The author thanks R. Boss, A. Cosandey, C. Fournier, I. Ivanovic, and J. Naskova for their excellent work.

1. aislados de Staphylococcus aureus <ol><li> Spread 10 l de S. aureus cultivo madre o una colonia crecida en un medio selectivo en placas de agar Columbia que contenían 5% de sangre de oveja (BA). </li><li> Incubar aeróbicamente a 37 ° C durante 18 horas a 24 horas. </li></ol> 2. Extracción de ADN <ol><li> Preparar tampón TEL que contiene Tris-HCl 10 y 10 mM Na 2 EDTA, pH 8,5. Preparar alícuotas y autoclave a 121 ° C durante 15 min. </li><li> Recoge 1 colonia de BA utilizando un asa estéril de plástico o un palillo de madera estéril y resuspender en 100 l TEL. Utilice tubos de 1,5 ml con tapones de rosca. </li><li> Incubar a 95 ° C durante 10 min. Después, colocar las muestras inmediatamente en hielo húmedo. </li><li> Diluir las muestras 1: 100 en H 2 O adecuado para PCR (= molde de ADN para la PCR). Las muestras no diluidas y diluidas pueden conservarse a -20 ° C durante 1 mes. </li></ol> 3. RS-PCR <ol><li&gt; Para cada pipeta de la muestra en un tubo de PCR 1.5 l H 2 O, 12.5 l Taq Master Mix, 2 l de cebador G1 10 M, 2 l L1 cebador 10 mM, y 7 l de plantilla de ADN que corresponde a aproximadamente 30 ng de pura 2 ADN. </li><li> Ejecute el siguiente programa de ciclos en un ciclador de PCR estándar: 15 min 95 ° C, seguido de 27 ciclos que comprenden 94 ° C durante 1 min, seguido de una rampa de 2 min y recocido a 55 ° C durante 7 min. Después de otros 2 min rampa, se extienden a 72 ° C durante 2 min. Terminar PCR por una extensión final a 72 ° C durante 10 min seguido de enfriamiento a 4 ° C. NOTA: Los productos de PCR se pueden almacenar a -20 ° C durante 5 días. </li><li> Para cada serie de RS-PCR incluye un control positivo, es decir, una muestra con un genotipo conocido (normalmente GTB), y un control negativo (H 2 O). </li></ol> 4. Electroforesis <ol><li> Utilice un kit de electroforesis miniaturizado comercial de ADN (kit MED) juntocon un Bioanalyzer conectado a un PC y el software instalado. Estudiar los manuales correspondientes del fabricante cuidadosamente. </li><li> Configurar la estación de cebado de chips y ajustar el clip de jeringa de acuerdo con el manual del fabricante. </li><li> Preparar la mezcla de gel colorante según el manual del fabricante. </li><li> Carga de la mezcla de gel colorante (de acuerdo con el manual del fabricante). <ol><li> Equilibrar la mezcla de gel de colorante a RT durante 30 min antes de su uso. </li><li> Ponga nuevo chip de ADN en el chip de la estación de cebado. Pipeta de 9 l de gel colorante en el G. bien marcado </li><li> Asegúrese de que el émbolo se posiciona a 1 ml y luego cerrar la estación de cebado de chips. Presione el émbolo hasta que se lleva a cabo por el clip de jeringa. Espere exactamente durante 30 segundos y luego suelte el clip de jeringa. </li><li> Espere a 5 seg. Tire lentamente el émbolo de 1 ml posición. </li><li> viruta abierta estación de cebado y pipeta de 9 l de gel colorante en ambos pozos marcados &quot;G&quot;. </li></ol></li><li> Carga del MArker <ol><li> Pipeta 5 l de marcador en los 12 pocillos de muestra y en la escalera bien. No deje ninguna de las 13 pozos vacíos. </li></ol></li><li> Cargando la escalera y las muestras. <ol><li> Pipeta de 1 l de escalera de ADN a la bien marcada con el signo escalera. </li><li> Pipeta de 1 l de muestra (pozos utilizados) o 1 l de agua desionizada (pocillos no utilizados). </li><li> Ponga el chip horizontalmente en el adaptador y de vórtice durante 1 min a 2400 rpm. </li><li> Ejecutar el chip en el Bioanalyzer dentro de 5 minutos. </li></ol></li><li> Ejecución del análisis <ol><li> Iniciar el Bioanalyzer y el ordenador conectado. Abra el software. </li><li> Abra la tapa de la Bioanalyzer y colocar el chip en el soporte de chip (chip encaja de una sola manera). Cierre la tapa con cuidado. </li><li> En el contexto del instrumento del software seleccione &quot;Electroforesis&quot;&gt; &quot;ADN de doble cadena&quot;&gt; &quot;ADN 7500&quot;. Aceptar el prefijo del archivo actual del software o modificarlo.</li><li> Haga clic en el botón de inicio presente en el software. Introduzca los nombres de las muestras en el software. Una vez finalizada la ejecución de chip (después de aproximadamente 30 min) el Fin del mensaje de ejecución aparece en el software Bioanalyzer. </li><li> Retirar el chip y la limpieza de los electrodos de la Bioanalyzer de acuerdo con las instrucciones del fabricante. </li></ol></li></ol> 5. Inferir el genotipo de la Reseña Electroforesis <ol><li> Cargar el software Mahal (software está disponible gratuitamente a partir de los autores). </li><li> datos de referencia de importación en el software Mahal: &quot;Archivo&quot;&gt; ​​&quot;Importar datos de referencia&quot; </li><li> Abra la electroforesis ejecutarse en el contexto de datos del software Bioanalyzer. Seleccionar un perfil de electroforesis de una muestra. </li><li> Compruebe si los picos relevantes. <ol><li> Utilice el software Mahal para comprobar si los picos relevantes: &quot;Herramientas&quot;&gt; &quot;media de fluorescencia&quot;. Insertar en el cuadro de la &quot;fluorescencia&quot;los valores de fluorescencia indican como unidades de fluorescencia (FU), ya que se pueden leer desde el software Bioanalyzer. Inserte los 4 (3) con picos de fluorescencia más elevada en orden ascendente y separados por comas. Ejemplo: 126 130 132 137. NOTA: Si no todos estos picos FU se indican mediante el software Bioanalyzer, tienen que ser estimado por la lectura de estas naciones de la trama. </li></ol></li><li> Presione el botón &quot;Calcular&quot;. Un pico es relevante si la fluorescencia observado es superior al valor indicado en el cuadro de &quot;pico de altura mínima&quot;. </li><li> Inferir el genotipo. <ol><li> Insertar en los Mahal cuadro de texto &quot;Picos de entrada (pb)&quot; a los tamaños en pares de bases de todos los picos pertinentes en orden ascendente y separados por comas. Ejemplo: 440.462.519.579.637. NOTA: Si no todos estos tamaños máximos se indican mediante el software Bioanalyzer, tienen que ser estimado por la lectura de estas naciones de la trama. </li></ol></li><li> Presione el botón &quot;Calcular&quot;. </li><li> En la listacaja, buscar la mínima distancia de Mahalanobis al cuadrado &quot;d2m&quot;. Si el valor de P correspondiente derecho a es ≥0.05 entonces la probabilidad es ≥5% de que la hipótesis nula H 0 se rechaza (H 0: el observado y el perfil de referencia son idénticos), que significa que el perfil observado se acepta ser idéntica a el perfil de referencia del genotipo indicada. </li><li> Si d2m es mínimo, pero el campo del valor P correspondiente está vacía, rechazar H 0 con una probabilidad &lt;5%. NOTA: Esto significa que los perfiles son desiguales aunque d2m es mínima. Tal resultado puede ser el resultado de condiciones que no sean promedio de electroforesis. <ol><li> Para corregir estas desviaciones, insertar en el cuadro de texto &quot;Observado Pico (pb)&quot; el ​​valor 395 y pulse el botón &quot;Calcular&quot;. Revisar una vez más d2m mínimo y un valor de p correspondiente ≥0.05. Si no tiene éxito, trate de los valores 405, 415, y 425. NOTA: A veces pasos más pequeños son aúnindicado. Si todavía no tiene éxito, el perfil observado no está presente en los datos de referencia, puede ser un nuevo genotipo o variante. </li></ol></li><li> Varios perfiles <ol><li> Repita los pasos 5.6 a 5.9 para cada perfil de electroforesis por separado. </li></ol></li></ol>

<ol>
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U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mastitis-related+subtypes+of+bovine+Staphylococcus+aureus+are+characterized+by+different+clinical+properties.">Mastitis-related subtypes of bovine Staphylococcus aureus are characterized by different clinical properties.</a> J.Dairy Sci. 92 (4), 1442-1451 (2009).</li><li>Heiniger, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kosten-Nutzen-Analyse+einer+Intervention+zur+Verbesserung+der+Eutergesundheit+in+Schweizer+Milchviehbetrieben.">Kosten-Nutzen-Analyse einer Intervention zur Verbesserung der Eutergesundheit in Schweizer Milchviehbetrieben.</a> Schweiz.Arch.Tierheilkd. 156 (10), 473-481 (2014).</li><li>Hamann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+the+physiological+cell+count+threshold+based+on+changes+in+milk+composition.">Definition of the physiological cell count threshold based on changes in milk composition.</a> IDF Mastitis Newsletter. 25, 9-12 (2003).</li><li>Hwang, S. Y., Park, Y. K., Koo, H. C., Park, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=spa+typing+and+enterotoxin+gene+profile+of+Staphylococcus+aureus+isolated+from+bovine+raw+milk+in+Korea.">spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea.</a> J.Vet.Sci. 11 (2), 125-131 (2010).</li><li>Sakwinska, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Link+between+genotype+and+antimicrobial+resistance+in+bovine+mastitis-related+Staphylococcus+aureus+strains,+determined+by+comparing+Swiss+and+French+isolates+from+the+Rhone+Valley.">Link between genotype and antimicrobial resistance in bovine mastitis-related Staphylococcus aureus strains, determined by comparing Swiss and French isolates from the Rhone Valley.</a> Appl.Environ.Microbiol. 77 (10), 3428-3432 (2011).</li><li>Rabello, R. 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The authors have nothing to disclose.

El paso más crítico de todo el procedimiento es cuando hay un exceso de ADN en RS-PCR (&gt; 30 ng de ADN / ensayo puro) 2. En general, la resolución de un perfil es óptimo si todos sus picos de inicio y fin en la línea de base. Si dos picos están demasiado juntos, la resolución es incompleta. En estas condiciones, el software Bioanalyzer puede conducir a la identificación de los picos apropiado de manera que se requiere la identificación manual. Todos los picos visiblemen...

Staphylococcus (S. aureus) se conoce como el patógeno más importante en todo el mundo responsable de la infección intramamaria (IMI) en el ganado 1. El estudio realizado por Fournier et al., 2 demostrado en las vacas suizas y los estudios realizados por Cosandey et al. 3 y de Boss et al. 4 en vacas de 12 países europeos que S. aureus aislado de bovino IMI es un grupo genéticamente heterogéneo. Mediante amplificación por PCR de la región espaciadora intergénica 16S-23S rRNA (RS-PCR), un total de 17 genotipos se detectaron inicialmente en 101 aislamientos epidemiológicamente independientes 2. El genotipo B y C de genotipo (GTC) fueron más comunes (80%), mientras que los otros 15 genotipos (SMT) se produjeron en raras ocasiones, cada uno haciendo del 1 al 4% de todos los aislamientos. A nivel europeo 3, GTB, GTC, GTF, GTI, y GTR fueron los genotipos más importantes que comprenden el 76% de todas las cepas analizadas (n = 456). GTB se encuentra en el centro de Europa wientras los otros genotipos se difundieron ampliamente. El 24% restante de las cepas incluyó 41 genotipos, muchos de ellos, sin embargo, se observaron sólo una vez. Los estudios realizados por Fournier et al., 2, Cosandey y col. 3, y Graber et al. 5 demostraron además que los genotipos fueron altamente asociados con su patrón de genes de virulencia, así como en el suizo como a nivel europeo. Los estudios revelaron diferencias más grandes en la prevalencia entre IMI S. aureus GTB, GTC y los otros genotipos 2,5: Teniendo en cuenta GTB, hasta el 87% de las vacas de un rebaño se infectaron por este genotipo. En el caso de GTC y de los otros genotipos, sin embargo, IMI fue sólo se encuentra en 1 a 2 vacas de un rebaño. IMI causada por S. aureus, normalmente resulta en la inflamación de las glándulas mamarias correspondientes y un aumento de células inflamatorias en la leche. Como consecuencia de ello, la co células somáticasunts en la leche (SCC), el indicador clave de la calidad de la leche en la mayoría de los países, se incrementa dando lugar a una disminución del precio de la leche o incluso detener la entrega. Además de la RS-PCR de Fournier et al. 2, se han descrito otros métodos de tipificación para subtipificación cepas bovinas de S. aureus 8-11. Dos de los más comunes incluyen la tipificación spa de 12 y Multi Locus Secuencia Typing (MLST) 13. El primero de ellos está basado en el ADN secuenciación de la región espaciadora variable del gen que codifica para la proteína de spa estafilocócica A, mientras que el segundo se requiere la secuenciación de los genes de limpieza 7. Esto significa que un 12 y siete PCRs 13, respectivamente, necesitan ser realizados, seguido de purificación amplicón, reacción de secuenciación y el análisis utilizando equipo específico. En comparación con RS-PCR, estos métodos requieren un esfuerzo adicional considerable en el laboratorio que resulta en un flujo de muestras de bajo y altos costos. losel rendimiento es especialmente baja en el PFGE. Teniendo en cuenta la resolución de estos métodos para las cepas de la especie bovina de S. aureus, RS-PCR es por lo menos tan bueno como spa escribir 4 y mejor que MLST 4 o PFGE 15. Todos estos métodos que incluyen la tipificación binaria 13 demostraron su eficacia en la obtención de una mayor comprensión de la patogénesis de la IMI bovina causada por S. aureus, pero debido a las restricciones mencionadas son menos apropiados para grandes investigaciones clínicas. Además, la genotipificación por RS-PCR como se describe por Fournier et al. 2 permite la predicción fiable de la epidemiológica y el potencial patógeno de S. aureus involucrado en el IMI bovina, dos valores clave en la medicina veterinaria clínica.

<table><tbody><tr><td>Tris(hydroxymethyl)-aminomethan</td><td>Merck</td><td>1.08382.0500</td><td>Mw = 121.14 g/mol</td></tr><tr><td>Titriplex III</td><td>Merck</td><td>1.08418.0250</td><td>Mw = 372.24 g/mol; Substance is equivalent to Na&lt;sub&gt;2&lt;/sub&gt;EDTA&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Hydrochloric acid 5 mol/L</td><td>Merck</td><td>1.09911.0001</td><td></td></tr><tr><td>Columbia agar+5% sheep blood</td><td>bioM&amp;eacute;rieux</td><td>43049</td><td></td></tr><tr><td>Agilent DNA7500 Kit</td><td>Agilent Technologies</td><td>5067-1504</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>Agilent 2100 expert sofware</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>HotStarTaq master mix&amp;nbsp;</td><td>Qiagen</td><td>203445</td><td></td></tr><tr><td>Primer L1</td><td>Mycrosinth</td><td></td><td>5&#039;CAA GGC ATC CAC CGT3&#039;</td></tr><tr><td>Primer G1</td><td>Mycrosinth</td><td></td><td>5&#039;GAA GTC GTA ACA AGG3&#039;</td></tr></tbody></table>

genotyping of staphylococcus aureus by ribosomal spacer pcr (rs-pcr)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Definición de un genotipo y sus variantes Un perfil electroforético que difieren en más de una banda de todos los genotipos identificados se considera como un nuevo genotipo. Nuevos genotipos se nombran y se extendieron acuerdo con Fournier et al. 5 que conduce a los genotipos de GTA a la GTZ, seguido de los genotipos GTAA a GTAZ, y GTBA a GTBZ, y al GTCA GTCC en la actualidad. Variación en sólo una ba...

Watch this Scientific Journal Video about Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR at JoVE.com

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

genotipado de<em&gt; Staphylococcus aureus</em&gt; por Ribosómica espaciador de PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

genotyping-of-staphylococcus-aureus-by-ribosomal-spacer-pcr-rs-pcr

Research

JoVE Journal

Immunology and Infection

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

9.6K vistas.  Agroscope. Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

Video: Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

Generación de un gen interrumpido Streptococcus mutans cepa sin gen clonación

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Investigación

Genética

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

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

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. ...

Inmunología e Infección

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Biosensor para la detección de antibióticos Las bacterias estafilococo resistentes

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Bioingeniería

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

Indice competitivo basado en PCR digital para el análisis de alta producción de la aptitud en Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

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 ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Metodología para el Estudio de la transferencia horizontal de genes en<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genoma escala supresiones de genes en<em&gt; Streptococcus sanguinis</em&gt; Por PCR de alto rendimiento

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Biología

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

Construcción de mutantes en el serotipo 1 Streptococcus pneumoniae cepa 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

La secuenciación de próxima generación de 16S ARN ribosomal genes Amplicones

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

Efficient Nucleic Acid Extracción y 16S rRNA genes de secuenciación para bacteriana Comunidad Caracterización

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

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