and S. epidermidis nasal carriage is low among Spanish podiatrists compared to other health professionals.

¿Está indicada la prescripción de antibióticos en la extracción del tercer molar retenido?: estudio comparativo entre patrones de prescripción / Is it necessary to prescribe antibiotics in impacted third molar surgical removal?: comparative study between prescribing patterns

Objetivo. Valorar si existe una diferencia significativa en tasa de infección tras cirugía de extracción dentaria en dos hospitales de Noruega y España, donde se aplican protocolos de profilaxis quirúrgica diferentes. Material y Métodos. Se realizó un estudio observacional analítico, tipo cohortes retrospectivo, analizando pacientes sanos, sin factores de riesgo, operados de tercer molar incluido en los servicios de maxilofacial de dos hospitales diferentes: St. Olav de Trondheim (Noruega) y Clínico San Carlos de Madrid (España). Se recogieron las variables: edad, número de piezas extraídas, tipo de anestesia, y observaciones recogidas en la historia clínica sobre el curso de la operación. Para valorar el desarrollo infección postoperatoria se recogieron los datos de los pacientes que escogieron el hospital como lugar de retirada de los puntos en el Hospital St. Olav, y en el Hospital Clínico San Carlos se llevó a cabo una encuesta telefónica para conocer el curso de la operación meses después. Resultados. El 11,1% de los pacientes operados en el Hospital St. Olav recibió pauta antibiótica durante una semana tras la operación, mientras que en el Hospital Clínico San Carlos fue del 100%. La tasa de infección tras ésta fue del 15% en el Hospital de St. Olav y del 7,5% en el Hospital Clínico siendo estas diferencias no estadísticamente significativas. Conclusiones. La administración sistemática de antibiótico a pacientes sanos sin factores de riesgo sometidos a extracción quirúrgica del tercer molar retenido es una práctica rutinaria en clínica que no parece estar justificada (AU)

Objective. To assess whether there is a significant difference in infection rate after surgery tooth extraction in two different hospitals from Norway and Spain where different surgical antimicrobial prophylaxis protocols are applied. Methods. An analytical observational study was conducted, retrospective cohorts type, analyzing healthy patients with no risk factors, who were third molar tooth operated in maxillofacial services of two different hospitals: St. Olav in Trondheim (Norway) and Clínico San Carlos in Madrid (Spain). The collected variables were: age, number of tooth removed, anesthesia type, and observations about the course of the operation registered in the clinical history. To assess the development of postoperative infection, patient’s data of those who chose the hospital as the place to remove the suture thread were collected in Norway, whereas in Spain a telephone survey was conducted to determine the course of the operation months later. Results. In St. Olav Hospital 11.1% of patients operated received antibiotic regimen after surgery, while in Hospital San Carlos were 100%. The infection rate was 15% in St.Olav Hospital and 7.5% in Hospital San Carlos. These differences were no statistically significant. Conclusions. The routine administration of antibiotics to healthy patients with no risk factors undergoing impacted third molar surgical removal is a common clinical practice which it does not seem to be justified (AU)

Bacterial translocation affects intracellular neuroinflammatory pathways in a depression-like model in rats.

Recent studies have suggested that depression is accompanied by an increased intestinal permeability which would be related to the inflammatory pathophysiology of the disease. This study aimed to evaluate whether experimental depression presents with bacterial translocation that in turn can lead to the TLR-4 in the brain affecting the mitogen-activated protein kinases (MAPK) and antioxidant pathways. Male Wistar rats were exposed to chronic mild stress (CMS) and the intestinal integrity, presence of bacteria in tissues and plasma lipopolysaccharide levels were analyzed. We also studied the expression in the prefrontal cortex of activated forms of MAPK and some of their activation controllers and the effects of CMS on the antioxidant Nrf2 pathway. Our results indicate that after exposure to a CMS protocol there is increased intestinal permeability and bacterial translocation. CMS also increases the expression of the activated form of the MAPK p38 while decreasing the expression of the antioxidant transcription factor Nrf2. The actions of antibiotic administration to prevent bacterial translocation on elements of the MAPK and Nrf2 pathways indicate that the translocated bacteria are playing a role in these effects. In effect, our results propose a role of the translocated bacteria in the pathophysiology of depression through the p38 MAPK pathway which could aggravate the neuroinflammation and the oxidative/nitrosative damage present in this pathology. Moreover, our results reveal that the antioxidant factor Nrf2 and its activators may be involved in the consequences of the CMS on the brain.

El aprendizaje activo de la microbiología en el Grado de Podología incrementa el rendimiento académico / Large increases in learning that result with the active teaching methods in Microbiology in Podiatry Degree

Se ha planteado como objetivo la mejora de la calidad de la docencia de la Microbiología mediante la actualización de la metodología docente, introduciendo como actividad docente el aprendizaje activo basado en preguntas (inquirybased learning:IBL) para conseguir mejorar las competencias que deberán adquirir los estudiantes como parte de su formación integral. En este estudio han participado 55 alumnos de Segundo Curso del Grado de Podología (Curso 2014-2015), y se ha calculado el porcentaje de alumnos que participaron en las 3 pruebas (3 IBL), en 2 (2 IBL), en 1 (1 IBL), y los que no participaron en ninguna y posteriormente se relacionó con las calificaciones obtenidas en la asignatura de Microbiología. Se incluyeron las preguntas IBL que se realizaron en clase en el campus virtual de la asignatura, pero sin incluir la corrección de las mismas. En los alumnos que realizaron alguna prueba IBL se obtuvieron calificaciones mejores en las preguntas diseñadas para analizar la síntesis de conocimientos y el análisis de datos que en aquellos que no habían participado en ninguna. Al finalizar la actividad se realizó un estudio transversal a través de un cuestionario autocumplimentado en el que se valoraba la opinión de los alumnos sobre el aprendizaje activo mediante IBL valorando positivamente esta actividad para medir el aprendizaje y mejorar la preparación del examen. Consideramos que el uso del campus virtual unido a la actualización en la metodología docente puede mejorar el rendimiento académico de los estudiantes de Microbiología (AU)

Our focus here is to improve teaching methods in University microbiology classes and the development of educational practices that explicit engage students while enhacing how they learn Microbiology. These practices including inquiry-based learning (IBL) in which students are actively engaged in the learning process for improving the skills to be acquired as part of their effective learning . This study included 55 Microbiology students of Second Year Degree in Podiatry (Academic Year 2014-2015), and calculated the percentage of students who used these resources 3 IBL, 2IBL, 1IBL, and that did not participate in any and subsequently related to the student performance in Microbiology exam. The IBL questions that were done in class ,were included in the virtual campus , but not including the correction. Students who prepared IBL scored higher en exam questions designed to test synthesis of knowledge and analysis of data. In order to assess the perceived impact of the active learning, the students were asked to fill out a questionnaire evaluating IBL positively considering that this activity can measure learning and improve exam preparation. We believe that the benefits of active learning methods over lecture instruction are consistent and can increases student performance in Microbiology (AU)

Genotypic and phenotypic diversity in Enterococcus faecalis: is agar invasion a pathogenicity score? / Diversidad genotípica y fenotípica en Enterococcus faecalis: ¿Es la invasión en agar un marcador de patogenicidad?

Objectives. The main objective of the present study is to analyze different genotypic and phenotypic traits related to virulence in Enterococcus faecalis, as well as evaluated the agar invasion phenotype in a collection of isolates with different clinical origins. Material and methods. Seventy-nine E. faecalis isolates, with invasive and non-invasive clinical origins, have been used in this work. Presence of cytolysin activator (cylA), gelatinase (gelE), surface protein (esp), aggregation substance (asa1), endocarditis antigen (efaA), and collagen-binding protein (ace) have been analyzed by PCR. Phenotypic characterization included gelatinase activity, haemolysin production, biofilm formation and agar invasion. Results. All the isolates tested harboured at least one of the virulence determinants. The 95.5% of isolates from haematologic samples were positive for agar invasion test, significantly higher than isolates from non-invasive diseases. A significant reduction in relative invasion area was observed in three selected agar-invasive strains after 15 serial passages. Conclusions. It has been observed a significant high prevalence of agar-invasion positive isolates among strains belonged to haematological samples. Agar invasiveness is reduced after adaptation of clinical isolates to laboratory conditions, showing that agar invasion phenotype can be modulate by culture conditions as other virulence factors observed in different bacterial species (AU)

Objetivos. El principal objetivo de este trabajo es la caracterización de determinantes de virulencia genotípicos y fenotípicos relacionados con patogenicidad en Enterococcus faecalis, evaluando además el fenotipo de invasión en agar en una colección de aislados clínicos de diversa procedencia. Material y métodos. Se han analizado 79 cepas de E. faecalis aisladas en infecciones invasivas y no invasivas. La detección de los principales determinantes asociados a la virulencia (cylA, gelE, esp, asa1, efaA y ace) se ha realizado mediante PCR. La caracterización fenotípica incluyó la detección de actividad gelatinasa, hemólisis, formación de biofilm y el test de invasión en agar. Resultados. Todos los aislados presentaron, al menos, un determinante de virulencia. El 95,5% de las cepas provenientes de hemocultivos resultaron positivas para el test de invasión en agar, significativamente superior a lo observado en cepas de origen clínico no invasivo. En tres cepas seleccionadas, positivas para el test de invasión en agar, se observó una reducción significativa del área relativa de invasión tras 15 pases seriados. Conclusiones. Se ha observado una alta prevalencia de cepas con alto grado de invasión en agar en los aislados hematológicos. Dicho grado de invasión disminuye significativamente al adaptar tres cepas al crecimiento en condiciones de laboratorio, sugiriendo una modulación en función de las condiciones de cultivo tal y como ocurre con otros determinantes asociados a virulencia en diferentes especies bacterianas (AU)

Impacto del aprendizaje activo de la Microbiología en el Grado de Podología / Impact of active learning: Teaching Microbiology in the degree of Podiatry

Con la adaptación al Espacio Europeo de Educación Superior, se ha planteado como objetivo la mejora de la calidad de la docencia de la Microbiología mediante la renovación de la metodología docente, introduciendo como actividad docente el aprendizaje activo basado en preguntas (inquiry-based learning:IBL) para conseguir mejorar las competencias que deberán adquirir los estudiantes como parte de su formación integral. En este estudio han participado 49 alumnos de Segundo Curso del Grado de Podología, y se ha calculado el porcentaje de alumnos que participaron en las 3 pruebas (3 IBL), en 2 (2 IBL), en 1 (1 IBL), y los que no participaron en ninguna y posteriormente se relacionó con las calificaciones obtenidas en la asignatura de Microbiología. En los alumnos que realizaron alguna prueba IBL se obtuvieron calificaciones mejores en las preguntas diseñadas para analizar la síntesis de conocimientos y el análisis de datos que en aquellos que no habían participado en ninguna. Al finalizar la actividad se realizó un estudio transversal a través de un cuestionario autocumplimentado en el que se valoraba la opinión de los alumnos sobre el aprendizaje activo mediante IBL valorando positivamente esta actividad para medir el aprendizaje y mejorar la preparación del examen. Consideramos que en líneas generales este tipo de estudios pueden optimizar la calidad de la docencia de la Microbiología en los estudios de Grado de Podología (AU)

In response to the Adaptation to the European Higher Education Area, one path to reform is the development of educational practices that explicit engage students while enhacing how they learn Microbiology. These practices including inquiry-based learning (IBL) in which students are actively engaged in the learning process for improving the skills to be acquired as part of their effective learning. This study included 49 Microbiology students of Second Year Degree in Podiatry, and calculated the percentage of students who used these resources 3 IBL, 2IBL, 1IBL, and that did not participate in any and subsequently related to the student performance in Microbiology exam. Students who prepared IBL scored higher en exam questions designed to test synthesis of knowledge and analysis of data. In order to assess the perceived impact of the active learning, the students were asked to fill out a questionnaire evaluating IBL positively considering that this activity can measure learning and improve exam preparation. We believe that this type of study can optimize the quality of teaching of Microbiology in podiatric studies (AU)

Toll-like 4 receptor inhibitor TAK-242 decreases neuroinflammation in rat brain frontal cortex after stress.

BACKGROUND: The innate immune response is the first line of defence against invading microorganisms and it is also activated in different neurologic/neurodegenerative pathological scenarios. As a result, the family of the innate immune toll-like receptors (TLRs) and, in particular, the genetic/pharmacological manipulation of the TLR-4 signalling pathway emerges as a potential therapeutic strategy. Growing evidence relates stress exposure with altered immune responses, but the precise role of TLR-4 remains partly unknown. METHODS: The present study aimed to elucidate whether the elements of the TLR-4 signalling pathway are activated after acute stress exposure in rat brain frontal cortex and its role in the regulation of the stress-induced neuroinflammatory response, by means of its pharmacological modulation with the intravenous administration of the TLR-4 specific inhibitor TAK-242. Considering that TLR-4 responds predominantly to lipopolysaccharide from gram-negative bacteria, we checked whether increased intestinal permeability and a resultant bacterial translocation is a potential regulatory mechanism of stress-induced TLR-4 activation. RESULTS: Acute restraint stress exposure upregulates TLR-4 expression both at the mRNA and protein level. Stress-induced TLR-4 upregulation is prevented by the protocol of antibiotic intestinal decontamination made to reduce indigenous gastrointestinal microflora, suggesting a role for bacterial translocation on TLR-4 signalling pathway activation. TAK-242 pre-stress administration prevents the accumulation of potentially deleterious inflammatory and oxidative/nitrosative mediators in the brain frontal cortex of rats. CONCLUSIONS: The use of TAK-242 or other TLR-4 signalling pathway inhibitory compounds could be considered as a potential therapeutic adjuvant strategy to constrain the inflammatory process taking place after stress exposure and in stress-related neuropsychiatric diseases.

Morphological plasticity of Streptococcus oralis isolates for biofilm production, invasiveness, and architectural patterns.

OBJECTIVE: Streptococcus oralis is an early coloniser of the oral cavity that contributes to dental plaque formation. Many different genotypes can coexist in the same individual and cause opportunistic infections such as bacterial endocarditis. However, little is known about virulence factors involved in those processes. The aim was to analyze the evolving growth of S. oralis colony/biofilm to find out potentially pathogenic features. DESIGN: Thirty-three S. oralis isolates were analyzed for: (1) biofilm production, by spectrophotometric microtiter plate assay; (2) colonial internal architecture, by histological methods and light and electron microscopy; (3) agar invasion, by a new colony-biofilm assay. RESULTS: S. oralis colonies showed two different growth patterns: (1) fast growth rate without invasion or minimally invasive; (2) slow growth rate, but high invasion ability. 12.1% of strains were biofilm non-producers and 24.2% not invasive, compared to 51.5% biofilm high-producers and 39.4% very invasive. Both phenotypic characteristics tended to be mutually exclusive. However, a limited number of strains (15%) co-expressed these features at the highest level. CONCLUSIONS: Morphological plasticity of S. oralis highlighted in this study may have important ecological and clinical implications. Coexistence of strains with different growth patterns could produce a synergic effect in the formation and development of subgingival dental plaque. Moreover, invasiveness might regulate dissemination and colonisation mechanisms. Simultaneous co-expression of high-invasive and high-biofilm phenotypes gives a fitness advantage during colonisation and may confer higher pathogenic potential.

[Structural dynamics of Legionella pneumophila and Legionella bozemanii colony/biofilm]. / Dinámica estructural de colonias/biofilm de Legionella pneumophila y Legionella bozemanii.

OBJECTIVES: The genus Legionella includes very pleomorphic species responsible for disease outbreaks in humans. The appearance of such has great importance to develop artificial biofilms in aquatic ecosystems. The aim of this work was to study the dynamics of growth and evolution of the internal structure of colonies of representative species of the genus as static biofilm model. METHODS: Isolated colonies of Legionella pneumophila and Legionella bozemanii grown in specific media for three and fifteen days were processed for histological methods and embedded in paraffin and epoxy resin for analysis by light microscopy, electron microscopy and image analysis. RESULTS. In colonies of both species were observed and defined specific architectural patterns, based on stratification and evolve over time. The strata differ in the amount of extracellular matrix, the morphology and population density and the proportion of dead cells. The internal structure of three days colonies showed large differences between L. pneumophila (two layers) and L. bozemanii (four layers). However, in the fifteen days colonies of both species evolved towards a common unique pattern formed by three layers. In both species the growth was also found within the culture medium, although this phenomenon was more intense in L. bozemanii with unique, central and larger invasions. CONCLUSIONS: Our results demonstrate that Legionella colonies on solid culture media are a good model of static biofilm with a complex structural dynamics characterized by the presence of morphological and functional subpopulations. We bring here an histological approach model, allowing, in further research, detailed studies in evolutionary adaptations in multicellular communities to adverse media and to antimicrobials in Legionella species of clinical interest.

Dinámica estructural de colonias/biofilm de Legionella pneumophila y Legionella bozemanii / Structural dynamics of Legionella pneumophila and Legionella bozemanii colony/biofilm

Objetivos. El género Legionella engloba especies muy pleomórficas responsables de brotes infecciosos en humanos. En la aparición de los mismos tiene gran importancia el desarrollo de biofilms en ecosistemas acuáticos artificiales. El objetivo de este trabajo fue estudiar la dinámica de crecimiento y la evolución de la estructura interna de colonias de especies representativas del género como modelo de biofilm estático. Material y métodos. Colonias aisladas de Legionella pneumophila y Legionella bozemanii crecidas en medios específicos durante tres y quince días fueron procesadas por métodos histológicos de inclusión en parafina y resina epoxi para su análisis mediante microscopía óptica, microscopía electrónica y análisis de imagen. Resultados. En las colonias de ambas especies se observaron patrones arquitecturales definidos y específicos, basados en la estratificación y que evolucionan en el tiempo. Los estratos se diferencian por la cantidad de matriz extracelular, la morfología y densidad poblacional y la proporción de células muertas. La estructura interna de las colonias de tres días presentaba grandes diferencias entre L. pneumophila (dos estratos) y L. bozemanii (cuatro estratos). Sin embargo, en las colonias de quince días ambas especies evolucionaron hacia un patrón único común formado por tres estratos. En ambas especies se comprobó también el crecimiento en el interior del medio de cultivo, aunque este fenómeno fue mucho más intenso en L. bozemanii, con invasiones únicas, centrales y de gran tamaño. Conclusiones. Nuestros resultados demuestran que las colonias de Legionella sobre medio de cultivo sólido son un buen modelo de biofilm estático, con una dinámica estructural compleja caracterizada por la presencia de subpoblaciones morfológicas y funcionales. La aproximación histológica empleada en este modelo permitirá estudiar adaptaciones evolutivas de comunidades multicelulares a medios hostiles, así como la respuesta a los antimicrobianos de las especies de Legionella de interés clínico (AU)

Objectives. The genus Legionella includes very pleomorphic species responsible for disease outbreaks in humans. The appearance of such has great importance to develop artificial biofilms in aquatic ecosystems. The aim of this work was to study the dynamics of growth and evolution of the internal structure of colonies of representative species of the genus as static biofilm model. Methods. Isolated colonies of Legionella pneumophila and Legionella bozemanii grown in specific media for three and fifteen days were processed for histological methods and embedded in paraffin and epoxy resin for analysis by light microscopy, electron microscopy and image analysis. Results. In colonies of both species were observed and defined specific architectural patterns, based on stratification and evolve over time. The strata differ in the amount of extracellular matrix, the morphology and population density and the proportion of dead cells. The internal structure of three days colonies showed large differences between L. pneumophila (two layers) and L. bozemanii (four layers). However, in the fifteen days colonies of both species evolved towards a common unique pattern formed by three layers. In both species the growth was also found within the culture medium, although this phenomenon was more intense in L. bozemanii with unique, central and larger invasions. Conclusions. Our results demonstrate that Legionella colonies on solid culture media are a good model of static biofilm with a complex structural dynamics characterized by the presence of morphological and functional subpopulations. We bring here an histological approach model, allowing, in further research, detailed studies in evolutionary adaptations in multicellular communities to adverse media and to antimicrobials in Legionella species of clinical interest (AU)

[Biofilm score: is it a differential element within gram negative bacilli?]. / Es la cuantificación del biofilm un elemento diferenciador en la patogenia de bacilos gramnegativos?

The aim of the study was to investigate biofilm formation in Gram negative bacteria and to quantify biofilm production applying a new developed technique that made possible to compare results about biofilm formation within the different Gram negative bacteria species. A total of 153 Gram negative strains corresponding to 12 different bacterium species were studied applying a variation of the optic density measurement technique reported by Stepanovic et al. Data obtained with optic density analysis allow to classify microorganisms in strong biofilm developers, moderate biofilm developers, weak biofilm developers and no biofilm developers. The results were expressed in two ways, using in both cases the same statistical method: without standardization, where controls were different depending on the day optic density measurements were performed, and standardized using a correction factor, using the same control for every strain of all our bacterium species in our study, which allows result homogenization. The obtained results in our study after data analysis and standardization show that over the 153 Gram negative strains in our study, 105 of them were no biofilm developers, representing 63.75% of all the studied bacterium genera. We consider that standardization and quantification of biofilm development in Gram negative bacteria can be useful in clinical practice, because biofilm development ability can lead or focus the gold treatment of pathologies produced by these microorganisms.

Qualitative and quantitative agar invasion test based on bacterial colony/biofilm.

Invasion of the culture medium is a feature frequently studied in yeasts, in which it has been related to a greater virulence, but it is practically unknown in bacteria. Recently, it has been demonstrated that several clinically relevant bacterial species were also able of invading agar media, so it was necessary to design a microbiological assay to study the expression of this character in bacteria. Accordingly, a bacterial agar invasion test based on colony/biofilm development was designed, which allows qualitative and quantitative characterization of bacterial growth into the agar culture medium. Once the culture conditions were optimized, the test was applied to 90 strains from nine bacterial species, validating its usefulness for differentiating invasive strains (positive) from those non invasive (negative). The test also allows sorting invasive strains according to agar invasion intensity (low, moderate, high) and topographic invasion pattern (peripheral, homogeneous, mixed). Moreover, an image analysis routine to quantify the invasion was developed. Implemented method enables direct measuring of two invasion parameters (invasion area and number of invasion dots), automated calculation of three relative variables (invasion relative area, invasion dots relative density, and invasion dot average area), and the establishment of strain specific frequency histograms. This new methodology is simple, fast, reproducible, objective, inexpensive and can be used to study a great number of specimens simultaneously, all of which make it suitable for incorporation to the routine of any microbiology laboratory. It could also be a useful tool for additional studies related to clinical aspects of bacterial isolates such as virulence and antimicrobial response.

¿Es la cuantificación del biofilm un elemento diferenciador en la patogenia de bacilos gramnegativos? / Biofilm score: is it a differential element within gram negative bacilli?

El objetivo del estudio fue investigar la formación de biofilms en bacterias gramnegativas y cuantificar la producción de biofilm mediante la aplicación de una técnica que permitiese una comparación de los resultados de la formación de biofilm entre las diferentes especies de gramnegativos. Se estudiaron un total de 153 cepas de bacilos gramnegativos correspondientes a 12 especies bacterianas por el método de la densidad óptica aplicando una modificación de la técnica descrita por Stepanovic et al. Los valores obtenidos mediante el análisis de la densidad óptica permiten clasificar a los microorganismos en formadores fuertes, moderados, débiles y no formadores. Los resultados obtenidos se han expresado de dos maneras, ambas utilizando el mismo método estadístico: sin estandarizar, donde los controles fueron diferentes dependiendo de los días en los que se realizaron las medidas; y estandarizados mediante un factor de corrección, utilizando el mismo control para todas las cepas de cada especie, lo que permite su homogeneización. Los resultados obtenidos en el estudio tras el análisis y estandarización establecen que de las 153 cepas de gramnegativos estudiados, 105 de ellas fueron no formadoras de biofilms, representando el 63,75% de los géneros estudiados. Consideramos que la estandarización y cuantificación de la producción de biofilm entre las bacterias gramnegativas puede resultar de utilidad en el ámbito clínico, ya que el conocimiento de la capacidad de producción de biofilm puede dirigir o enfocar el tratamiento de elección de las patologías producidas por dichos microorganismos(AU)

The aim of the study was to investigate biofilm formation in Gram negative bacteria and to quantify biofilm production applying a new developed technique that made possible to compare results about biofilm formation within the different Gram negative bacteria species. A total of 153 Gram negative strains corresponding to 12 different bacterium species were studied applying a variation of the optic density measurement technique reported by Stepanovic et al. Data obtained with optic density analysis allow to classify microorganisms in strong biofilm developers, moderate biofilm developers, weak biofilm developers and no biofilm developers. The results were expressed in two ways, using in both cases the same statistical method: without standardization, where controls were different depending on the day optic density measurements were performed, and standardized using a correction factor, using the same control for every strain of all our bacterium species in our study, which allows result homogenization. The obtained results in our study after data analysis and standardization show that over the 153 Gram negative strains in our study, 105 of them were no biofilm developers, representing 63.75% of all the studied bacterium genera. We consider that standardization and quantification of biofilm development in Gram negative bacteria can be useful in clinical practice, because biofilm development ability can lead or focus the gold treatment of pathologies produced by these microorganisms(AU)

Influence of the MBC/MIC ratio on the antibacterial activity of vancomycin versus linezolid against methicillin-resistant Staphylococcus aureus isolates in a pharmacodynamic model simulating serum and soft tissue interstitial fluid concentrations reported in diabetic patients.

OBJECTIVES: To explore serum and tissue pharmacodynamics of linezolid versus vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates with different MBC/MIC ratios. METHODS: Five strains (vancomycin MIC/MBCs, mg/L) were used: TOL-1 (2/≥64), TOL-2 (1/16), LT-1 and LT-2 (1/8) and NT (1/2). The linezolid MIC/MBC for all strains was 2/≥64 mg/L. A two-compartment dynamic computerized device was used (inocula 10(7) cfu/mL). Free concentrations obtained in serum and interstitial fluid with twice-daily regimens of 1 g of vancomycin or 600 mg of linezolid were simulated over 48 h. ABBCs (differences between control growth curves and killing curves of bacteria exposed to antibiotics; log10 cfu × h/mL) and log10 reductions in initial inocula were calculated. RESULTS: In serum simulations, vancomycin (AUC0-24/MIC = 251.8 for TOL-1 and 503.6 for the remaining strains) was bacteriostatic against strains with MBC/MIC ≥8, but bactericidal against NT. In interstitial fluid simulations (AUC0-24/MIC = 54.6 for TOL-1 and 109.2 for the remaining strains), initial inocula grew in all cases. Linezolid, both in serum (AUC0-24/MIC = 87.0) and in interstitial fluid (AUC0-24/MIC = 130.6) simulations, reduced initial inocula ≥2.2 log10 for all strains (apart from LT-1 in serum simulations that showed a bacteriostatic profile). ABBCs were similar in serum and interstitial fluid with linezolid, but significantly lower in interstitial fluid simulations with vancomycin. CONCLUSIONS: From the pharmacodynamic perspective (serum concentrations), vancomycin tolerance should include MBC/MIC ≥8 since strains exhibiting this ratio showed bacteriostatic profiles similar to those obtained with isolates with MBC/MIC ratios of 16 or 32. Insufficient concentrations of vancomycin at the simulated infected site were linked to bacteriological failure. Free concentrations of linezolid at the infection site pharmacodynamically covered MRSA.

Histological approach to Bacillus subtilis colony-biofilm: evolving internal architecture and sporulation dynamics.

Bacillus subtilis has been used as a classic model to study biofilm formation and sporulation process. Colonies of wild-type strains usually have a complex external morphology, but the details of their internal architecture are still undisclosed. Since bacterial biofilms fulfill the criteria to be considered tissues, the aim of this work was to analyse B. subtilis colony-biofilm internal architecture evolution and sporulation dynamics using histological techniques. Transversal sections of colony-biofilms incubated from 24 hours up to 20 days were stained using histochemical techniques to analyse the internal structure by light and electron microscopy. A morphometric study of the different structural biofilm components was performed by image analysis, and an application to quantify spores was developed. Internal biofilm architecture was characterised by a stratified pattern, which evolved from 3 strata at 24 hours, up to 5 strata at 20 days. At 48 hours, strata at the central area of the biofilm was folded, resulting in elevated structures (vein-like structures) that could reach up to 465 µm in height. Sporulation started at 48 hours, at the top of the vein-like structures, at the interface between the two uppermost strata. At 20 days spores formed a continuous central layer, representing 7.5% of the total biofilm. In summary, our results demonstrate that B. subtilis colony-biofilm has a complex and organized internal architecture, evolving over time, and taking place in different cell subpopulations with different functionalities. Furthermore, in situ spore quantification described in this work could be a good alternative to the classical chamber counting.