Este trabajo fue apoyado por el azm Profileringsfonds (PF245).

PARTE I: la identificación de patógenos 1. Preparación de la muestra Nota: El flujo de trabajo molecular que se describe en el siguiente protocolo se debe realizar de acuerdo a las recomendaciones para garantizar la calidad en el diagnóstico molecular 3. <ol><li> Añadir una alícuota de 0,1 ml de cultivo de sangre a 0,9 ml de NaCl al 0,9% en un tubo de 1,5 ml de reacción para convertirse en una muestra diluida 1:10. (Diluir con el fin de prevenir la inhibición qPCR). </li><li> Centrifugar la muestra a 13400 xg durante 5 minutos para sedimentar el ADN bacteriano. </li><li> Resuspender el sedimento bacteriano en 100 l estéril desmineralizada H 2 O. </li><li> Almacenar la muestra de ADN a 4 ° C hasta su uso posterior. </li></ol> 2. Ensayo de identificación: en tiempo real PCR 16S rDNA <ol><li> Preparar las mezclas de reacción de la siguiente manera. El ensayo consiste en cuatro reacciones separadas por muestra. Cada mezcla incluye 12,50 maestro de lmezcla, 0,9 mM cebador (5-TCCTACGGGAGGCAGCAGT-3) 7, cebador 0,6 mM inversa (5-GGACTACCAGGGTATCTAATCCTGTT-3) 8 y un panel de sondas. La cantidad de sondas se da para cada una de las cuatro reacciones separadas en adelante. </li><li> La primera reacción incluye: <ul><li> 0,2 mM universal de la sonda (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) 8 </li><li> 0,2 mM p aeruginosa sonda (5-JOE-CCAAAACTACTGAGCTAGAGTACG-3-BHQ1) </li></ul></li><li> La segunda reacción incluye: <ul><li> 0,2 mM E. la sonda coli (5-JOE-GGAGTAAAGTTAATACCTTTGCTCATT-3-BHQ1) </li><li> 0,2 mM de Pseudomonas spp. de la sonda (5-NED-CCTTCCTCCCAACTTAAAGTGCTT-3-MGBNFQ) </li></ul></li><li> La tercera reacción incluye: <ul><li> 0,2 mM Staphylococcus spp. de la sonda (5-NED-AATCTTCCGCAATGGGCGAAAGC-3-MGBNFQ) </li><li> 0,2 mM de S. la sonda aureus (5-FAM-AGATGTGCACAGTTACTTACACATAT-3-BHQ1) </li><li> 0,2 mM de Enterococcus spp. (5-JOE-TCCTTGTTCTTCTCTAACAACAGAG-3-BHQ1) </li></ul></li><li> La reacción cuarto incluye: <ul><li> 0,2 mM universal de la sonda (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) </li><li> 0,3 mM Streptococcus spp. de la sonda (5-NED-CCAGAAAGGGACSGCTAACT-3-MGBNFQ) </li><li> 0,2 mM de S. pneumoniae de la sonda (5-JOE-CCAAAGCCTACTATGGTTAAGCCA-3-BHQ1) </li></ul></li><li> Añadir desmineralizada estéril, H 2 O para llegar a un volumen total de 20 l. Añadir 20 l de cada mezcla de reacción a los pocillos de una placa de 96 pocillos de PCR. </li><li> Añadir 5 l de muestra a cada pocillo. </li><li> Utilizar una película adhesiva para sellar la placa 96-así PCR. </li><li> Ejecutar la placa en el ABI PRISM 7900HT Sistema de Tiempo Real PCR utilizando los siguientes ciclos térmicos condiciones óptimas: <ul><li> Pre-calentamiento a 50 ° C durante 10 min </li><li> Desnaturalización a 95 ° C durante 15 minutos </li><li> 42 ciclos de <ul type="circle"><li> Desnaturalización a 95 ° C durante 15 s </li><li> El recocido a 60 ° C durante 1 minuto </li></ul></li></ul></li></ol> 3. Análisis de los resultados <ol><li> Ajuste el umbral del análisis de Ct a 0,1 en la pestaña Configuración de análisis. Limitar las configuraciones de referencia para empezar (Ciclo): 6 y el fin (del ciclo): 15. </li><li> Registre el ciclo umbral (Ct), el valor de todas las muestras. El valor de corte para considerar un resultado positivo por PCR como se puede ajustar a un valor de CT-35. La cantidad de bacterias presentes en los cultivos de sangre osciló entre 07 al 10 10 11 UFC / ml, la generación de Ct valores por debajo de 35. </li></ol> PARTE II: pruebas de susceptibilidad antimicrobiana 4. El aislamiento de bacterias de cultivos de sangre positivos 9 <ol><li> Aspirar 5 ml de caldo de una botella de hemocultivo positivo y la transfiere en un tubo separador de suero. </li><li> Centrifugar el tubo separador de suero a 2000 xg durante 10 min. </li><li> Se desecha el sobrenadante del tubo separador de suero. </li><li> Transferencia de bacbacterias de la capa de gel del tubo con una torunda de algodón estéril en solución salina al 0,9% hasta un 0,5 McFarland suspensión estándar se obtiene. </li></ol> 5. La inoculación de las placas Micro Titre <ol><li> Diluir la suspensión 0,5 McFarland en doble concentrado caldo Mueller Hinton II para formar una suspensión de 5 x 10 5 UFC / ml. </li><li> Añadir esta suspensión a los pocillos de una placa de micro título que contiene una selección de antibióticos (Tabla 1). </li><li> Se incuba la placa micro título a 37 ° C durante 6 h. </li><li> Almacenar una alícuota de la suspensión a 4 ° C (como testigo de crecimiento negativo). </li><li> Después de 6 horas de incubación, transferir el contenido de cada pocillo en un tubo estéril, así como la muestra de control de crecimiento negativo que se almacenó a 4 ° C. </li><li> Centrifugar los tubos a 16000 xg durante 5 min. </li><li> Retire con cuidado el sobrenadante, sin perturbar el sedimento bacteriano. </li><li> Resuspender el precipitado en H desmineralizada estéril <sub&gt; 2 O. </li><li> Diluir las muestras de 10 veces en estériles desmineralizada H 2 O. </li></ol> 6. En tiempo real de PCR del rDNA 16S 10 <ol><li> Preparar la mezcla de PCR como sigue: <ul><li> 12,50 l iQ SYBR Green Supermix </li><li> 0,5 mM cebador 16S-1 (5-TGGAGAGTTTGATCCTGGCTCAG-3) 11 </li><li> 0.25 16S-2 micras de imprimación inversa (5-TACCGCGGCTGCTGGCAC-3) 11 </li><li> estéril desmineralizada H 2 O hasta un volumen total de 20 l </li></ul></li><li> Añadir 20 l de mezcla de PCR a los pocillos de una placa de 96 pocillos de PCR. </li><li> Añadir 5 l de muestra a cada pocillo. </li><li> Utilizar una película adhesiva para sellar la placa 96-así PCR. </li><li> Ejecutar la placa en el MyiQ un solo color en tiempo real del sistema de detección por PCR, utilizando las siguientes condiciones óptimas ciclos térmicos: <ul><li> Desnaturalización a 95 ° C durante 4 minutos </li><li> Inicial recocido a 65 ° C durante 30 s </li><li> 35 ciclos de <ul><li> Desnaturalización a 95 ° C durante 15 s </li><li> El recocido a 60 ° C durante 1 minuto </li></ul></li><li> Fundir análisis de la curva (60 a 95 ° C en 20 minutos con incrementos de 0,57 ° C) </li></ul></li></ol> 7. Análisis de los resultados <ol><li> Calcular el valor de corte de Ct utilizando una de las fórmulas siguientes (dependiendo del tipo de antibiótico). <ul><li> En general: De corte el valor Ct = Ct de control de valor de un crecimiento positivo + 0,5 x (control de Ct valor de crecimiento negativo - el control de Ct valor de crecimiento positivo) </li><li> Piperacilina, piperacilina / tazobactam y ceftazidima en bacilos Gram-negativos, amoxicilina, oxacilina y trimetoprim / sulfametoxazol en el S. aureus, Enterococcus spp amoxicilina en:. De corte Ct = valor de control de valor de Ct crecimiento positivo + 0,25 x (crecimiento Ct valor negativo - Control Ct valor de crecimiento positivo) </li></ul></li><li> Use la muestra se incuba con agua estéril desmineralizada H 2 O, como testigo de crecimiento positivo.</li><li> Dependiendo del microorganismo, utiliza el control apropiado de crecimiento negativo: <ul><li> Gram-negativos Muestra varillas se incubaron con la mezcla de antibióticos </li><li> Enterococcus spp. Muestra almacenada a 4 ° C </li><li> Ejemplo de S. aureus se almacena a 4 ° C </li></ul></li><li> Determinar la susceptibilidad (S) o resistencia (R) de la cepa para el antibiótico ensayó como sigue: <ul><li> Un valor Ct superior al valor de corte de Ct indica la susceptibilidad </li><li> Un valor de Ct inferior al valor de corte de Ct indica que la resistencia </li></ul></li></ol> 8. Los resultados representativos Dos organismos modelo, es decir, una E. Gram-negativos coli y S. una Gram-positivas aureus, se eligen para visualizar el procedimiento combinado para la detección e identificación de bacterias patógenas y la determinación de su perfil antimicrobiano. La primera parte del protocolo comprende la identificación de patógenos. Spesondas específicas están diseñadas para la detección de ocho microorganismos clínicamente relevantes. En presencia de un objetivo incluido en el panel bacteriana, curvas de amplificación se generan y Ct valores se calculan (Figura 2). El valor de corte para considerar un resultado positivo por PCR como se establece en un valor de Ct de 35 años. En la Figura 2A, el perfil de identificación de un cultivo de sangre infectada con E. coli se muestra. La sonda 16S universal está incluida en dos mezclas de reacción separadas y, en consecuencia genera dos curvas de amplificación (Ct de 25,20 y 25,95). La tercera señal se deriva de la sonda específica para E. E. (Ct de 27.04). La identificación de una S. aureus infectada cultivo de sangre se muestra en la Figura 2B. La sonda 16S universal tiene señales de amplificación de 33,35 y 33,71. Las dos señales restantes se derivan de las sondas específicas para Staphylococcus spp. y S. aureus (Ct de 32,48 y 30,59). La figura 3 es un ejemplo de un gráfico de amplificación pruebas de susceptibilidad a los antibióticos, en representación de la cepa de E. coli que se mostró también en la Figura 2A. Cada línea representa un antibiótico que la muestra bacteriana se incubó con. Una muestra con un bajo valor de CT es una muestra en la que se ha producido crecimiento en presencia de una resistencia a los antibióticos, lo que indica que el antibiótico probada. Por el contrario, un alto valor Ct representa una muestra en la que no hay crecimiento se ha producido por el funcionamiento efectivo de la susceptibilidad a los antibióticos, lo que indica que el antibiótico probada. La Tabla 1 ilustra la determinación del perfil de los antimicrobianos de la E. coli y S. aureus aislados. Todos los valores de Ct se informan y, utilizando las fórmulas mencionadas en el texto del protocolo (7,1), dos de corte los valores de Ct se calcularoncionado a distinguir entre la resistencia y susceptibilidad. La cepa es resistente a los antibióticos, si el valor de Ct informado es más bajo que el calculado valor de corte de Ct (y viceversa). <img alt="Figura 1" src="/files/ftp_upload/3254/3254fig1.jpg" /> Figura 1. Diagrama de flujo del procedimiento de identificación de patógenos y la susceptibilidad a los antibióticos mediante pruebas en tiempo real PCR del rDNA 16S. <img alt="Figura 2" src="/files/ftp_upload/3254/3254fig2.jpg" /> Figura 2 Ensayo de identificación:. Parcelas de amplificación y los valores de ciclo umbral (Ct valores) Un cultivo de sangre positivo se detecta por lo universal de la sonda de ADNr 16S, mientras que las sondas específicas se utilizan para la identificación del agente causal.. Parcela A. amplificación de cultivo de sangre que contiene E. coli; parcela B. amplificación de la cultura de la sangre que contiene S. aureus, Pseudomonas ae, Pseudomoncomo P. aeruginosa, uni, 16S universal de la sonda; Ecoli, Escherichia coli de la sonda, Pseudomonas sp, Pseudomonas spp. la sonda, S. pneumoniae, Streptococcus pneumoniae sonda; Strep sp, Streptococcus spp. la sonda, Entero, Enterococcus spp. la sonda, S. aureus, la sonda de Staphylococcus aureus, estafilococo sp, Staphylococcus spp. sonda. <img alt="Figura 3" src="/files/ftp_upload/3254/3254fig3.jpg" /> Figura 3. Amplificación parcela de las pruebas de sensibilidad antibiótica de una E. E. aislar (muestra 1). Cada curva representa un antibiótico que la cepa se incubó con. Una señal temprana es causada por una alta carga bacteriana, lo que significa que la cepa ha crecido en presencia del antibiótico probado y es por lo tanto resistentes al antibiótico. Señales tardías indican que la cepa no ha crecido en presencia del antibiótico, en otras palabras, es susceptible. <table border="1"><tbody&gt; <tr><td> Ejemplo 1: E. E. </td><td></td><td></td><td> Ejemplo 2: S. Staphylococcus </td><td></td><td></td></tr><tr><td> AST </td><td> Ct </td><td> R / S </td><td> AST </td><td> Ct </td><td> R / S </td></tr><tr><td> La amoxicilina 8 mg / l </td><td> 16,83 </td><td> R </td><td> La amoxicilina 0,25 mg / l </td><td> 21,03 </td><td> R </td></tr><tr><td> La amoxicilina-ácido clavulánico 4.8 mg / L </td><td> 17,36 </td><td> R </td><td> Oxacilina 2 mg / l </td><td> 25,80 </td><td> S </td></tr><tr><td> Piperacilina 16 mg / l </td><td> 16,67 </td><td> R </td><td> La vancomicina 2 mg / l </td><td> 25,20 </td><td> S </td></tr><tr><td> Piperacilina-tazobactam 16 /4 mg / l </td><td> 24,15 </td><td> S </td><td> La gentamicina 4 mg / l </td><td> 25,86 </td><td> S </td></tr><tr><td> La ciprofloxacina 1 mg / l </td><td> 29,72 </td><td> S </td><td> Trimetoprim-sulfametoxazol 2/38 mg / L </td><td> 24,62 </td><td> S </td></tr><tr><td> La ceftazidima 1 mg / l </td><td> 24,03 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> La ceftazidima 8 mg / l </td><td> 26,58 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> La gentamicina 4 mg / l </td><td> 29,83 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Trimetoprim-sulfametoxazol 2/38 mg / L </td><td> 27,60 </td><td> S </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Control de crecimiento negativo (mezcla de antibióticos) </td><td> 30,41 </td><td></td><td> Testigo de crecimiento negativo (muestra almacenada a 4 ° C) </td><td> 27,42 </td><td></td></tr><tr><td> Control de crecimiento positivo </td><td> 16,90 </td><td></td><td> Control de crecimiento positivo </td><td> 20,22 </td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> De corte CT-valor de 1 * </td><td> 21,76 </td><td></td><td> De corte CT-valor 1 *** </td><td> 23,82 </td><td></td></tr><tr><td> De corte CT-valor 2 ** </td><td> 18,75 </td><td></td><td> De corte CT-valor 2 **** </td><td> 22,02 </td><td></td></tr><tr><td> * Para la amoxicilina, amoxicilina-clavulánico, ciprofloxacino, gentamicen, trimetoprim-sulfametoxazol ** Para piperacilina, piperacilina-tazobactam, ceftazidime </td><td></td><td></td><td> *** Para vancomicina y gentamicina **** Para amoxicilina, oxacilina y trimetoprim-sulfametoxazol </td><td></td><td></td></tr></tbody></table> Tabla 1. Determinación de las pruebas de sensibilidad antibiótica de las dos muestras (E. coli y S. aureus). Ct valores de la PCR-ensayo fueron copiados a este archivo de Excel, ya que automáticamente se calcula las dos línea de corte alores Ct del control de crecimiento positivo y negativo, utilizando las fórmulas que aparecen en el texto del protocolo. Si un antibiótico muestra un valor de Ct menor que el valor de corte Ct, la cepa es resistente a los antibióticos, si el valor de CT fue mayor que la de corte, la cepa es susceptible.

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No tenemos nada que revelar.

El protocolo aquí descrito permite la identificación rápida de patógenos y ofrece un perfil funcional a los antimicrobianos que puedan conducir a la administración precoz de antibióticos adecuados, mejorando así el pronóstico de los pacientes con infecciones del torrente sanguíneo. Dependiendo de las condiciones exigidas de una prueba, es decir, costo bajo, alto rendimiento, un mínimo de tiempo de vuelta, las condiciones de prueba se puede ajustar. Todo el procedimiento se puede realizar en un día de trabajo. Además, las dos partes del protocolo puede realizarse simultáneamente, lo cual reduce el tiempo de giro alrededor de manera significativa. Por lo aquí expuesto, el panel de identificación es una selección de las bacterias con mayor relevancia clínica en nuestro hospital. Desde el principio principal se dirige a la región del gen 16S, las sondas específicas para otros microorganismos pueden ser diseñados y se añade a la del ensayo. El ensayo completo fue originalmente pensado para el análisis rápido de los cultivos de sangre, pero también pueden ser utilizados para el procesamiento de Otros materiales de la muestra. Este es también el caso de los antibióticos que se utilizaron para las pruebas de susceptibilidad a los antibióticos: los antibióticos más o se pueden agregar otros, con base en los patrones de resistencia locales y directrices.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> De cloruro de sodio (NaCl) </td><td> Merck Chemicals </td><td> 106404 </td><td> 0,9% en agua </td></tr><tr><td> Vacutainer SST suero tubo separador 5 ml </td><td> BD Diagnostic Systems </td><td> 367986 </td><td></td></tr><tr><td> Mueller Hinton II caldo de </td><td> BD Diagnostic Dystems </td><td> 212322 </td><td> 44 g / l en agua </td></tr><tr><td> Centrifugar Rotixa 50 rs </td><td> Andreas Hettich GmbH &amp; Co. KG </td><td> 4910 </td><td></td></tr><tr><td> Centrífuga 5415 D </td><td> Eppendorf </td><td> Suspendido </td><td></td></tr><tr><td> Primers </td> &lt;td&gt; Sigma-Aldrich </td><td> na </td><td></td></tr><tr><td> Sondas </td><td> Sigma-Aldrich/Applied Biosystems </td><td> na </td><td></td></tr><tr><td> TaqManEnvironmental maestro de la mezcla 2,0 </td><td> Applied Biosystems </td><td> 4396838 </td><td></td></tr><tr><td> iQ SYBRGreen Supermix </td><td> Bio-Rad Laboratories BV </td><td> 170-8880 </td><td></td></tr><tr><td> MicroAmp óptica de 96 pocillos de reacción </td><td> Applied Biosystems </td><td> N8010560 </td><td></td></tr><tr><td> Película MicroAmp adhesivo óptico </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> iQ PCR de 96 pocillos placas </td><td> Bio-Rad Laboratories BV </td><td> 223-9441 </td><td></td></tr><tr><td> Microseal B burletes adhesivos en </td><td> Bio-Rad Trabajoatories BV </td><td> MSB-1001 </td><td></td></tr><tr><td> PCR en tiempo real del sistema de detección </td><td> Applied Biosystems </td><td> ABI PRISM 7900HT </td><td></td></tr><tr><td> PCR en tiempo real del sistema de detección </td><td> Bio-Rad Laboratories BV </td><td> MyiQ un solo color </td><td></td></tr></tbody></table>

one-day workflow scheme for bacterial pathogen detection and antimicrobial resistance testing from blood cultures

Infecciones del torrente sanguíneo se asocia con altas tasas de mortalidad a causa de la manifestación probable de sepsis, sepsis grave y shock séptico 1. Por lo tanto, la administración rápida de la terapia antibiótica adecuada es de importancia principal en el tratamiento de infecciones del torrente sanguíneo. El elemento crítico en este proceso es el tiempo, depende en gran medida los resultados de la identificación de bacterias y pruebas de susceptibilidad a los antibióticos. Ambos de estos parámetros son rutinariamente obtenida por cultivo basado en pruebas, que consume tiempo y necesita una media de 24-48 horas 2, 4. El objetivo del estudio fue desarrollar ensayos basados ​​en el ADN para la identificación rápida de infecciones del torrente sanguíneo, así como pruebas rápidas de susceptibilidad antimicrobiana. El primer ensayo es un 16S rDNA eubacterial real basado en PCR en tiempo complementadas con especies del género-o sondas específicas 5. El uso de estas sondas, bacterias Gram-negativas, incluyendo Pseudomonas spp., Pseudomonas aeruginosay Escherichia coli, así como las bacterias Gram-positivas como Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp. y Streptococcus pneumoniae pueden ser distinguidos. El uso de este ensayo de Multiprobe, una primera identificación del microorganismo causal se da después de 2 horas. En segundo lugar, hemos desarrollado un ensayo molecular para el semi-pruebas de sensibilidad antibiótica de S. aureus, Enterococcus spp. y (facultativo) aerobios bacilos Gram-negativos 6. Este ensayo se basa en un estudio en el que la PCR se utilizó para medir el crecimiento de bacterias 7. Las bacterias recolectadas directamente de los cultivos de sangre se incubaron durante 6 horas con una selección de los antibióticos, y después de un SYBR Green-tiempo real basado en el ensayo de PCR determina la inhibición del crecimiento. La combinación de estos dos métodos podría dirigir la elección de una terapia antibiótica apropiada, en el mismo día (Figura 1). En conclusión, El análisis molecular de ambos identificación y susceptibilidad a los antibióticos ofrece una alternativa más rápida para la detección de patógenos, y podría mejorar el diagnóstico de infecciones del torrente sanguíneo.

Watch this Scientific Journal Video about One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures at JoVE.com

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

El diseño de un esquema de flujo de trabajo sencillo de un día para el diagnóstico de patógenos bacterianos permite el reconocimiento rápido de infecciones del torrente sanguíneo. La inclusión de los ocho objetivos bacterianos clínicamente relevantes y sus perfiles de resistencia a los antibióticos ofrece al clínico una visión inicial en el mismo día, que puede conducir a una terapia más adecuada.

Un día de flujo de trabajo Método de detección de patógenos bacterianos y pruebas de resistencia a los antimicrobianos de cultivos de sangre

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

one-day-workflow-scheme-for-bacterial-pathogen-detection

Research

JoVE Journal

Immunology and Infection

25.1K vistas.  Maastricht University Medical Center. El diseño de un esquema de flujo de trabajo sencillo de un día para el diagnóstico de patógenos bacterianos permite el reconocimiento rápido de infecciones del torrente sanguíneo. La inclusión de los ocho objetivos bacterianos clínicamente relevantes y sus perfiles de resistencia a los antibióticos ofrece al clínico una visión inicial en el mismo día, que puede conducir a una terapia más adecuada.

Video: One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Un procedimiento de 1,5 horas para la identificación de Enterococcus Especies directamente los cultivos de sangre

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Investigación

Inmunología e Infección

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

La invasión de las células humanas por un patógeno bacteriano

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

De alto rendimiento Método de detección de virus de la influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

La saliva, la glándula salival, y la Colección hemolinfa de las garrapatas Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

Nanomecánica de Interacciones con Medicamentos objetivo y detección de resistencia a los antibacterianos

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR Ensayo para la tipificaci

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Preparación de una Cultura de pellets de sangre para Rapid bacteriana Identificación y Prueba de Susceptibilidad a los antibióticos

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isotérmico de amplificación (LAMP) Ensayos Loop mediada para la detección específica de las especies de<em&gt; Eimeria</em&gt; Que infectan pollos

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.