El autor desea agradecer a Li Zhang, Xian-Zhi Li, Aaron Hinz y Clayton Hall por la ayuda editorial con este manuscrito. Este ensayo se desarrolló inicialmente en el laboratorio de George O'Toole, de la Escuela de Medicina Geisel en Dartmouth. La investigación en el laboratorio del Dr. Mah está respaldada por subvenciones del Consejo de Investigación de Ciencias Naturales e Ingeniería de Canadá y Cystic Fibrosis Canada.

1. MBC-B
<ol><li>
Cultivo de un biofilm (adaptado de O'Toole9).
	<ol>
<li>Cultivar un cultivo de tipo salvaje cepa de interés y cepa mutante durante 16 horas en un medio rico a 37 °C.</li>
		<li>Diluir los cultivos saturados durante la noche 1:100 en un medio fresco para ensayos de resistencia a los antibióticos. Un medio estándar para P. aeruginosa es el medio mínimo M63 suplementado con sulfato de magnesio y arginina (ver Tabla 1). Este medio estimula la formación de un biofilm más robusto.</li>
		<li>Añadir 100 μl de dilución por pozo en una antena parabólica de microtiter de 96 pozos (ver Tabla 1). Dado que estos ensayos se realizan normalmente por triplicado para cada cepa, debe haber 24 pozos de cada cepa.</li>
		<li>Incubar la placa de microter durante 24 horas a 37 °C.</li>
	</ol>
</li>
	<li>
Exponer la biopelícula preformada a un gradiente de concentración de antibióticos
	<ol>
<li>Prepare una serie de dilución de 10x de antibióticos para 7 pozos. Ejemplo: para el antibiótico tobramicina, las concentraciones finales en los pozos son 0,4, 0,2, 0,1, 0,05, 0,025, 0,0125 y 0,006 mg/ml 5-8. A partir de un stock de 25 mg/ml, prepare 10x diluciones de 4, 2, 1, 0,5, 0,25, 0,125 y 0,06 mg/ml. Dejar en el hielo.</li>
		<li>Retire el sobrenadante gastado (que contiene células planctónicas) utilizando una pipeta multicanal (véase la Tabla 1).</li>
		<li>Agregue 90 μl M63 (Mg/Arg) a todos los pozos.</li>
		<li>Añadir 10 μl de cada 10x concentración de antibióticos para lograr las concentraciones finales deseadas. Añadir 10 μl de agua (sin control antibiótico) al pozo final para cada réplica y cepa.</li>
		<li>Incubar la placa de microter durante 24 horas a 37 °C.</li>
	</ol>
</li>
	<li>
Refrescar los medios y permitir el desprendimiento de células vivas.
	<ol>
<li>Retire el sobrenadante gastado (que contiene células planctónicas) utilizando una pipeta multicanal.</li>
		<li>Agregue 115 μl M63 (Mg/Arg) a todos los pozos.</li>
		<li>Incubar la placa de microter durante 24 horas a 37 °C.</li>
	</ol>
</li>
	<li>
Ensayo para células vivas.
	<ol>
<li>Etiquete dos placas de agar LB por placa de microtíter de 96 pozos.</li>
		<li>Esterilice el dispositivo multipróngo (ver Tabla 1)sumergiendo las puntas en etanol al 100% y pasando las puntas a través de la llama abierta de un quemador Bunsen. repetir. Deja que las puntas se enfríen ligeramente. Usando el dispositivo multiprong, transfiera ~3 μl (cantidad que normalmente se retiene en las puntas de las puntas) de cultivo planctónico desde cada pozo de la placa de microter a la superficie de una placa de agar LB.</li>
		<li>Incubar las placas de agar LB durante 16 horas a 37 °C.</li>
		<li>Determinar la concentración bactericida mínima del antibiótico identificando, a ojo, el corte para el crecimiento bacteriano (Figura 1).</li>
	</ol>
</li>
</ol>2. MBC-P
<ol><li>
Preparación de cepas bacterianas
	<ol>
<li>Cultivar un cultivo de tipo salvaje cepa de interés y cepa mutante durante 16 horas en un medio rico a 37 °C.</li>
		<li>Diluir los cultivos saturados durante la noche 1:100 en un medio fresco para ensayos de resistencia a los antibióticos. Un medio estándar para P. aeruginosa es el medio mínimo M63 suplementado con sulfato de magnesio y arginina (ver Tabla 1).</li>
		<li>Añadir 90 μl de dilución por pozo en una antena parabólica de 96 kódteres (ver Tabla 1). Dado que estos ensayos se realizan normalmente por triplicado para cada cepa, debe haber 24 pozos de cada cepa.</li>
	</ol>
</li>
	<li>
Exposición de las células planctónicas a un gradiente de concentración de antibióticos
	<ol>
<li>Preparar 10x series de dilución de antibióticos para 7 pozos. Ejemplo: para el antibiótico tobramicina, las concentraciones finales en los pozos son 0,032, 0,016, 0,008, 0,004, 0,002, 0,001 y 0,0005 mg/ml. A partir de un stock de 25 mg/ml, preparar diluciones de 0,32, 0,16, 0,08, 0,04, 0,02, 0,01 y 0,005 mg/ml. Dejar en el hielo.</li>
		<li>Añadir 10 μl de cada 10x concentración de antibióticos para lograr las concentraciones finales deseadas. Añadir 10 μl de agua (sin control antibiótico) al pozo final para cada réplica y cepa.</li>
		<li>Incubar la placa de microter durante 24 horas a 37 °C.</li>
	</ol>
</li>
	<li>
Ensayo para células vivas.
	<ol>
<li>Etiquete dos placas de agar LB por placa de microtíter de 96 pozos.</li>
		<li>Esterilice el dispositivo multipróngo (ver Tabla 1)sumergiendo las puntas en etanol al 100% y pasando las puntas a través de la llama abierta de un quemador Bunsen. repetir. Deja que las puntas se enfríen ligeramente. Usando el dispositivo multiprong, transfiera ~3 μl (cantidad que normalmente se retiene en las puntas de las puntas) de cultivo planctónico desde cada pozo de la placa de microter a la superficie de una placa de agar LB.</li>
		<li>Incubar las placas de agar LB durante 16 horas a 37 °C.</li>
		<li>Determinar la concentración bactericida mínima del antibiótico mediante la identificación, a simple vista, del corte para el crecimiento bacteriano (Figura 2).</li>
	</ol>
</li>
</ol>

<ol>
	<li>Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int. J. Antimicrob. Agents. 35 (4), 322-332 (2010).</li><li>Mah, T. F., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+biofilm+resistance+to+antimicrobial+agents.">Mechanisms of biofilm resistance to antimicrobial agents.</a> Trends Microbiol. 9 (1), 34-39 (2001).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Zhang, L., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Involvement+of+a+Novel+Efflux+System+in+Biofilm-Specific+Resistance+to+Antibiotics.">The Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics.</a> J. Bacteriol. 190 (13), 4447-4452 (2008).</li><li>Zhang, L., Hinz, A. J., Nadeau, J. P., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+tssC1+Links+Type+VI+Secretion+and+Biofilm-specific+Antibiotic+Resistance.">Pseudomonas aeruginosa tssC1 Links Type VI Secretion and Biofilm-specific Antibiotic Resistance.</a> J. Bacteriol. 193 (19), 5510-5513 (2011).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biofilm-Specific+Antibiotic+Resistance+Gene,+ndvB,+is+Important+for+Expression+of+Ethanol+Oxidation+Genes+in+Pseudomonas+aeruginosa+Biofilms.">The Biofilm-Specific Antibiotic Resistance Gene, ndvB, is Important for Expression of Ethanol Oxidation Genes in Pseudomonas aeruginosa Biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+Dish+Biofilm+Formation+Assay.">Microtiter Dish Biofilm Formation Assay.</a> J. Vis. Exp. (47), e2437 (2011).</li><li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+tolerance+of+biofilms+and+persister+cells.">Multidrug tolerance of biofilms and persister cells.</a> Curr. Top. Microbiol. Immunol. 322, 107-131 (2008).</li><li>Merritt, J. H., Kadouri, D. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+and+Analyzing+Static+Biofilms.">Growing and Analyzing Static Biofilms.</a> Curr. Protoc. Microbiol. 1, 1B.1.1-1B.1.17 (2005).</li><li>Ramey, B. E., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.+Growing+and+analyzing+biofilms+in+fermenters.">Chapter 1. Growing and analyzing biofilms in fermenters.</a> Curr. Protoc. Microbiol. 1, 1B.3.1-1B.3.14 (2005).</li></ol>

La autora declara que no tiene intereses financieros contrapuestos.

La resistencia a los antibióticos en las células planctónicas se define como un aumento en la concentración inhibitoria mínima (MIC) de un antibiótico debido a un cambio permanente en las células(por ejemplo, una mutación). Los mecanismos de resistencia o tolerancia específicos de la biopelícula que se han identificado hasta la fecha son el resultado de la expresión de genes de tipo salvaje dentro de las biopelículas. Por lo tanto, la definición clásica de resistencia no se aplica a las biopelícul...

Los ensayos de resistencia a los antibióticos se desarrollaron inicialmente para probar la resistencia de los cultivos planctónicos (de natación libre) de bacterias. Dado que muchas infecciones bacterianas involucran biopelículas (células adheridas a la superficie), estábamos interesados en desarrollar un método para ensayar la resistencia a los antibióticos específicos de la biopelícula. Sin embargo, la mayoría de los ensayos de resistencia a los antibióticos son poco adecuados para medir la resistencia de las biopelículas. Por ejemplo, la determinación de la concentración inhibitoria mínima (MIC) es el patrón oro para determinar la resistencia a los antibióticos de los cultivos bacterianos planctónicos 1. Este ensayo implica mezclar un cultivo planctónico diluido con una serie de dilución de antibióticos.  La concentración de antibiótico que inhibe el crecimiento visible de las células planctónicas es el MIC. Dado que este ensayo se basa en la inhibición del crecimiento, por definición, no puede funcionar con cultivos de biopelículas, lo que requiere examinar la sensibilidad a los antibióticos de las células en una biopelícula precrecida. En lugar de medir la inhibición del crecimiento, el ensayo MBC-B descrito aquí determina la concentración de antibiótico que mata las células que ya existen en una biopelícula. Por lo tanto, este ensayo tiene como objetivo imitar los tratamientos antibióticos de las infecciones por biopelículas establecidas, y proporcionar una visión más relevante de la resistencia bacteriana a los antibióticos in vivo.
Dado que las biopelículas son generalmente más resistentes a los antibióticos que los cultivos planctónicos 2-4, fue necesario idear un método que relacione directamente la resistencia a los antibióticos de una biopelícula con la de un cultivo planctónico. Por lo tanto, otro objetivo de este método es poder comparar directamente el nivel de resistencia a los antibióticos entre las células planctónicas y de biopelículas. Los ensayos MBC-P y MBC-B descritos aquí hacen esto posible porque las células se cultivan en condiciones similares. Hemos utilizado este método para estudiar varios genes que son importantes para la resistencia a antibióticos específicos de biopelículas 5-8.

<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td style="width:160px;">
			1x&#160;M63
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			Prepare as a 5x&#160;M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature.&#160; Dilute 5x&#160;stock 1:5, autoclave, cool, then add the desired components.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			KH2PO4
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			P285-500
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			K2HPO4
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			P288-500
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			(NH4)2SO4
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			A5132
			</td>
			<td style="width:160px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Magnesium sulfate
			</td>
			<td style="width:160px;">
			Fisher
			</td>
			<td style="width:160px;">
			M63-500
			</td>
			<td style="width:160px;">
			Add to 1 mM final concentration.&#160; Prepare as a 1 M stock in water and autoclave.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Tobramycin
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			
			</td>
			<td style="width:160px;">
			Prepare 50 mg/m&#160;stock. Aliquot and store at -20&#176;C.
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Arginine
			</td>
			<td style="width:160px;">
			Sigma
			</td>
			<td style="width:160px;">
			A5131
			</td>
			<td style="width:160px;">
			Add to 0.4% final concentration.&#160; Prepare as a 20% stock in water and filter sterilize.&#160; This alternative carbon/energy source can replace glucose and casamino acids
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			96-well microtiter plates
			</td>
			<td style="width:160px;">
			Corning
			</td>
			<td style="width:160px;">
			3595
			</td>
			<td style="width:160px;">
			Sterile, flat-bottom, low evaporation
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Tranferpette (multichannel pipette)
			</td>
			<td style="width:160px;">
			BrandTech
			</td>
			<td style="width:160px;">
			2703610
			</td>
			<td style="width:160px;">
			8-channel, 20-200 &#956;l
			</td>
		</tr>
		<tr>
			<td style="width:160px;">
			Multiprong device
			</td>
			<td style="width:160px;">
			Dan-Kar
			</td>
			<td style="width:160px;">
			MC48
			</td>
			<td style="width:160px;">
			48 prongs fit into &#189; of a 96-well microtiter plate
			</td>
		</tr>
	</tbody>
</table>

establishing the minimal bactericidal concentration of an antimicrobial agent for planktonic cells (mbc-p) and biofilm cells (mbc-b)

Este protocolo permite una comparación directa entre la resistencia planctónica y la de biopelícula para una cepa bacteriana que puede formar una biopelícula in vitro. Las bacterias se inoculan en los pozos de una placa de microtíter de 96 pozos. En el caso del ensayo planctónico, se añaden diluciones en serie del agente antimicrobiano de elección a las suspensiones bacterianas. En el ensayo de biopelícula, una vez inoculadas, las bacterias se dejan formar una biopelícula durante un período de tiempo determinado. Las células no unidas se eliminan de los pocillos, se reponen los medios y se añaden diluciones en serie del agente antimicrobiano de elección. Después de la exposición al agente antimicrobiano, las células planctónicas se ensayan para el crecimiento. Para el ensayo de biopelícula, el medio se actualiza con medios frescos que carecen del agente antimicrobiano y las células de la biopelícula se dejan recuperar. La viabilidad de las células de biopelículas se ensaya después del período de recuperación. El MBC-P para el agente antimicrobiano se define como la concentración más baja de droga que mata las células en el cultivo planctónico.  En contraste, el MBC-B para una cepa se determina exponiendo biopelículas preformadas a concentraciones crecientes de agente antimicrobiano durante 24 horas. El MBC-B se define como la concentración más baja de agente antimicrobiano que mata las células en el biofilm.

Se realizaron ensayos MBC-P y MBC-B, comparando la sensibilidad del tipo salvaje PA14 con PA14 ∆ndvB. La tobramicina se utilizó como antibiótico. Se presentan los resultados correspondientes a los pasos 1.4.4 (Figura 1) y 2.3.4 (Figura 2). PA14 y ∆ndvB fueron inoculados en los ensayos MBC-P y MBC-B por triplicado. Después de completar los pasos 1.0-1.4 del protocolo MBC-B y los pasos 2.0-2.3 del protocolo MBC-P, las células viables fueron plateadas sobre una pla...

Watch this Scientific Journal Video about Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B at JoVE.com

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

Este protocolo permite una comparación directa entre la resistencia planctónica y la de biopelícula para una cepa bacteriana que puede formar una biopelícula in vitro utilizando una placa de microtíter de 96 pozos. Las bacterias planctónicas o de biopelículas están expuestas a diluciones en serie del agente antimicrobiano de elección. La viabilidad se ensa prueba por el crecimiento en placas de agar.

Establecimiento de la concentración bactericida mínima de un agente antimicrobiano para las células planctónicas (MBC-P) y las células de biopelícula (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

establishing-minimal-bactericidal-concentration-an-antimicrobial

Research

JoVE Journal

Immunology and Infection

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

19.8K vistas.  University of Ottawa. Este protocolo permite una comparación directa entre la resistencia planctónica y la de biopelícula para una cepa bacteriana que puede formar una biopelícula in vitro utilizando una placa de microtíter de 96 pozos. Las bacterias planctónicas o de biopelículas están expuestas a diluciones en serie del agente antimicrobiano de elección. La viabilidad se ensa prueba por el crecimiento en placas de agar.

Video: Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa y Biofilm Saccharomyces cerevisiae en las celdas de flujo

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Investigación

Inmunología e Infección

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

En la formación de biofilm in vitro en una diapositiva Cámara de 8 pocillos

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Determinación de Biofilm Iniciación, el virus de las células infectadas por bacterias y hongos

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Desarrollo y validación de una sola molécula ultrasensible matriz Digital análisis de enzima-ligado del inmunosorbente para interferón-α humano

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Cuantificación de basados en citometría de flujo multicolor de las mitocondrias y los lisosomas en las células T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un alto rendimiento Shigella-ensayo bactericida específico

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Producción y caracterización de macrófagos humanos a partir de células madre pluripotentes

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

Establecer un modelo de córnea ex vivo porcina para estudiar tratamientos farmacológicos contra la queratitis bacteriana

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.