O autor gostaria de agradecer li Zhang, Xian-Zhi Li, Aaron Hinz, e Clayton Hall para ajuda editorial com este manuscrito. Este ensaio foi inicialmente desenvolvido no laboratório de George O'Toole, Geisel School of Medicine em Dartmouth. A pesquisa no laboratório do Dr. Mah é apoiada por bolsas do Conselho de Pesquisa em Ciências Naturais e Engenharia do Canadá e da Fibrose Cística do Canadá.

1. MBC-B
<ol><li>
Crescimento de um biofilme (adaptado de O'Toole9).
	<ol>
<li>Cresça uma cultura do tipo selvagem de interesse e tensão mutante por 16 horas em um meio rico a 37 °C.</li>
		<li>Diluir as culturas saturadas durante a noite 1:100 em meio fresco para ensaios de resistência a antibióticos. Um meio padrão para P. aeruginosa é m63 médio mínimo suplementado com sulfato de magnésio e arginina (ver Tabela 1). Este meio estimula a formação de um biofilme mais robusto.</li>
		<li>Adicione 100 μl da diluição por poço em um prato de microtiter de 96 poços (ver Tabela 1). Uma vez que esses ensaios são tipicamente realizados em triplicado para cada cepa, deve haver 24 poços de cada cepa.</li>
		<li>Incubar a placa de microtútmetro por 24 horas a 37 °C.</li>
	</ol>
</li>
	<li>
Expondo o biofilme pré-formado a um gradiente de concentração de antibiótico
	<ol>
<li>Prepare uma série de diluição de 10x de antibiótico para 7 poços. Exemplo: para a tobramicina antibiótico, as concentrações finais nos poços são 0,4, 0,2, 0,1, 0,05, 0,025, 0,0125 e 0,006 mg/ml 5-8 . A partir de um estoque de 25 mg/ml, prepare diluições de 10x de 4, 2, 1, 0,5, 0,25, 0,125 e 0,06 mg/ml. Deixe no gelo.</li>
		<li>Remova o supernatante gasto (contendo células planctônicas) usando uma pipeta multicanal (ver Tabela 1).</li>
		<li>Adicione 90 μl M63 (Mg/Arg) a todos os poços.</li>
		<li>Adicione 10 μl de cada concentração de antibiótico de 10x para alcançar as concentrações finais desejadas. Adicione 10 μl de água (sem controle de antibióticos) ao poço final para cada réplica e tensão.</li>
		<li>Incubar a placa de microtútmetro por 24 horas a 37 °C.</li>
	</ol>
</li>
	<li>
Refrescar a mídia e permitir o desprendimento de células vivas.
	<ol>
<li>Remova o supernatante gasto (contendo células planctônicas) usando uma pipeta multicanal.</li>
		<li>Adicione 115 μl M63 (Mg/Arg) a todos os poços.</li>
		<li>Incubar a placa de microtútmetro por 24 horas a 37 °C.</li>
	</ol>
</li>
	<li>
Ensaio para células vivas.
	<ol>
<li>Rotule duas placas de ágar LB por placa de microtiter de 96 poços.</li>
		<li>Esterilize o dispositivo multifaceto (ver Tabela 1) mergulhando as pontas em 100% etanol e passando as pontas através da chama aberta de um queimador de Bunsen. repetir. Deixe as pontas esfriarem ligeiramente. Usando o dispositivo multifacetado, transfira ~3 μl (quantidade que normalmente é retida nas pontas das pontas) da cultura planctônica de cada poço da placa de microtítmetro para a superfície de uma placa de ágar LB.</li>
		<li>Incubar as placas de ágar LB por 16 horas a 37 °C.</li>
		<li>Determine a concentração bactericida mínima do antibiótico identificando, por olho, o corte para o crescimento bacteriano(Figura 1).</li>
	</ol>
</li>
</ol>2. MBC-P
<ol><li>
Preparando cepas bacterianas
	<ol>
<li>Cresça uma cultura do tipo selvagem de interesse e tensão mutante por 16 horas em um meio rico a 37 °C.</li>
		<li>Diluir as culturas saturadas durante a noite 1:100 em meio fresco para ensaios de resistência a antibióticos. Um meio padrão para P. aeruginosa é m63 médio mínimo suplementado com sulfato de magnésio e arginina (ver Tabela 1).</li>
		<li>Adicione 90 μl da diluição por poço em um prato de microtiter de 96 poços (ver Tabela 1). Uma vez que esses ensaios são tipicamente realizados em triplicado para cada cepa, deve haver 24 poços de cada cepa.</li>
	</ol>
</li>
	<li>
Expondo células planctônicas a um gradiente de concentração de antibiótico
	<ol>
<li>Prepare a série de diluição de 10x de antibiótico para 7 poços. Exemplo: para a tobramicina antibiótico, as concentrações finais nos poços são 0,032, 0,016, 0,008, 0,004, 0,002, 0,001 e 0,0005 mg/ml. A partir de um estoque de 25 mg/ml, prepare diluições de 0,32, 0,16, 0,08, 0,04, 0,02, 0,01 e 0,005 mg/ml. Deixe no gelo.</li>
		<li>Adicione 10 μl de cada concentração de antibiótico de 10x para alcançar as concentrações finais desejadas. Adicione 10 μl de água (sem controle de antibióticos) ao poço final para cada réplica e tensão.</li>
		<li>Incubar a placa de microtútmetro por 24 horas a 37 °C.</li>
	</ol>
</li>
	<li>
Ensaio para células vivas.
	<ol>
<li>Rotule duas placas de ágar LB por placa de microtiter de 96 poços.</li>
		<li>Esterilize o dispositivo multifaceto (ver Tabela 1) mergulhando as pontas em 100% etanol e passando as pontas através da chama aberta de um queimador de Bunsen. repetir. Deixe as pontas esfriarem ligeiramente. Usando o dispositivo multifacetado, transfira ~3 μl (quantidade que normalmente é retida nas pontas das pontas) da cultura planctônica de cada poço da placa de microtítmetro para a superfície de uma placa de ágar LB.</li>
		<li>Incubar as placas de ágar LB por 16 horas a 37 °C.</li>
		<li>Determinar a concentração bactericida mínima do antibiótico identificando, por olho, o corte para o crescimento 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>

A autora declara que não tem interesses financeiros concorrentes.

A resistência a antibióticos em células planctônicas é definida como um aumento na concentração inibitória mínima (MIC) de um antibiótico devido a uma mudança permanente nas células (por exemplo, uma mutação). Os mecanismos de resistência ou tolerância específicas de biofilmes que foram identificados até hoje são o resultado da expressão de genes do tipo selvagem dentro de biofilmes. Assim, a definição clássica de resistência não se aplica a biofilmes. No entanto, outro conjunto de defin...

Ensaios de resistência a antibióticos foram inicialmente desenvolvidos para avaliar a resistência de culturas planctônicas (natação livre) de bactérias. Como muitas infecções bacterianas envolvem biofilmes (células ligadas à superfície), estávamos interessados em desenvolver um método para avaliar a resistência a antibióticos específicos do biofilme. No entanto, a maioria dos ensaios de resistência a antibióticos são pouco adequados para medir a resistência de biofilmes. Por exemplo, determinar a concentração inibitória mínima (MIC) é o padrão-ouro para determinar a resistência a antibióticos das culturas bacterianas planctônicas 1. Este ensaio implica misturar uma cultura plantiônica diluída com uma série de diluição de antibióticos.  A concentração de antibiótico que inibe o crescimento visível das células planctônicas é o MIC. Uma vez que este ensaio se baseia na inibição do crescimento, por definição, ele não pode trabalhar com culturas de biofilme, o que requer examinar a sensibilidade antibiótico das células em um biofilme pré-cultivado. Em vez de medir a inibição do crescimento, o ensaio MBC-B descrito aqui determina a concentração de antibiótico que mata células já existentes em um biofilme. Assim, este ensaio visa imitar tratamentos antibióticos de infecções por biofilmes estabelecidas, e fornecer uma visão mais relevante da resistência a antibióticos bacterianos in vivo.
Uma vez que os biofilmes são geralmente mais resistentes a antibióticos do que as culturas planctônicas 2-4, foi necessário elaborar um método que relaciona diretamente a resistência a antibióticos de um biofilme com o de uma cultura planctônica. Assim, outro objetivo deste método é ser capaz de comparar diretamente o nível de resistência a antibióticos entre células planctônicas e biofilmes. Os ensaios MBC-P e MBC-B descritos aqui tornam isso possível porque as células são cultivadas em condições semelhantes. Utilizamos este método para estudar vários genes que são importantes para a resistência a antibióticos específicas de biofilme 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 uma comparação direta entre a resistência planctônica e biofilme para uma cepa bacteriana que pode formar um biofilme in vitro. Bactérias são inoculadas nos poços de uma placa de microtismo de 96 poços. No caso do ensaio plantônico, diluições seriais do agente antimicrobiano de escolha são adicionadas às suspensões bacterianas. No ensaio de biofilme, uma vez inoculado, as bactérias são deixadas para formar um biofilme durante um determinado período de tempo. Células nãoectadas são removidas dos poços, a mídia é reabastecida e diluições seriais do agente antimicrobiano de escolha são adicionadas. Após a exposição ao agente antimicrobiano, as células planctônicas são avaliadas para o crescimento. Para o ensaio de biofilme, a mídia é atualizada com mídia fresca sem o agente antimicrobiano e as células de biofilm são deixadas para se recuperar. A viabilidade da célula biofilm é avaliada após o período de recuperação. O MBC-P para o agente antimicrobiano é definido como a menor concentração de droga que mata as células na cultura planctônica.  Em contrapartida, o MBC-B para uma cepa é determinado expondo biofilmes pré-formados ao aumento das concentrações de agente antimicrobiano por 24 horas. O MBC-B é definido como a menor concentração de agente antimicrobiano que mata as células do biofilme.

Foram realizados ensaios MBC-P e MBC-B, comparando a sensibilidade do tipo selvagem PA14 com o PA14 ∆ndvB. A tobramicina foi usada como antibiótico. São apresentados resultados correspondentes à etapa 1.4.4 (Figura 1) e à etapa 2.3.4 (Figura 2). PA14 e ∆ndvB foram inoculados nos ensaios MBC-P e MBC-B em triplicado. Após a conclusão das etapas 1.0-1.4 do protocolo MBC-B e as etapas 2.0-2.3 do protocolo MBC-P, as células viáveis foram emplacar em uma placa de ...

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 uma comparação direta entre a resistência planctônica e biofilme para uma cepa bacteriana que pode formar um biofilme in vitro usando uma placa de microtismo de 96 poços. Bactérias planctônicas ou biofilmes são expostas a diluições seriais do agente antimicrobiano de escolha. A viabilidade é avaliada pelo crescimento em placas de ágar.

Estabelecendo a Concentração Bactericidal Mínima de um Agente Antimicrobiano para Células Planktônicas (MBC-P) e Células Biofilm (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 ...

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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)

19.9K visualizações.  University of Ottawa. Este protocolo permite uma comparação direta entre a resistência planctônica e biofilme para uma cepa bacteriana que pode formar um biofilme in vitro usando uma placa de microtismo de 96 poços. Bactérias planctônicas ou biofilmes são expostas a diluições seriais do agente antimicrobiano de escolha. A viabilidade é avaliada pelo crescimento em placas de ágar.

Vídeo: 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 e Saccharomyces cerevisiae Biofilme em Células de Fluxo

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

Imunologia e Infecção

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

In vitro a formação de biofilme em um slide Câmara de 8 poços

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.

Seguindo o destino celular, em<em&gt; E. coli</em&gt; Após a infecção por um fago 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.

Determinação do biofilme Iniciação em células infectadas por vírus por bactérias e fungos

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.

Desenvolvimento e validação de uma única molécula ultra-sensível de matriz Digital ensaio imunossorvente ligado à enzima para humana Interferon-α

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.

Quantificação de baseados em citometria de fluxo multicolor de mitocôndrias e lisossomos em 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.

Um do elevado-throughput Shigella-ensaio 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.

Produção e Caracterização de Macrófagos Humanos de Células-Tronco 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.

Estabelecendo um modelo de córnea porcina ex vivo para estudar tratamentos medicamentosos contra ceratite 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.