ES2609612T3 - Una composición para detectar biofilms en tejidos viables - Google Patents
Una composición para detectar biofilms en tejidos viables Download PDFInfo
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- ES2609612T3 ES2609612T3 ES11805905.4T ES11805905T ES2609612T3 ES 2609612 T3 ES2609612 T3 ES 2609612T3 ES 11805905 T ES11805905 T ES 11805905T ES 2609612 T3 ES2609612 T3 ES 2609612T3
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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Abstract
Una composición de tinte para uso en la producción de biofilm detectable en tejido viable, en donde la composición tiñe preferentemente el biofilm en lugar del tejido viable y comprende 0,1 a 2,0% en peso de EDTA, 0,1 a 1,0% en peso de un surfactante, y 0,0001% a 1% en peso, preferentemente 0,0025% a 0,025% en peso de un agente de tinción para teñir dicho biofilm y hacerlo detectable, caracterizada por que la composición es un líquido, el agente de tinción es Rosa Bengala y el surfactante se selecciona de cloruro de benzalconio o cloruro de bencetonio.
Description
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DESCRIPCION
Una composicion para detectar biofilms en tejidos viables.
Esta invencion se refiere a una composicion que puede aplicarse a tejidos viables tales como heridas cronicas (v.g. ulceras de las piernas, ulceras de presion, ulceras de pie diabetico), heridas agudas (v.g. cortes, abrasiones, quemaduras), piel y hueso para la deteccion de biofilms microbianos como un indicador precoz de alarma para tejido en riesgo de infeccion. Mas particularmente, la invencion se refiere a una composicion capaz de producir biofilms en tejidos viables observables al mismo tiempo que se evitan la irritacion de la herida y la piel y el retardo de la curacion.
Los tejidos viables estan colonizados a menudo por una diversidad de microorganismos, algunos de los cuales pueden causar infeccion. Esta siendo cada vez mas aceptado que la curacion de heridas cronicas y agudas se ve dificultada por la presencia de microorganismos que colonizan la herida. Esta surgiendo una evidencia convincente de que estos microorganismos pueden existir en las heridas fundamentalmente en forma de biofilms. Durante la colonizacion, bacterias y otros microorganismos tales como levaduras y hongos, se fijan firmemente al tejido y forman biofilm por la secrecion de una matriz extracelular de sustancias polfmeras. Este modo de crecimiento imparte cierto grado de proteccion a los microorganismos dentro del biofilm en forma de proteccion ffsica contra los agentes antimicrobianos topicos y sistemicos debido a la matriz circundante. Se cree tambien que los microorganismos en el interior de los biofilms tienen fenotipos y genotipos alterados comparados con sus homologos planctonicos que nadan libremente. Es sabido que los microorganismos del biofilm son menos activos metabolicamente y, debido a ello, pueden proporcionar tambien cierto grado de resistencia a los metodos antimicrobianos tradicionales tales como los antibioticos, que se sabe actuan contra las bacterias metabolicamente activas. Adicionalmente, la presencia de biofilms en las heridas inhibe tambien el sistema inmune del hospedador en el aclaramiento de los microbios inflamatorios, y las fases de granulacion y re-epitelializacion, del proceso normal de curacion de las heridas.
Como consecuencia, existe necesidad de desarrollar metodos o dispositivos que detecten rapidamente la presencia de biofilm en los tejidos viables antes y despues de protocolos de tratamiento seleccionados. Esto podna ayudar a los investigadores a comprender si existen microorganismos vivos en el estado del biofilm en la piel y las heridas, y permitir a los mismos seguir la maduracion o aclaramiento de tales comunidades de biofilm. Un metodo o dispositivo de deteccion de biofilm podna permitir tambien que los investigadores desarrollen estrategias anti-biofilm eficaces y protocolos de cuidado eficaces para curacion de las heridas, y en la practica clmica, podna guiar la seleccion de vendajes apropiados de las heridas y ayudar a la monitorizacion de la eficacia de un protocolo de tratamiento dado por el profesional de cuidado de las heridas. Los biofilms estan constituidos tfpicamente por bacterias encerradas dentro de una sustancia (o matriz) exopolfmera que consiste en polisacaridos de cadena larga con p-enlaces complejos tales como enlaces hexosa unidos en 1,3 o 1,4 (ejemplos de algunos polisacaridos comunes de biofilm son acido teicoico, piruvato enlazados a cetal, N- acetil-glucosamina, y los acidos uronicos: acidos D-guluronico, D-galacturonico y manuronico), proterna (algunas de las cuales pueden jugar un papel estructural), DNA (extracelular, algunos de los cuales pueden tener un papel estructural), lfpidos, iones metalicos (Ca2+, Mg2+, Fe3+, etc.) y agua. Los biofilms pueden estar asociados tambien con tejidos desvitalizados del hospedador tales como escara y tejido necrotico.
A las sustancias polfmeras extracelulares (EPS) del biofilm se hace referencia tambien como matriz extracelular del biofilm o tejido derivado de bacterias. Mientras que el biofilm es principalmente agua en peso y las bacterias pueden constituir solo 10-20% del biofilm, el EPS constituye la mayor parte de la biomasa/peso seco del biofilm.
biofilms tales como los encontrados en los dientes en la forma de placa dental, son a menudo faciles de visualizar a simple vista debido a su grosor y su color y a la naturaleza del sustrato sobre el que se forman. La visualizacion del biofilm en, por ejemplo, una herida cronica o aguda no es simple debido a los colores presentes en la herida y los contenidos de la herida. Las heridas cronicas y agudas son usualmente complejas en el sentido de que contienen tejido muerto o desvitalizado (escara), exudado, pus, sangre, medicamentos, componentes del vendaje, ademas de bacterias y biofilm. Por ello, puede ser dificil detectar la presencia de un biofilm en una herida, dado que la visualizacion de los biofilms de la herida a simple vista es dificil. Por tanto, existe necesidad de un medio para ayudar a la deteccion del biofilm, por ejemplo por una composicion que sea capaz de tenir preferentemente los biofilms de heridas de tal modo que los mismos puedan ser visualizados. Una vez visualizado, el biofilm puede tratarse de modo adecuado. Sorprendentemente, se ha encontrado que es posible tenir preferentemente los biofilms por el uso de una composicion que comprende un tinte que permite la deteccion del biofilm.
Composiciones para tincion de biofilms se describen, por ejemplo, en la publicacion de patente internacional WO 2010/070292 A1; sin embargo, las composiciones descritas se encuentran predominantemente en forma de gel.
De acuerdo con ello, un primer aspecto de la invencion proporciona una composicion lfquida de tincion para uso en la produccion de biofilm en tejido viable detectable segun las reivindicaciones adjuntas, en donde la composicion tine preferentemente el biofilm. Con preferencia, el biofilm se hace detectable a simple vista o en asociacion con iluminacion y/o dispositivos opticos.
El agente de tincion es Rosa Bengala.
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Por la expresion “tine preferentemente se entiende que el agente de tincion se fija selectivamente al biofilm en lugar de fijarse al tejido viable del hospedador. De esta manera, el agente de tincion puede utilizarse simplemente para detectar el biofilm por revelado de la presencia y localizacion del biofilm. El agente de tincion queda fijado o adsorbido a las moleculas de la matriz del biofilm extracelular ademas de fijarse o adsorberse a y/o ser capturado por las celulas bacterianas del biofilm en lugar de tejido de la herida. Preferiblemente, la tincion del biofilm por el agente de tincion revela la presencia de biofilm por hacerlo detectable a simple vista. Alternativamente, puede hacerse que el biofilm tenido emita fluorescencia, por ejemplo por iluminacion con una fuente luminosa. La fluorescencia puede hacer mas visible el biofilm tenido. La fuente luminosa se selecciona de modo que emita luz de una longitud de onda apropiada tal que el agente de tincion absorbe energfa luminosa en la forma de fotones que excitan el agente de tincion y causan que el mismo emita fluorescencia. La observacion de la fluorescencia puede mejorarse utilizando filtros opticos apropiados que excluyen las longitudes de onda no fluorescentes del agente de tincion, por ejemplo en la forma de lentes provistas de filtros opticos.
Las composiciones segun un primer aspecto de la invencion comprenden Rosa Bengala como el agente de tincion capaz de tenir preferentemente los biofilms.
El agente de tincion se incluye preferiblemente en la composicion a un nivel que oscila desde 0,0001% a 1% en peso, mas preferiblemente 0,0025% a 0,025% en peso, y aun mas preferiblemente 0,0025% a 0,01% en peso.
Las composiciones de la presente invencion pueden encontrarse en una forma que se adhiere ligeramente a los tejidos y pueden eliminarse facilmente por lavado despues de un breve periodo de tiempo para ayudar a la visualizacion del biofilm tenido. Por ejemplo, una solucion lfquida basada en agua o glicerol (en la forma de un aplicador, nebulizador u hoja), podna proporcionar contacto mtimo con un lecho de herida. En cualquier sistema de suministro de este tipo, la formulacion debena eliminarse preferiblemente con facilidad de los tejidos viables por lavado utilizando un irrigador estandar tal como solucion salina, durante unos cuantos segundos.
La composicion de la invencion puede comprender tambien un humectante tal como propilenglicol (PG), glicerol, polietilenglicol, polidextrosa, sorbitol, trietanolamina, ciclometicona, lactato de amonio o glicerol-ester. Preferiblemente, la composicion comprende desde 5% a 15% en peso de un humectante, y muy preferiblemente PG.
La composicion de la invencion comprende excipientes para optimizar la fijacion del tinte al biofilm, a saber, sal tetrasodica de acido etilenodiamina-tetraacetico (EDTA) a un nivel de 0,1 a 2,0% en peso, y un agente para ayudar a la penetracion del agente de tincion en el biofilm, a saber, el surfactante cloruro de benzalconio (BaCl) o cloruro de bencetonio (BeCl) a un nivel de 0,1 a 1,0% en peso. La adicion de un surfactante puede actuar tambien como agente espumante.
Preferiblemente, la composicion de la invencion tiene un pH comprendido en el intervalo de 5 a 7, y muy preferiblemente alrededor de 5,5. Con preferencia, la composicion de la invencion se encuentra la forma de una solucion aplicada a la herida desde una jeringuilla, bolsita, frasco de nebulizacion, frasco de aerosol, frasco dispensador sin propelente, pincel u hoja de gel, film u oblea que se disuelve.
La formulacion podna esterilizarse finalmente por tratamiento en autoclave o irradiacion gamma. Alternativamente, la formulacion podna ser una solucion preservada que contenga por ejemplo conservantes tales como DMDM-hidantoma o parabenes tales como metil-, etil- o propil-paraben.
La composicion de la presente invencion se utilizara fundamentalmente en tejidos viables que muestren signos de infeccion clmica (inflamacion, olor desagradable, exudado purulento, hipoxia, etc.), puedan encontrarse en riesgo de infeccion, parezcan encontrarse en presencia de escara (tejido derivado del hospedador) o biofilm (tejido derivado de bacterias), o sean reacios en general. La composicion podna utilizarse tambien en caso de cambio de vendaje, a fin de detectar biofilm, y tambien para monitorizar la eficacia del regimen de tratamiento y dirigir el tratamiento futuro por reduccion en el biofilm detectado.
En un ejemplo de uso tfpico, una composicion segun la invencion se aplica a toda la herida o a regiones deseadas a fin de conseguir una capa de composicion fina pero consistente, por ejemplo de 0,1 a 0,5 cm de grosor. La composicion se deja in situ durante 0,1 a 15 min, mas preferiblemente 0,5 a 2 min. Antes de cualquier iluminacion, la formulacion puede dejarse in situ, o mas preferiblemente el exceso de formulacion se elimina por lavado de la herida utilizando una solucion de irrigacion de heridas adecuada. La eliminacion del exceso de formulacion por lavado puede permitir que la tincion muestre donde se encuentra el biofilm en la herida, a fin de que estas areas puedan tratarse por ejemplo mediante raspado.
La herida se examina luego en cuanto a la presencia de biofilm tenido preferentemente. Esto puede hacerse a simple vista, o la herida puede iluminarse con una fuente luminosa a una distancia comprendida en el intervalo de 1 a 50 cm. Con preferencia, la fuente luminosa se encuentra a una distancia de 5 a 20 cm. La senal espectral de la fuente luminosa se selecciona de modo que corresponda al espectro de emision de fluorescencia del agente de tincion; a saber, para Rosa Bengala, que tiene un maximo de fluorescencia de 575 nm, una fuente luminosa adecuada podna emitir longitudes de onda de 550-600 nm. Esto causa que el agente contenido en el biofilm tenido emita fluorescencia. Preferiblemente, el
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usuario lleva gafas para observar la herida u observa la herida a traves de una lente integrada en la fuente luminosa. Las gafas o la lente tienen un sistema de filtracion de luz que excluye las longitudes de onda de luz por debajo del intervalo de emision de fluorescencia del agente de tincion y permite que cualquier fluorescencia se observe de modo notablemente claro y espedfico y por tanto, la deteccion de cualquier biofilm presente.
Tfpicamente, el tratamiento debena tener lugar en los cambios de vendaje subsiguientes. La herida puede examinarse ulteriormente en cuanto a presencia y reduccion del biofilm - a simple vista o con la fuente luminosa y las gafas o la lente integrada. La herida puede vendarse luego con un vendaje primario y secundario apropiado.
Lo que sigue es una breve descripcion de las figuras y tablas:
Figura 1. Seleccion inicial de colorantes seleccionados como tintes de biofilm utilizando biofilms desarrollados en el CDFF (n = 6).
Figura 2a. Seleccion de colorantes seleccionados como tintes de biofilm utilizando biofilms desarrollados en el reactor de biofilm CDC (n = 9).
Datos de absorcion.
Figura 2b. Seleccion de colorantes seleccionados como tintes de biofilm utilizando biofilms desarrollados en el reactor CDC (n = 9).
Datos de concentracion.
Figura 3. Rosa bengala con muestras tenidas (A-C) con EDTA y BaCl (RBEB) y muestras de control (DE) de carne que contema biofilms mixtos de S. aureus y P. aeruginosa.
Figura 4. Edad de un biofilm de S. aureus frente a concentracion de colorante.
Figura 5. Edad de un biofilm de P. aeruginosa frente a concentracion de Rosa Bengala.
Los ejemplos siguientes son ilustrativos de la presente invencion.
Ejemplo 1
Evaluacion de las tinciones de biofilm utilizando el fermentador de biofilm de altura constante
Se utilizo un fermentador de biofilm de altura constante (CDFF) para cultivar biofilms mixtos de 4 dfas de Staphylococcus aureus y Pseudomonas aeruginosa. Resumidamente, se formaron biofilms en rebajos en 15 cubetas de PTFe alrededor del borde de una mesa rotativa de acero sobre la cual se dejaba gotear continuamente inoculo o medio de crecimiento esteril. Una barra rascadora distribrna el inoculo bacteriano o medio sobre las cubetas a medida que giraba la mesa rotativa, manteniendo los biofilms en una altura constante. Cada cubeta contema 5 muestras amovibles, de 4 mm de diametro, que estaban rebajadas hasta una altura de 300 pm. Los biofilms resultantes que credan en los rebajos de las muestras eran reproducibles en terminos de aspecto y composicion microbiana, y podfan retirarse del CDFF por una abertura de muestreo utilizando instrumentos esteriles. Por duplicado, se retiraron del CDFF cubetas que conteman el biofilm, se lavaron una sola vez por inmersion en solucion salina esteril durante 5 segundos, y se incubaron luego en volumenes de 10 mL de los tintes potenciales de biofilm siguientes a concentracion 100 pM en agua desionizada a no ser que se indique otra cosa, en la oscuridad durante 2 minutos: Eritrosina; Rosa Bengala; Verde Rapido; Rosa Bengala con 2% peso/volumen de EDTA y 1% peso/volumen de cloruro de benzalconio (BaCI) (RBEB); Rosa Bengala con 2% peso/volumen de EDTA tetrasodico y 1% peso/volumen de cloruro de benzalconio en 2% peso/volumen de hidroxietilcelulosa y 10% peso/volumen de gel de propilenglicol (Gel-RBEB). Despues de otro lavado en solucion salina, se incubaron durante una noche 6 muestras para cada tinte a 37°C en volumenes de 6 mL de dodecilsulfato de sodio (SDS) al 2% en agua para digestion. Las muestras se centrifugaron luego 13.000 rpm durante 10 min para separar el sobrenadante de los residuos celulares digeridos. Los espectros de absorcion de los sobrenadantes aspirados se midieron luego en un espectrofotometro y estos espectros se compararon con espectros de concentraciones conocidas (100 pM) de cada tinte en SDS al 2%.
La Figura 1 muestra que Verde Rapido pareda ser el tinte mas eficaz en los biofilms desarrollados en el CDFF, seguido de cerca por Rosa Bengala con EDTa y BaCl en forma lfquida. La adicion de los excipientes EDTA y BaCl pareda mejorar la captura de Rosa Bengala en los biofilms.
Ejemplo 2
Evaluacion de las tinciones de biofilm utilizando el reactor de biofilm CDC
Se utilizo un reactor de biofilm del Centro para Control de las Enfermedades (CDC) para cultivar biofilms mixtos de 48 horas de Staphylococcus aureus y Pseudomonas aeruginosa. Resumidamente, se formaron biofilms sobre probetas en bastoncillos de reactor que se mantuvieron dentro de la vasija del reactor que contema un cultivo mezclado continuamente de S. Aureus y P. aeruginosa a 35° C. Las probetas que conteman el biofilm se retiraron de los bastoncillos, se lavaron una sola vez por inmersion en solucion salina esteril durante 5 segundos, y se incubaron luego en volumenes de 10 mL de los tintes de biofilm siguientes a concentracion 100 pM en agua desionizada, en la oscuridad durante 2 min: Eritrosina; Rosa Bengala; Azul Alcian; Rodamina B; Rodamina 123; Rosa Bengala con 2% peso/volumen de EDTA y 1%
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peso/volumen de BaCI (RBEB). Despues de otro lavado en solucion salina, se anadieron 9 probetas para cada tincion a volumenes de 2 mL de SDS al 2% para rechazo a ajuste 'alto' durante 1 minuto y se incubaron luego durante una noche en la oscuridad a 37°C, para digestion. Las muestras se centrifugaron luego 13.000 rpm durante 5 min para separar el sobrenadante de los residuos celulares digeridos. Los espectros de absorcion de los sobrenadantes aspirados se midieron luego en un espectrofotometro y estos espectros se compararon con espectros de concentraciones conocidas (100 jM) de cada tinte en SDS al 2%.
La figura 2a muestra como, en terminos de absorcion absoluta o 'brillo', RBEB era el tinte de biofilm mas eficiente, seguido por el tinte de carbohidrato, Azul Alcian. El EDTA y/o BaCl paredan mejorar la captura de Rosa Bengala mas de 60%. Cuando se expresaron los mismos datos como una ratio de absorcion medida: absorcion a 100 jM (es decir concentracion), Azul Alcian pareda ser el tinte de biofilm mas eficaz.
Ejemplo 3
Evaluacion de tintes de biofilm utilizando un modelo de biofilm de panza de cerdo
Se utilizo un modelo de biofilm de panza de cerdo para evaluar mas los tintes de biofilm. Se cortaron trozos de panza de cerdo utilizando un taladro de 20 mm y una barrena de 6 mm para crear indentaciones en el centro de las muestras. Las muestras se esterilizaron luego por irradiacion gamma. Las muestras se inocularon con volumenes de 10 jl de una suspension mixta de ~1 x 107 unidades formadoras de colonias/mililitro de S. aureus y P. aeruginosa y se incubaron luego a 35° C en placas de Petri selladas con Parafilm durante 72-96 horas. Las muestras que paredan tener biofilms visibles unicamente en el orificio central del taladro se tineron luego por inmersion de las muestras en volumenes de 10 mL de Rosa Bengala 100 jM con 2% peso/volumen de EDTA y 1% peso/volumen de BaCl (RBEB) durante 2 minutos con lavado en solucion salina antes y despues de la tincion. Las muestras de control no se tineron y por tanto se sumergieron unicamente en solucion salina durante 2 minutos. Las muestras se fotografiaron luego, ejemplos de las cuales se representan en Fig. 3. Las tres muestras (A a C) mostraban que estaban tenidas con RBEB, demostrando claramente la captura selectiva del tinte por los biofilms que estaban contenidos dentro de las indentaciones. Las tres muestras de control demuestran que sin esta tincion es dificil averiguar si y donde esta presente biofilm en las muestras
Ejemplo 4
Tincion con Rosa Bengala de biofilms desarrollados utilizando un modelo de biofilm en filtro de membrana
Se utilizo un modelo de biofilm en filtro de membrana para estudiar el efecto de la concentracion de Rosa Bengala sobre la eficacia de la tincion del biofilm. Resumidamente, se anadio un volumen de 5 jL de una suspension de 5 * 105 unidades formadoras de colonias/mililitro de S. aureus o P. aeruginosa al centro de discos de filtros de membrana esteriles (tamano de poro 0,2 jm; Anodisc, Whatman) que se pusieron en volumenes de 7 mL de Caldo Tnptico de Soja esteril en placas de 6 pocillos tapadas. Las muestras se incubaron durante 4 horas (biofilms inmaduros), y 24 y 48 horas (biofilms maduros), y el exceso de celulas planctonicas se retiro de los filtros por lavado. Los biofilms que se encontraban en los filtros se tineron por pipeteado de volumenes de 2 mL de Rosa Bengala (60 jM o 300 jM) o solucion salina (control negativo) durante 30 segundos, seguido por lavado. El Rosa Bengala se recupero de las muestras de biofilm por rechazo y digestion durante una noche en dodecilsulfato de sodio al 2%, centrifugacion, y medicion posterior de los espectros de absorcion en un espectrofotometro UV-vis. La captura de Rosa Bengala por muestra se determino por comparacion de los valores de absorcion con una curva estandar de Rosa Bengala en SDS al 2%.
Las Tablas 1 y 2 muestran que los biofilms de S. aureus y P. aeruginosa capturaban Rosa Bengala de una manera dependiente de la concentracion de colorante y la edad del biofilm. Unicamente a las concentraciones maximas de 300 jM paredan tenirse los biofilms inmaduros de 4 horas, aunque esto se debfa probablemente a cierta tincion de los filtros propiamente dichos. Las Figuras 4 y 5 muestran como los biofilms maduros de 48 horas de S. aureus y P. aeruginosa capturaban mas Rosa Bengala que los biofilms de 24 horas, y tambien que Rosa Bengala 300 jM daba como resultado una tincion significativamente mayor de los biofilms que Rosa Bengala 60 jM. Este metodo simple para cuantificar la captura de Rosa Bengala por los biofilms utiliza los espectros de absorcion del colorante. La utilizacion de los espectros de absorcion para cuantificar el colorante es similar a la deteccion de la fluorescencia utilizando una fuente luminosa con filtros opticos. La captura podna observarse alternativamente de modo cualitativo a simple vista o por observacion de la emision de fluorescencia del Rosa Bengala en conjuncion con una fuente luminosa y un filtro optico.
Tabla 1
- Edad (h)
- [RB] (uM) 1 2 3 Media S.D.
- 4
- 0 0 0 0 0 0
- 4
- 60 0 0 0 0 0
- 4
- 300 z,59 1,45 1,64 1,56 0,10
- 24
- 0 0 0 0 0 0
- 24
- 60 1,66 1,72 2 ,17 1,85 0,28
- 24
- 300 4,82 4,55 3,95 4,44 0,45
- 48
- 0 0 0 0 0 0
- 48
- 60 1,22 1,12 1,37 1,24 0,12
- 48
- 300 6,26 5,18 5,71 5,72 0,54
Tabla 2
- Edad (h)
- [RB] (uM) 1 2 3 Media S.D.
- 4
- 0 0 0 0 0 0
- 4
- 60 0 0 0 0 0
- 4
- 300 1,52 1,76 1,88 1,72 0,18
- 24
- 0 0 0 0 0 0
- 24
- 60 2,04 1,76 2,02 1,94 0,15
- 24
- 300 5,05 5,84 4,41 5,10 0,72
- 48
- 0 0 0 0 0 0
- 48
- 60 3,15 1,66 2,37 2,39 0,74
- 48
- 300 6,20 9,01 6,69 7,30 1,50
Claims (12)
- 5101520253035404550REIVINDICACIONES1. Una composicion de tinte para uso en la produccion de biofilm detectable en tejido viable, en donde la composicion tine preferentemente el biofilm en lugar del tejido viable y comprende 0,1 a 2,0% en peso de EDTA, 0,1 a 1,0% en peso de un surfactante, y 0,0001% a 1% en peso, preferentemente 0,0025% a 0,025% en peso de un agente de tincion para tenir dicho biofilm y hacerlo detectable, caracterizada por que la composicion es un lfquido, el agente de tincion es Rosa Bengala y el surfactante se selecciona de cloruro de benzalconio o cloruro de bencetonio.
- 2. Una composicion para uso segun la reivindicacion 1, caracterizada por que la composicion tine preferentemente el biofilm por fijacion selectiva al biofilm, siendo la composicion opcionalmente capaz de tenir preferentemente los biofilms por fluorescencia y deteccion visual a simple vista o utilizando una fuente luminosa y filtros opticos correspondientes a los espectros de emision de fluorescencia del agente de tincion.
- 3. Una composicion para uso segun la reivindicacion 1 o la reivindicacion 2, caracterizada por que la composicion permite la deteccion del biofilm por tincion y revelado preferentes del mismo, preferiblemente a simple vista.
- 4. Una composicion para uso segun la reivindicacion 1 o 2, caracterizada por que el agente de tincion absorbe luz de longitudes de onda que van desde 380 nm a 720 nm.
- 5. Una composicion para uso segun cualquiera de las reivindicaciones anteriores, caracterizada por que la composicion permite la deteccion del biofilm por tincion y revelado preferentes del mismo cuando se ilumina este con luz que incluye longitudes de onda que causan que el mismo emita fluorescencia.
- 6. Una composicion para uso segun cualquiera de las reivindicaciones anteriores, caracterizada por que la composicion es una solucion lfquida basada en agua o glicerol (en forma de un aplicador, nebulizador u hoja).
- 7. Una composicion para uso segun cualquiera de las reivindicaciones anteriores, caracterizada por que la composicion comprende ademas al menos un agente adicional seleccionado del grupo constituido por un humectante y un agente de rotura de biofilms.
- 8. Un kit de partes para uso en la deteccion de biofilms en tejido viable, comprendiendo el kit:una composicion segun la reivindicacion 1 y una fuente luminosa capaz de causar que el agente de tincion del biofilm emita fluorescencia.
- 9. Un kit para uso segun la reivindicacion 8, caracterizado por que la fuente luminosa emite luz para hacer que el agente de tincion emita fluorescencia.
- 10. Un kit para uso segun las reivindicaciones 8 o 9, caracterizado por que el mismo comprende gafas o lentes para uso en la deteccion del biofilm, en donde las gafas o lente actuan para excluir todas las longitudes de onda de luz excepto aquellas a los cuales el agente de tincion emite fluorescencia.
- 11. Un kit para uso segun las reivindicaciones 8, 9 o 10 que comprende adicionalmente una solucion de irrigacion de herida.
- 12. Uso de una composicion segun la reivindicacion 1 para deteccion de un biofilm en una herida, que comprende los pasos de:(a) aplicar dicha composicion;(b) examinar el tejido respecto a la presencia de biofilm tenido a simple vista o con una fuente luminosa que causa que el biofilm tenido emita fluorescencia;(c) detectar la fluorescencia emitida por el biofilm tenido.
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CA2819303A1 (en) | 2012-06-07 |
AU2017201084A1 (en) | 2017-03-09 |
HUE032884T2 (hu) | 2017-11-28 |
CN108330157A (zh) | 2018-07-27 |
BR112013013662A2 (pt) | 2016-09-06 |
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CA2819303C (en) | 2022-04-19 |
RU2650803C2 (ru) | 2018-04-17 |
AU2011334682B2 (en) | 2016-11-17 |
LT2646566T (lt) | 2017-01-25 |
JP2014500495A (ja) | 2014-01-09 |
AU2017201084B2 (en) | 2019-04-18 |
HK1258842A1 (zh) | 2019-11-22 |
DK2646566T3 (en) | 2017-01-23 |
JP2017062238A (ja) | 2017-03-30 |
EP2646566A1 (en) | 2013-10-09 |
AU2011334682A1 (en) | 2013-07-18 |
US20210369874A1 (en) | 2021-12-02 |
CN103328651A (zh) | 2013-09-25 |
SI2646566T1 (sl) | 2017-02-28 |
RU2013129866A (ru) | 2015-01-10 |
GB201020236D0 (en) | 2011-01-12 |
MX2013006090A (es) | 2013-07-15 |
NZ612573A (en) | 2015-05-29 |
JP2018189655A (ja) | 2018-11-29 |
US11135315B2 (en) | 2021-10-05 |
US20140161728A1 (en) | 2014-06-12 |
US20190216953A1 (en) | 2019-07-18 |
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