These experiments have been supported by research grants from Merck and Co. and Summit, PLC.

1. Isolamento de C. difficile a partir de diferentes fontes ambientais ou clínicas <ol><li> Isolados ambientais: Utilizando um indicador de algodão pré-esterilizado (ligeiramente molhado com NaCl a 0,85%), limpe a superfície de qualquer área de interesse (chão, porta, pega, prateleira, etc. ). 8 Certifique-se de usar luvas estéreis e coloque o cotonete em um tubo esterilizado após concluído. </li><li> Isolados clínicos (fezes): Placa de 10 a 100 mg de amostras clínicas de fezes em cefoxitina-cicloserina-frutose ágar (CCFA), utilizando um ciclo de inoculação e incubar sob condições anaeróbicas rigorosas durante 48-72 h. Armazenar colônias isoladas de C. difficile estoque em cryovials a -80 ° C para análises adicionais. 7 , 9 , 10 </li><li> Enriqueça as amostras de esfregaços ambientais em infusão de coração cerebral (BHI) com 0,05% de taurocolato de sódio e coloque em câmara anaeróbica a 37 ° CDurante 5 dias. Centrifugar 1 mL da cultura a 10.000 xg e ressuspender o sedimento em 100 mL de etanol. </li><li> Plantar as células ressuspensas (50 μL) em placas de cicloserinecefoxitina frutose agar (CCFA) e incubar numa câmara anaeróbica a 37 ° C durante 40-48 h. Armazenar colônias isoladas de C. difficile estoque em cryovials a -80 ° C para análises adicionais. </li><li> Teste as colônias suspeitas de C. difficile usando o reagente de aglutinação de látex ou PCR. </li></ol> 2. Culturing C. difficile e Killing Kinetic Procedures <ol><li> Cultivar estirpes ambientais ou clínicas de C. difficile em placas de agar de sangue numa câmara anaeróbica a 37 ° C durante 48 h. </li><li> Tomar uma colónia isolada com um ciclo de inoculação, transferi-la para 5 mL de meio BHI num tubo de 15 mL e crescer durante 24 h numa câmara anaeróbica a 37 ° C. </li><li> Diluir as pré-culturas 1: 100 a aproximadamente 10 6 unidades formadoras de colónias(CFU) / mL em BHIS pré-reduzido fresco (BHI mais 5 g / L de extracto de levedura e 1% de L-cisteína) suplementado com taurocolato de sódio a 0,1% e a concentração apropriada de antibiótico (T0). </li><li> Recolher uma amostra de 1 mL com uma pipeta em cada ponto de tempo (T0, T6, T24, T48) e espalhar uma pequena alíquota (100 μL em diluições em série) numa placa de agar de sangue. Deixar as células crescerem sobre a placa de agar de sangue durante 48 h numa câmara anaeróbica a 37 ° C e contar o número resultante de colónias para determinar CFU. </li></ol> 3. Preparação de amostras para Microscopia Eletrônica de Varredura <ol><li> Recolher 1 mL de células de cada ponto de tempo em tubos de microcentrífuga e centrifugar a 10.000 xg durante 10 min. Descartar o sobrenadante e lavar as células em PBS. </li><li> Centrifugar as amostras a 10 000 xg durante 10 min novamente e descartar o sobrenadante. Re-diluir as células em 1 mL de paraformaldeído a 4% e incubar durante 1 h à temperatura ambiente. </li><li> Centrifugar novamente as amostras durante 10 min a 10,000 xg e descartar sobrenadante. Lavar as células duas vezes com água destilada e redilutar em 100 μL de água destilada. Ajuste o volume dependendo da turbidez da solução. NOTA: Fazer várias diluições em série é uma boa idéia. </li><li> Rotule os lamínulas e adicione 40 μL da amostra nele. Incubar durante 15 min em uma capa de fluxo para evaporar o líquido e permitir que as células aderem à lamela. Se ainda houver líquido, use um ventilador para remover o líquido. </li><li> Coloque lamínulas rotuladas com células em uma máquina de pulverização de mesa e fita para baixo. Secure ouro puro na máquina de pulverização. Ligue a máquina e comece a pulverização a baixa pressão (50 mTorr). Revestir as células durante 30 s a 80 mA, o que se traduz em 20 nm de revestimento de ouro. </li><li> Transferência de células revestidas para o microscópio eletrônico de varredura. </li></ol> 4. Imagens de células C. difficile em um microscópio eletrônico de varredura <ol><li> Ventilar o microscópio eletrônico de varredura (SEM) corretamente por prEssing o botão de ventilação no software do computador. </li><li> Usando fita de carbono, fixe os lamínulas revestidas para o estágio de metal. Uma vez que o SEM é ventilado, a porta deve abrir facilmente. Bloqueie o estágio de metal na câmara de SEM, aparafusando-o. </li><li> Clique no botão PUMP no software do computador. O SEM será utilizável quando o sistema lê &quot;Vac OK&quot;. </li><li> Clique no detector SE na guia de detectores. Ligue o feixe clicando no botão que exibe a tensão. Iniciar imagens com uma tensão mais baixa (5 kV) antes de aumentar a tensão (até 15 kV). Uma imagem será exibida após o feixe estar ligado. </li><li> Usando a função de rastreamento, encontre uma área nos lamínulas revestidos para a imagem. Zoom para a região e encontrar em forma de haste estruturas; Estes são clostridium difficile . </li><li> Amplie e focalize a imagem para calibrar o sistema. Isto deve ser feito em várias distâncias de trabalho: 15, 9 e 5 mm. Imagem a uma distância de trabalho de 5 mm. </li><li> Ligue o uLtra modo de imagem de alta resolução e começar a focar e otimizar o astigmatismo. </li><li> Comece a focar em alta ampliação. Utilize os botões de foco fino e grosseiro para isso. Ajustar o astigmatismo também para obter uma imagem mais clara. <ol><li> Ajuste o astigmatismo com alta ampliação. Para fazer isso, ajuste as alternâncias de astigmatismo (elas se parecem com as alternâncias de foco fino / grosseiro) e verifique a imagem para obter clareza digitando zoom no software do computador. </li></ol></li><li> A função de leitura lenta para coletar uma imagem de alta qualidade. Salve a imagem coletada como um arquivo .TIFF, que será usado para análise. Certifique-se de que a barra de dados esteja selecionada se as medições forem feitas durante a análise. </li><li> Colecione imagens em diferentes ângulos (até 52 °, girando manualmente o ângulo diretamente no SEM.) As imagens angulares tendem a revelar mais informações de profundidade. </li><li> Altere a tensão do feixe dependendo das células que estão sendo visualizadas. Manter este principalmente entre 5 - 15 kV para todas as experiências. </li&gt; <li> Após completar a imagem, desligue o feixe e aumente a distância de trabalho para 20 mm. A câmara pode então ser ventilada e a fase pode ser removida. Copie todas as imagens em uma unidade para uma análise mais aprofundada. </li></ol> 5. Processamento e Análise de Imagens <ol><li> Faça o download e instale o software disponível gratuitamente, FIJI (http://fiji.sc), no computador. </li><li> Abra o arquivo de imagem em FIJI. </li><li> Usando a função de linha, traçar com precisão a barra de escala. </li><li> Clique na guia Analisar no programa FIJI e finalmente selecione a função Selecionar Escala. Aparecerá uma janela que exigirá a definição da distância conhecida com base na barra de escala. Altere também a unidade de comprimento e clique em OK. </li><li> Agora que o programa é calibrado para a distância, use a função de linha para medir o comprimento da célula. Para obter o comprimento, use a função de linha para rastrear a célula na sua totalidade. Selecione a guia Analisar novamente e clique em Medir. O comprimento deve serNas unidades indicadas anteriormente. </li></ol>

<ol>
	<li>Lessa, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+Clostridium+difficile+infection+in+the+United+States.">Burden of Clostridium difficile infection in the United States.</a> N Engl J Med. 372 (9), 825-834 (2015).</li><li>Vickers, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ridinilazole:+a+novel+therapy+for+Clostridium+difficile+infection.">Ridinilazole: a novel therapy for Clostridium difficile infection.</a> Int J Antimicrob Agents. 48 (2), 137-143 (2016).</li><li>Shah, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+update+on+emerging+antibiotic+treatment+options+and+antibiotic+resistance.">Clostridium difficile infection: update on emerging antibiotic treatment options and antibiotic resistance.</a> Expert Rev Anti Infect Ther. 8 (5), 555-564 (2010).</li><li>Ambrose, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+EMA+guideline+for+antimicrobial+development.">New EMA guideline for antimicrobial development.</a> Lancet Infect Dis. 12 (4), 265-266 (2012).</li><li>Bass&#232;res, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+on+toxin+production+and+cell+morphology+in+Clostridium+difficile+by+ridinilazole+(SMT19969),+a+novel+treatment+for+C.+difficile+infection.">Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.</a> J Antimicrob Chemother. 71 (5), 1245-1251 (2016).</li><li>Endres, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+imaging+the+pharmacological+effects+of+antibiotic+treatment+on+Clostridium+difficile.">A novel method for imaging the pharmacological effects of antibiotic treatment on Clostridium difficile.</a> Anaerobe. 40, 10-14 (2016).</li><li>Endres, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Effects+of+Surotomycin+Treatment+on+Clostridium+difficile+Toxin+A+and+B+Production,+Immune+Response,+and+Morphological+Changes.">Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes.</a> Antimicrob Agents Chemother. 60 (6), 3519-3523 (2016).</li><li>Alam, M. J., Anu, A., Walk, S. T., Garey, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+potentially+pathogenic+Clostridium+difficile+contamination+in+household+environs.">Investigation of potentially pathogenic Clostridium difficile contamination in household environs.</a> Anaerobe. 27, 31-33 (2014).</li><li>Aitken, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+the+Endemic+Setting,+Clostridium+difficile+Ribotype+027+Is+Virulent+But+Not+Hypervirulent.">In the Endemic Setting, Clostridium difficile Ribotype 027 Is Virulent But Not Hypervirulent.</a> Infect Control Hosp Epidemiol. , 1-6 (2015).</li><li>Basseres, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+on+toxin+production+and+cell+morphology+in+Clostridium+difficile+by+ridinilazole+(SMT19969),+a+novel+treatment+for+C.+difficile+infection.">Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.</a> J Antimicrob Chemother. 71 (5), 1245-1251 (2016).</li><li>Walters, B. A., Roberts, R., Stafford, R., Seneviratne, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapse+of+antibiotic+associated+colitis:+endogenous+persistence+of+Clostridium+difficile+during+vancomycin+therapy.">Relapse of antibiotic associated colitis: endogenous persistence of Clostridium difficile during vancomycin therapy.</a> Gut. 24 (3), 206-212 (1983).</li><li>Chilton, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+oritavancin+on+Clostridium+difficile+spore+germination,+outgrowth+and+recovery.">Evaluation of the effect of oritavancin on Clostridium difficile spore germination, outgrowth and recovery.</a> J Antimicrob Chemother. 68 (9), 2078-2082 (2013).</li><li>Ofosu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+a+review+of+current+and+emerging+therapies.">Clostridium difficile infection: a review of current and emerging therapies.</a> Ann Gastroenterol. 29 (2), 147-154 (2016).</li><li>McDonald, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epidemic,+toxin+gene-variant+strain+of+Clostridium+difficile.">An epidemic, toxin gene-variant strain of Clostridium difficile.</a> New Eng J Med. 353 (23), 2433-2441 (2005).</li></ol>

KWG has received past and current research support from Merck &amp; Co. and Summit, PLC.

O objetivo do presente estudo foi criar um método de alto rendimento para isolar C. difficile e testar a susceptibilidade aos antibióticos usando microscopia eletrônica de varredura (MEV) como meio para uma caracterização mais completa da ação farmacológica do antibiótico. Usando os protocolos aqui delineados, demonstrámos que imaginar a resposta fenotípica da célula ao tratamento com antibióticos pode revelar uma visão da acção farmacológica do fármaco. No total, a porção de imagem deste pr...

Clostridium difficile é uma bactéria gram-positiva, formadora de esporos, causando cerca de 500.000 infecções anualmente nos EUA e é considerada um patógeno urgente de ameaça pelos Centros de Controle e Prevenção de Doenças (CDC), o maior nível de risco. A década passada tem visto um desenvolvimento considerável de fármacos em antimicrobianos com atividade contra C. difficile . 2 , 3 Os estudos in vitro são um componente necessário do processo de desenvolvimento do fármaco. 4 Tradicionalmente, estudos de susceptibilidade in vitro e morte são usados ​​para validar futuros estudos animais e outros in vivo . Embora estes métodos desempenhem um papel importante na avaliação da acção de morte, não capturam a resposta fenotípica das células ao tratamento farmacológico. Ao incorporar microscopia eletrônica de varredura (MEV) comD matando estudos cinéticos, uma caracterização mais completa dos efeitos diretos do antibiótico é possível. 5 , 6 , 7 Aqui, apresentamos um método em que o SEM é utilizado como um meio para perfilar a eficácia do tratamento antibiótico.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>cotton gauze&#160;</td>
			<td>Caring</td>
			<td>PRM21408C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Macron</td>
			<td>7532</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI)&#160;</td>
			<td>Criterion</td>
			<td>C5141</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Alfa Aesar</td>
			<td>A10389</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Criterion</td>
			<td>C741</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium taurocholate</td>
			<td>Alfa Aesar</td>
			<td>A18346</td>
			<td></td>
		</tr>
		<tr>
			<td>anaerobic chamber</td>
			<td>Coy</td>
			<td></td>
			<td>vinyl anaerobic chamber</td>
		</tr>
		<tr>
			<td>cycloserinecefoxitin fructose agar (CCFA) plates</td>
			<td>Anaerobe systems</td>
			<td>AS-213</td>
			<td></td>
		</tr>
		<tr>
			<td>blood agar plates</td>
			<td>Hardy diagnostics</td>
			<td>A-10</td>
			<td></td>
		</tr>
		<tr>
			<td>latex agglutination reagent</td>
			<td>Oxoid</td>
			<td>DR1107A</td>
			<td>C. diff test kit</td>
		</tr>
		<tr>
			<td>microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>222363204</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-259-98</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>J. Melvin freed brand</td>
			<td>7525M</td>
			<td>75x25mm</td>
		</tr>
		<tr>
			<td>flow hood</td>
			<td>Labconco</td>
			<td>Class II type A2&#160;</td>
			<td>biosafety cabinet</td>
		</tr>
		<tr>
			<td>desk sputtering machine</td>
			<td>Denton Vacuum</td>
			<td>Desk II</td>
			<td></td>
		</tr>
		<tr>
			<td>tape</td>
			<td>Plastic Core</td>
			<td>05072-AB</td>
			<td>SPI Double Sided Adhesive Carbon Tape</td>
		</tr>
		<tr>
			<td>gold</td>
			<td>Denton Vacuum</td>
			<td>TAR001-0158</td>
			<td>2.375&#8221; Diameter x .002&#8221; Thick Gold foil</td>
		</tr>
		<tr>
			<td>scanning electron microscope</td>
			<td>FEI</td>
			<td>XL-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to characterize the morphological changes of clostridium difficile in response to antibiotic treatment

A avaliação da ação antibiótica com o desenvolvimento de novos fármacos voltados para as bactérias anaeróbias é difícil e tecnicamente exigente. Para se ter uma ideia do possível MOA, as alterações morfológicas associadas à exposição aos antibióticos podem ser visualizadas utilizando microscopia electrónica de varrimento (SEM). Integrar a imagem de SEM com as curvas tradicionais de morte pode melhorar nossa visão sobre a ação da droga e avançar no processo de desenvolvimento da droga. Para testar esta premissa, as curvas de morte e os estudos de SEM foram conduzidos utilizando fármacos com MOA conhecidos mas diferentes (vancomicina e metronidazol). As células de C. difficile (R20291) foram cultivadas com ou sem a presença de antibiótico até 48 h. Ao longo do intervalo de 48 h, as células foram recolhidas em múltiplos pontos de tempo para determinar a eficácia de antibióticos e para imagens no SEM. De acordo com os relatórios anteriores, a vancomicina eo metronidazol apresentaram uma actividade bactericida significativa após 24 h de tratamento, tal como medido pela unidade de formação de colónias (UFC)Ting Usando a imagem SEM, determinou-se que o metronidazol teve efeitos significativos sobre o comprimento celular (&gt; 50% de redução no comprimento celular para cada antibiótico, P &lt;0,05) em comparação com os controles e vancomicina. Embora a resposta fenotípica ao tratamento com fármaco não tenha sido previamente documentada desta forma, são consistentes com o MOA do fármaco demonstrando a versatilidade e fiabilidade da imagem e medições e a aplicação desta técnica para outros compostos experimentais.

Clostridium difficile é uma bactéria formadora de esporos e, portanto, é essencial para determinar as diferenças morfológicas entre células vegetativas e esporos antes de qualquer análise funcional. A Figura 1 demonstra imagens representativas de células vegetativas que foram capturadas durante a fase exponencial da curva de crescimento e células de esporos. Conforme ilustrado, as células vegetativas são longas, lisas, em forma de haste estru...

Watch this Scientific Journal Video about A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment at JoVE.com

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Protocolo para Caracterizar as Alterações Morfológicas<em&gt; Clostridium difficile</em&gt; Em resposta ao tratamento antibiótico

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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Immunology and Infection

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

8.9K visualizações.  University of Houston College of Pharmacy. Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Vídeo: A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

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

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Uma abordagem comparativa para caracterizar a paisagem de Patógeno-Hospedeiro interações proteína-proteína

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Cultivar e Manter<em&gt; Clostridium difficile</em&gt; Em um ambiente anaeróbico

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

In Vitro Análise do mediada por Myd88 Cellular resposta imune a vírus do Nilo Ocidental Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Phenomics fago: Abordagens fisiológicas para Caracterizar Novel Proteínas Virais

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Materiais biomiméticos para caracterizar as interações bactérias hospedeiras-

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

tratados com Cefoperazone Rato Modelo de clinicamente relevante<em&gt; Clostridium difficile</em&gt; R20291 Strain

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

Exame de fenótipos de acolhimento em<em&gt; Gambusia affinis</em&gt; Após o tratamento antibiótico

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Ensaios de caracterização de romance endoteliais reguladores envolvidos na resposta inflamatória de triagem

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sobreexpressão e purificação do ser humano<em&gt; Cis</em&gt; -preniltransferase em<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Protocolo para Caracterizar as Alterações Morfológicas

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