We thank Saran Kupul for technical assistance. This work was supported in part by NIH grant R01-AI104952 to FW. Further support was provided by the University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH funded program (P30 AI027767) that was made possible by the following NIH Institutes: NIAID, NIMH, NIDA, NICHD, NHLDI, NIA.

1. biofilme Iniciação <ol><li> Crescer uma cultura de biofilme organismo produtor num meio rico em nutrientes. Inocular Staphylococcus aureus estirpe Newman, em 10 ml de meio de Mueller-Hinton a partir de um stock de glicerol. Realizar todos os trabalhos envolvendo a manipulação de S. aureus com luvas e dentro de um gabinete de biossegurança. </li><li> Incubar durante 16 horas a 37 ° C num agitador rotativo (100-200 rpm). </li><li> Determinar DO600 da cultura utilizando um espectrofotómetro e uma cuvete padrão (comprimento do caminho: 1 centímetro). </li><li> Calcular o volume (V) de cultura necessário para preparar 20 ml de inoculo com a DO600 (inoculo) de 0,1 em chelexed meios de Roswell Park Memorial Institute (cRPMI) 9 utilizando a fórmula: [V = 20 ml * 0,1 / OD 600 (cultura )] Nota: Referência 9 fornece detalhes sobre a preparação cRPMI. <ol><li> Adicionar o volume calculado de cultura (de acima) a um tubo de centrífuga e as células de pelotas por centrifugação durante 1 min a 10.000 x g. para prepare inoculo biofilme, aspirar o sedimento de células e meio de crescimento gasto ressuspender em 20 ml de cRPMI. </li></ol></li><li> Pour inoculo para um reservatório de 25 ml. </li><li> Remova cuidadosamente a tampa de uma placa de peg 96 poços estéril, puxando-o para cima. Nota: É preciso ter cuidado para não contaminar os pinos de suspensão para baixo da tampa. A tampa pode ser colocado de cabeça para baixo em uma superfície limpa dentro de uma cabine de segurança biológica. </li><li> Usando uma pipeta de 200 uL de multi-canal, adicionar 125 uL de inoculo em cada cavidade de filas A a G da placa de fundo. </li><li> Encha meio cRPMI 125 ul estéril em cada poço de uma fila H como o controle de esterilidade. </li><li> Leve o peg-tampa e mantenha-o acima da parte inferior da placa com os pinos voltados para baixo. Alinhe cuidadosamente os pinos individuais com seus respectivos poços. Evitar o contacto físico entre a placa e as estacas. Uma vez alinhadas, abaixe a tampa com cuidado para baixo. Evitar distorções como reajustes realizados após as estacas ter tocado a placa de fundo pode contcontroles de esterilidade aminar em H. linha </li><li> Selar a tampa para a placa com película de parafina para evitar a evaporação e coloque em um saco plástico lacrado, selando firmemente. Manter o volume de ar dentro do saco o mais baixo possível para minimizar a evaporação. </li><li> Incubar sem agitação a 37 ° C durante 48 horas numa incubadora de CO2 a 5%. Nota: 48 horas de crescimento tem sido óptimo para S. aureus, mas irá variar com base no organismo utilizado. Nota: Embora incubação estática é um bom ponto de partida para optimizar o ensaio, agitando a 150 rpm num agitador de microplacas é uma variação possível que pode aumentar a formação de biofilme de alguns S. aureus. </li></ol> 2. Preparação da placa de biofilme Desafio <ol><li> No dia do desafio biofilme tomar uma placa de 96 poços frescas e adicionar 100 ul de cRPMI, equilibrada à temperatura ambiente, a cada poço de linhas B de H. A fim de evitar resultados inconsistentes, utilizar uma placa de 96 poços que pode conter 200 uldos meios de comunicação, quando as cavilhas são para dentro, e apresenta cavidades com dimensões idênticas às da placa de iniciação biofilme. </li><li> Diluir até 4 compostos de teste separadamente em 1,0 ml de cRPMI para 2x a concentração final desejada, de modo a representar uma diluição final no passo 2.7. Nota: Um bom ponto de partida é de 8 vezes acima da concentração inibitória das células planctônicas. </li><li> Adicionar 200 mL de composto 1 em poços A1 para A3, composto 2 em poços A4 a A6, composto 3 em poços A7 a A9 e composto 4 em poços A10 a A12. </li><li> Diluir em série os compostos de teste, utilizando uma pipeta de canais múltiplos e transferir 100 ul da linha A para a linha B e misturar. </li><li> Desse modo, continuam a transferir 100 ul poço a poço. Misturar após cada transferência e parar na linha F. </li><li> Após a mistura, expulsar 100 ul de linha F em um recipiente de resíduos. Não transferir qualquer líquido linhas G e H. </li><li> Adicionar 100 ul de cRPMI a cada poço da placa utilizando uma pipeta de canais múltiplos paraperfazer o volume de 200 uL. Veja a Figura 1 para o layout final da placa. </li></ol> 3. biofilme Desafio <ol><li> Adicionar 200 ul de solução salina tamponada com fosfato (PBS) a uma placa de 96 poços estéril fresco que apresenta cavidades com dimensões idênticas às da placa de iniciação biofilme. </li><li> Retire o saco de plástico e filme de parafina a partir de chapa de iniciação biofilme incubadas. </li><li> Levante a tampa para cima. Evitar o contacto entre as estacas e parte inferior da placa, pois isso pode danificar o biofilme. Manter parte inferior para posterior análise de OD 600 (ver 3.8). </li><li> Enxaguar as células planctônicas, colocando o PEG-tampa sobre a placa contendo PBS (do passo 3.1). Submergir estacas por 5 segundos, levante fora da PBS, e submergir mais uma vez, tomando cuidado para não bater as estacas contra os lados também. </li><li> Coloque a tampa peg-lavado na placa de desafio. Evite contato desnecessário entre pinos e placa de fundo, pois isso irá danificar o biofilme levando aresultados inconsistentes. </li><li> placa de vedação com filme de parafina e coloque em um saco plástico lacrado como descrito no passo 1.10. </li><li> Incubar a placa sem agitação durante 24 h a 37 ° C numa incubadora de CO2 a 5%. </li><li> Tome parte inferior da placa de iniciação biofilme de 3,3. Ressuspender as bactérias em cada poço utilizando uma pipeta de canais múltiplos e medir a OD 600 com um leitor de microplacas adequado para confirmar o crescimento ocorre em todos os poços mas os controlos de esterilidade em linha H. </li></ol> 4. Determinação de biofilme <ol><li> Usar um leitor de placa equipado com uma câmara de incubação e capaz de tomar leituras de fluorescência para a detecção de resazurina cinéticas de conversão. Defina-se uma medição da fluorescência cinética 24 h usando uma excitação de 530 nm e 590 nm de emissão. </li><li> Definir a temperatura da câmara para 37 ° C e tem a placa lida a cada 20 min. Definir o leitor de placas para medir a partir do fundo da placa de acordo com as instruções de fabricanteções. </li><li> Prepare a placa de lavagem através da adição de 200 ul de PBS com uma pipeta de canais múltiplos para uma placa de 96 cavidades estéreis. </li><li> Preparar o meio de recuperação de biofilme pela adição de 400 ul de 0,8 mg / ml de solução stock de resazurina para 20 ml de meio cRPMI a uma concentração final de 16 ug / mL de resazurina. Nota: resazurina é um irritante da pele conhecido. Use uma bata de laboratório, óculos contra respingos e luvas durante o manuseio. CRPMI como os meios de recuperação de biofilme foi usado porque ele proporciona o menor sinal de fundo, mas pode ser substituído por meios de Mueller Hinton se for caso disso. </li><li> Adicionar 150 ul de meio de recuperação de biofilme com uma pipeta de canais múltiplos para cada poço de uma placa de 96 poços frescas. </li><li> Transferir a placa desafio da incubadora em uma cabine de segurança biológica e remova cuidadosamente saco de plástico e filme de vedação. </li><li> Remover o peg-tampa da placa de desafio e enxaguar células planctônicas em PBS, conforme descrito no ponto 3.4. Manter a parte inferior da placa de desafio subsequente por OD 600 analise (ver 4.10). </li><li> Após a lavagem, coloque o peg-tampa na parte inferior da placa contendo o meio de recuperação do biofilme. </li><li> Enrole a lado da placa com película de parafina, tendo o cuidado de não obstruir o fundo dos poços de perímetro. Imediatamente após o término do prato, coloque-o no leitor de placas e começar uma leitura cinética durante um período de 24 horas iniciando a feita anteriormente resazurina protocolo cinética a partir do passo 4.1. </li><li> Para quantificar o crescimento das células planctônicas que podem ou não podem ocorrer durante o desafio, ler a OD 600 de toda a parte inferior da placa desafio usando um leitor de microplacas. Siga as instruções dadas no passo 3.8. Nota: ressuspender cuidadosamente o conteúdo de cada poço como células derramamento fora do biofilme tendem a crescer em aglomerados na parte inferior do poço. Quaisquer bolhas dentro do bem irá interferir fortemente com leituras OD 600 e deve ser evitado ou removidos antes da leitura. </li></ol>

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The authors have nothing to disclose.

Aqui, nós descrevemos um ensaio de biofilme modificado para determinar a actividade de inibidores testados em S. biofilmes aureus com foco no estado metabólico das células do biofilme associado. Enquanto a iniciação biofilme descritos e procedimentos de desafio recomendações do fabricante principalmente imitava, a utilização de resazurina corante para detectar e quantificar células associada-biofilme que sobrevivem a um desafio inibidor 24 h simplifica significativamente o procedimento recome...

Micróbios patogênicos podem formar biofilmes in vivo que levam a infecções crónicas não agudos 1. Infecções associadas aos biofilmes é um fator de risco grave de procedimentos médicos que envolvem a implantação de objetos estranhos (por exemplo, substitutos ósseos artificiais, implantes de mama) ou a instalação de tubos traqueais ou cateteres urinários 2. Nestes contextos, a terapia anti-infecciosa é quase sempre necessário, como infecções baseados em biofilme raramente são apuradas por conta própria, mesmo em indivíduos imunocompetentes. Staphylococcus aureus é um dos patógenos mais frequentemente observadas implicados em complicações relacionadas com o biofilme que ocorrem durante o uso de dispositivos médicos invasivos 3. Infelizmente, a própria natureza do biofilme como uma barreira de protecção torna-as mais resistentes ao tratamento do que as células planctônicas de 1,4, e a avaliação da eficácia clínica predito é uma parte crítica de ambos inicialdesenvolvimento de medicamentos, bem como a vigilância da resistência de droga. Há um crescente reconhecimento de que condições de laboratório, com foco em culturas planctônicas, podem não representar fielmente a doença no mundo real 5. Para agravar ainda mais o problema, a replicação de fenótipos de biofilme é difícil, e os modelos de biofilmes existentes são tediosos e sofrem de elevada variabilidade inter- e intra-ensaio de seis. Assim, muitos pesquisadores, por necessidade, o padrão para ensaios de células planctônicas de sensibilidade às drogas, assim potencialmente negligenciar um aspecto importante da virulência bacteriana e doenças. Aqui descrevemos o protocolo para o ensaio de bactérias, especificamente S. aureus, em biofilmes pré-cultivadas utilizando uma base 7,8 sistema biofilme de 96 poços. Enquanto o protocolo para a formação de biofilme e desafio segue essencialmente a recomendação do fabricante, apresentamos uma metodologia alternativa rico em informações para quantificar a viabilidade do BIOFilm após o desafio. Em resumo, as bactérias são cultivadas na placa de PEG, onde formam biofilmes sobre as cavilhas salientes ligados a tampa da placa. Após a formação do biofilme, os pinos são suavemente mergulhado em poços de uma placa fresca cheio com PBS para remover as células planctônicas. O PEG-tampa, com biofilmes em anexo, é transferido para uma nova placa de desafio, contendo várias concentrações de antibiótico a ser ensaiado. Após uma segunda incubação, as tampas são novamente removidos, lavados e transferidos para uma placa de recuperação de corante contendo resazurina, onde são submetidos a uma incubação final. conversão resazurina pode ser gravado cineticamente ou tomado como uma leitura nó de extremidade após um período de recuperação definido. Este método à base de corantes de quantificação da viabilidade do biofilme difere consideravelmente de CFU (unidades formadoras de colónias) tediosas metodologia baseada em contar descrito no protocolo original 7. OD 600 medições da placa desafio de drogas e resazurina cinética de conversão servir como leitura viabilidadeouts de células planctônicas e biofilme, respectivamente, oferecendo um ensaio rápido, confiável e tecnicamente simples rico em informações para a sobrevivência do biofilme.

<table><tbody><tr><td>&amp;nbsp;Mueller-Hinton Medium</td><td>Oxoid</td><td>CM0405</td><td>Follow recommendations of manufacturer</td></tr><tr><td>RPMI-medium</td><td>Corning&amp;nbsp;</td><td>17-105-CV</td><td></td></tr><tr><td>CRPMI</td><td>Ref 9</td><td></td><td>RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td></tr><tr><td>Chelex 100 Resin</td><td>Bio Rad</td><td>142-2822</td><td></td></tr><tr><td>MBEC-plates</td><td>Innovotech</td><td>19111</td><td></td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>800 &amp;micro;g/ml in DI water&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Filter sterile</td></tr><tr><td>Micro Plate Shaking Platform&amp;nbsp;</td><td>Heidolph Titramax 1000</td><td></td><td></td></tr><tr><td>Cytation 3 Plate Reader</td><td>Biotek</td><td></td><td></td></tr><tr><td>Gen5 software</td><td>Biotek</td><td></td><td>Recording and analysis of resazurin conversion</td></tr><tr><td>Neocuproine</td><td>Sigma</td><td>N1501&amp;nbsp;</td><td>prepare 10 mM stock in 100% Ethanol, store at -80 &amp;ordm;C</td></tr><tr><td>Copper sulfate</td><td>Acros Organics</td><td>7758-99-8</td><td>prepare a 100 mM stock solution in water, store at 4 &amp;ordm;C</td></tr><tr><td>Cu-Neocuproine</td><td>Self-Made</td><td></td><td>Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G3632-1G</td><td></td></tr></tbody></table>

targeting biofilm associated staphylococcus aureus using resazurin based drug-susceptibility assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

O ensaio resazurina é sensível o suficiente para detectar com segurança muito poucas células viáveis, capazes de se replicar em meio livre de drogas após o desafio. Nesta metodologia, define-se a concentração mínima de biofilme erradicação como a concentração mais baixa à qual nenhuma conversão resazurina é observada após 24 h. O ensaio baseia-se resazurina em moléculas oxidativos encontrados em células metabolicamente activas, que convertem a cor do corante de azul pa...

Watch this Scientific Journal Video about Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay at JoVE.com

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Segmentação de biofilme Associated<em&gt; Staphylococcus aureus</em&gt; Usando resazurina Baseado Ensaio Drogas susceptibilidade

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

targeting-biofilm-associated-staphylococcus-aureus-using-resazurin

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

13.0K visualizações.  University of Alabama at Birmingham. Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. 

Vídeo: Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

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.

Biossensor para detecção de bactérias Staphylococcus resistente a antibióticos

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

Bioengenharia

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de meio de escarro Artificial para testar a eficácia antibiótica contra<em&gt; Pseudomonas aeruginosa</em&gt; Em condições mais relevantes para o Pulmão Fibrose Cística

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Imunologia e Infecção

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

Teste de suscetibilidade de antibiótico induzido por estresse em um chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

A Plataforma de Ensaios Anti-biofilme adequado para a Exploração de bibliotecas de compostos naturais

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Sistema de Eficácia e citotoxicidade Rastreio de inibidores Segmentação intracelular<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

Teste de Eficácia de Antibióticos em um Modelo Ex vivo de Pseudomonas aeruginosa e Staphylococcus aureus Biofilms no Pulmão da Fibrose Cística

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

Ensaios In vitro padronizados para visualizar e quantificar interações entre neutrófilos humanos e biofilmes de Staphylococcus aureus

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

Um ensaio de redução solúvel à base de tetrazólio para avaliar o efeito de anticorpos em biofilmes de Candida tropicalis

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Ensaio de proteção de enzimas melhorada para estudar a internalização staphylococcus aureus e eficácia intracelular de compostos antimicrobianos

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

Biologia

An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

Source: Schaaf, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55453">A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis.</a> J. Vis. Exp. (2017)
This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.

Um ensaio para avaliar a eficácia do medicamento contra um modelo de infecção por Mycobacterium tuberculosis

1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain ...

Segmentação de biofilme Associated

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