Agradecemos aos técnicos do laboratório de bacteriologia do Hospital Central de Lausanne University por sua ajuda para implementar as técnicas no laboratório.

Este protocolo foi desenvolvido e validado seguindo os processos de pesquisa e desenvolvimento e as normas éticas da nossa instituição antes de ser implementado como uma ferramenta de rotina. 1 Preparação de uma Cultura de sangue bacteriana Pellet por Amônio Procedimento Chloride Erythrocyte-lise <ol><li> Preparação de uma hemocultura positiva para posterior centrifugação. <ol><li> Esterilizar a tampa hemocultura. Adicionar 70% de etanol na tampa da garrafa e queimá-lo. NOTA: Não execute este passo debaixo de uma capela de fluxo laminar. </li><li> Mover o frasco para uma câmara de fluxo laminar. Recolha 5 ml da cultura de sangue com uma seringa 20 G. </li><li> Adicionar os 5 ml de cultura de sangue em um tubo de centrífuga de 50 ml contendo 45 ml de água estéril (H 2 O) e agitar a amostra. </li></ol></li><li> Preparação de uma cultura de sangue pelete bacteriana por centrifugação a lise. <ol><li> Centrifuga-se a amostra a 1000 xg durante 10 min à temperatura ambiente. </li><li> Remove-se o sobrenadante (que contém principalmente H2O e células do sangue) com uma bomba de vácuo de laboratório. </li><li> Ressuspender o sedimento em 1 ml de cloreto de amónio caseira solução (0,15 M NH 4 Cl, 1 mM KHCO 3) de lise. Quebrar o sedimento por raspagem e em vortex do tubo e centrifugar a amostra a 140 xg durante 10 min à temperatura ambiente. </li><li> Rejeitar o sobrenadante, que contém principalmente detritos de células vermelhas do sangue. <ol><li> Se o sedimento permaneceu hemorrágico, ressuspender o sedimento em 2 ml de solução estéril destilada e H 2 0 para lisar os glóbulos vermelhos mais residuais e para lavar o sedimento. Centrifuga-se a amostra a 140 xg durante 10 min à temperatura ambiente e desprezar o sobrenadante. </li></ol></li><li> Ressuspender o sedimento em 200 ul de H2O </li></ol></li><li> Subcultura da cultura de sangue em vários meios de ágar seletivo e não seletivo. <ol><li> Recolha de 1 ml da cultura de sangue com uma seringa 20 G. </li><li> Inocular uma gota de hemocultura into quatro quadrantes em ágar-sangue, ágar chocolate, ágar McConkey e ágar Schaedler. </li><li> Incubar a 37 ° C, o agar de sangue e as placas de ágar de chocolate em uma atmosfera de CO2 a 5%, o agar McConkey em atmosfera normal e a placa de ágar Schaedler em condições anaeróbicas. </li><li> Siga o crescimento bacteriano na mídia diferente. </li><li> Verifique se há pureza bacteriana. </li></ol></li></ol> 2 Identificação por MALDI-TOF MS <ol><li> Identificação direta do sedimento sangue. <ol><li> Transferir 1 mL da pelota de sangue em uma placa-alvo MALDI e deixe secar sobre uma plataforma de aquecimento de 42 ° C. </li><li> Sobreponha a amostra com ácido fórmico 1 ml de 70% e deixe secar sobre uma plataforma de aquecimento de 42 ° C. </li><li> Sobreponha a amostra com 1 ml de matriz MALDI e deixe secar sobre uma plataforma C de aquecimento 42 ° (solução de α-ciano-4-hidroxicinâmico em 50% de acetonitrilo-2.5% de ácido trifluoroacético saturada). </li><li> Vá para a MALDI-TOF MS a análise com um sistema de MALDI-TOF MS, como descrito pelos fabricantes. </li><li> Se a pontuação de identificação não é válido a nível de espécie (por exemplo, com um score &lt;2), realizar uma extração de proteínas da amostra de sedimento sangue. </li></ol></li><li> Identificação após a extracção de proteínas a partir do sedimento da cultura do sangue. <ol><li> Misturar 20 ul do sedimento com 1 ml de etanol a 70% e centrifuga-se a 13.000 xg durante 2 min à temperatura ambiente. </li><li> Descartar o sobrenadante e adicionar 25 ul de ácido fórmico a 70% e 25 mL de 100% de acetonitrilo ao sedimento. Vortex vigorosamente, para voltar a suspender as pelotas. Ressuspender pelotas duras ao dividi-los com pontas de pipeta antes de vórtex. </li><li> Centrifuga-se a 13000 xg durante 2 min à temperatura ambiente. Transferir 1 mL do sobrenadante (que contém proteínas extraídas) em uma placa-alvo MALDI e deixe secar sobre uma plataforma de aquecimento de 42 ° C. Proceda como descrito no ponto 2.1.2. </li></ol></li></ol> 3. bacteriana Identificação e Antibiótico Suscetibilidade Testes com um sistema automatizado Microbial <ol><li> Preparação de uma suspensão bacteriana para a identificação de bactérias e a AST, com um sistema automatizado microbiana. <ol><li> Preparar um tubo de plástico contendo 3 ml de solução estéril de 0,45% de NaCl compatível com o sistema microbiano automatizado. </li><li> Adicionar um volume suficiente da pelete da cultura de sangue em solução de NaCl a 0,45% para se obter uma suspensão bacteriana correspondente para uma turbidez de McFarland de 0,6 a 0,8. Medir a turvação, utilizando um densitómetro de máquina. Nota: O volume do sedimento bacteriano, cultura do sangue varia de 50 a 200 ul para atingir uma turbidez de McFarland de 0,6 a 0,8. </li></ol></li><li> Inocular as bactérias Gram-positivas (GP) ou Gram-negativa (GN) cartões automáticos de identificação e os cartões AST automatizadas para testes de susceptibilidade antimicrobiana de cocos Gram-positivos e Gram-negativas, como descrito pelo fabricante. NOTA: identification com GP ou cartões de GN não é aplicada se MALDI-TOF MS valor pontuação identificação é&gt; 2. Verifique a pureza da suspensão bacteriana por subcultura a suspensão bacteriana em agar de sangue (GP e GN) e ágar de MacConkey (GN) placas. </li></ol> 4. Antibiótico Teste à Susceptibilidade pelo Disk Diffusion Assay O ensaio de difusão em disco é descrito pelo Comité Europeu para os testes de susceptibilidade antimicrobiana (EUCAST, versão 3.0, em abril de 2013, www.eucast.org). <ol><li> Preparação de uma suspensão bacteriana para realizar testes de susceptibilidade antimicrobiana pelo método de difusão em disco. <ol><li> Preparar um tubo de vidro contendo 3 ml de solução estéril de NaCl a 0,9%. </li><li> Adicionar um volume suficiente da pelete da cultura de sangue em solução de NaCl 0,9% para obter uma suspensão bacteriana correspondente para uma turbidez de McFarland de 0,5. Medir a turvação, utilizando um densitómetro de máquina. NOTA: O volume da cultura de sanguesedimento bacteriano varia de 50 a 200 ul para atingir uma turbidez de McFarland de 0,6 a 0,8. </li><li> Vortex a suspensão bacteriana. </li></ol></li><li> Inoculação de suspensão bacteriana em agar Mueller-Hinton (MH) ou de Mueller-Hinton agar-agar de organismos fastidiosos (MHF) (MH suplementado com 5% de sangue de cavalo desfibrinado e 20 mg / L de β-nicotinamida adenina dinucleótido). <ol><li> Mergulhar uma mecha de algodão estéril na suspensão bacteriana e cuidadosamente remover o excesso de fluido, rodando a mecha na parede interior do tubo de vidro. </li><li> Espalhar a suspensão bacteriana uniformemente sobre toda a superfície da placa de agar MH / MHF por raspagem em três direcções, ou por meio de um rotor placa automatizado. </li></ol></li><li> Aplicação de discos antimicrobianos em placas de ágar inoculadas. <ol><li> Aplicar perto os discos na superfície das placas de agar inoculadas secas manualmente ou utilizando um dispensador de disco de sensibilidade. </li><li> Incubar a placa na tempe apropriadoratura e atmosfera, conforme descrito no teste de suscetibilidade método de difusão em disco EUCAST antimicrobiana (Versão 3.0, em abril de 2013, www.eucast.org). </li></ol></li></ol> Baseado em PCR 5. automatizado de teste de diagnóstico para a detecção do MRSA <ol><li> Usando uma micropipeta, transferir 50 ul do sedimento de cultura de sangue para dentro do frasco de reagente de amostra fornecido com o MRSA baseada em PCR kit de teste de diagnóstico e automatizado vórtice durante 20 seg. </li><li> Usando uma micropipeta 1 ml, distribuir a amostra na porta espécime do cartucho. Inserir o cartucho dentro do sistema de PCR automatizado e iniciar o ensaio, tal como descrito pelo fabricante. </li></ol>

<ol>
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Os autores declaram que não têm interesses financeiros concorrentes.

Comparado aos métodos diagnósticos hemocultura positiva convencionais, a rápida aquisição de um sedimento de bactérias, utilizando o cloreto de amônio lise abordagem centrifugação reduzir o tempo para relatar a identificação por 16 a 24 horas eo tempo para relatar AST por 24-48 horas (Figuras 1 e 3). A rápida introdução de antibioticoterapia adequada é fundamental para melhorar a evolução dos pacientes que sofrem de infecções da corrente sanguínea. Assim,...

Infecções da corrente sanguínea e septicemia em pacientes hospitalizados são uma das principais causas de morbidade e mortalidade. Assim, a mortalidade relacionada com a corrente sanguínea infecções é observada em cerca de 14% a 37% dos pacientes hospitalizados e pode aumentar para 35% em unidades de terapia intensiva de pacientes 1-3. A rápida identificação do agente infeccioso é fundamental para orientar o tratamento antimicrobiano ideal e aumentar o êxito da terapia antimicrobiana 4,5. A análise rápida da coloração Gram da hemocultura positiva já tem um impacto significativo sobre a adaptação da terapia antimicrobiana 6,7, mas a identificação precisa do agente infeccioso é obrigada a fornecer o tratamento antibiótico mais adequado às pacientes. Por exemplo, diferentes regimes de tratamento antibiótico devem ser implementadas após bacteremia com enterococos e estreptococos que são difíceis de distinguir pela coloração de Gram. Do mesmo modo, a identificação da espécie level é necessária para detectar bactérias Gram enterobactérias negativa que codifica um gene cromossómico ampC que conferem uma maior resistência à β-lactâmicos 8. Com uma hemocultura positiva, a abordagem convencional de diagnóstico é a subcultura do agente infeccioso em diferentes placas de ágar, que requer várias horas de incubação adicional identificação prévia com várias abordagens, incluindo testes bioquímicos, o crescimento em diferentes meios seletivos e sistemas de identificação microbiana automatizadas. O tempo para os resultados de um método de diagnóstico convencional é de cerca de 1 a 3 dias. O surgimento da tecnologia de matriz assistida por laser de dessorção / ionização por espectrometria de massa de tempo-de-voo (MALDI-TOF), para a rápida identificação de microrganismos forneceu uma nova ferramenta para identificar rapidamente os microrganismos a partir de colónias que cresceram em placas de agar como também directamente a partir de sangue positiva culturas (Figura 1), 9-12. The uso de MALDI-TOF para identificar um agente infeccioso a partir de culturas de sangue tem reduzido significativamente o tempo para resultados de alguns minutos em vez de horas e dias exigidos por métodos tradicionais. Como discutido por Croxatto et al. 13, a eficiência de identificação de MALDI-TOF depende de diferentes parâmetros, incluindo a quantidade e pureza do microrganismo. Estes dois critérios são facilmente obtidos a partir de colónias discretas cultivadas em placas de agar, mas é necessária uma pré-tratamento analítico de enriquecimento bacteriana e purificação de amostras complexas, tais como a cultura de sangue, que contêm vários componentes celulares e de proteínas que podem interferir com a identificação de MALDI-TOF. Vários métodos de isolamento de microrganismos de cultura do sangue tenham sido utilizados num certo número de estudos, incluindo a saponina ou outro método de detergentes suaves para a extracção 9,14 bacteriana, método de separação de soro de 10, a lise métodos de centrifugação 12 </sse&gt; e soluções comercialmente, tais como o kit sepsityper. O nosso laboratório de diagnóstico bacteriológico desenvolveu um simples sangue-cultura preparação sedimento bacteriano à base de cloreto de amónio-lise dos eritrócitos, que permitem uma identificação rápida de bactérias e levedura por MALDI-TOF e sistemas de identificação automáticos (Figura 2) 15. Esta preparação de pelotas de sangue-cultura também fornecer uma amostra para outras aplicações diretas jusante, tais como coloração de Gram, testes de diagnóstico automatizado com base na PCR, como POCT-PCRs para a detecção rápida de resistente à meticilina Staphylococcus aureus (MRSA), e os testes de sensibilidade aos antibióticos com sistemas automatizados de AST e / ou por ensaios de difusão de disco em placas de agar (Figura 3). Neste trabalho, nós descrevemos os diferentes passos para a preparação do sedimento bacteriano sangue-cultura, tal como explicado por Prod&#39;hom et al. 15 (Figura 4). Nós também irá deescriba os protocolos de três das principais aplicações que podem ser executadas no pellet hemocultura: Identificação por MALDI-TOF 15, a identificação (ID) e teste de sensibilidade aos antibióticos (AST) com os sistemas automatizados 16 de Enterobacteriaceae e estafilococos e PCR-automatizado teste de diagnóstico baseado na detecção de MRSA 17.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">20 needle gauge&#160;</td>
			<td style="width:268px;">Terumo, Leuven, Belgium</td>
			<td style="width:239px;">NN-2038R</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">50 ml Falcon tube</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>352070</td>
			<td>50 ml centrifuge tubes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ammonium chlorure</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>101145</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Potassium hydrogen carbonate</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>60340</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>F0507</td>
			<td>Flammable, corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">a-Cyano-4-hydroxycinnamic acid</td>
			<td>Fluka, St. Louis, MO, USA&#160;</td>
			<td>70990</td>
			<td>Acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Acetonitrile</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>271004</td>
			<td>Flammable, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Trifluoroacetic acid</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA&#160;</td>
			<td>T6508</td>
			<td>Corrosive, acute toxicity</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 60 instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27202</td>
			<td>automated microbial system instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-positive (GP) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21342</td>
			<td>automated GP&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-P580 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>22233</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 Gram-negative (GN) card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>21341</td>
			<td>automated GN&#160; identification card</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vitek 2 AST-N242 card</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>413391</td>
			<td>automated microbial AST system</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Xpert MRSA</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>&#160;GXMRSA-100N-10</td>
			<td>nucleic acid amplification technology MRSA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">GeneXpert IV instrument</td>
			<td>Cepheid, Sunnyvale, Ca, USA</td>
			<td>GXIV-4-D</td>
			<td>nucleic acid amplification technology instrument</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Microflex LT MALDI-TOF MS instrument</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>BDAL microflex LT/SH</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MSP 96 target&#160; steel BC</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>280799</td>
			<td>MALDI target plate</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Densitometer Densicheck instrument</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>27208</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MALDI Sepsityper kit 50</td>
			<td>Bruker Daltonics, Bremen, Germany</td>
			<td>8270170</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mac Conkey agar</td>
			<td>Biolife, Milano, Italy</td>
			<td>4016702</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Mueller-Hinton agar</td>
			<td>Oxoid, Hampshire, England</td>
			<td>CM0337</td>
			<td>Mueller-Hinton agar (MH)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHF agar</td>
			<td>Biom&#233;rieux, Marcy-l&#39;Etoile, France</td>
			<td>43901</td>
			<td>Mueller-Hinton agar-fastidious organisms agar (MHF)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD columbia III agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254071</td>
			<td>blood agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD chocolate agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254089</td>
			<td>chocolate agar</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">BD schaedler agar</td>
			<td>BD, Franklin Lakes, NJ, USA</td>
			<td>254084</td>
			<td>Schaedler agar</td>
		</tr>
	</tbody>
</table>

preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing

Infecções da corrente sanguínea e septicemia são uma das principais causas de morbidade e mortalidade. O êxito de pacientes que sofrem de bacteremia depende de uma rápida identificação do agente infeccioso para orientar o tratamento antibiótico ideal. A análise de manchas Gram de hemocultura positiva pode ser rapidamente realizada e já impactar significativamente o regime de antibióticos. No entanto, a identificação precisa do agente infeccioso ainda é necessário para estabelecer o melhor tratamento direcionado. Apresentamos aqui uma preparação pellet bacteriano simples e rápido de uma hemocultura positiva que pode ser usado como um exemplo para várias aplicações a jusante essenciais, como a identificação por MALDI-TOF MS, teste de sensibilidade aos antibióticos (AST) pelo método de difusão de disco ou sistemas automatizados AST e por PCR automatizados baseados em testes de diagnóstico. O desempenho desses sistemas de identificação e AST diferentes aplicados diretamente sobre as culturas de sangue pelotas bacterianas é muito siMilar para o desempenho, normalmente obtido a partir de colónias isoladas cultivadas em placas de agar. Em comparação com métodos convencionais, a rápida aquisição de um sedimento bacteriano, reduz significativamente o tempo de identificação e relatam tanto AST. Assim, seguindo a positividade da hemocultura, identificação por MALDI-TOF podem ser relatados em menos de 1 hora enquanto que os resultados de AST por sistemas automatizados ou AST difusão em disco dentro de 8 a 18 horas, respectivamente. Da mesma forma, os resultados de um ensaio baseado em PCR rápida podem ser comunicadas para os clínicos menos do que 2 horas após o relatório de uma bacteremia. Juntos, estes resultados demonstram que a preparação rápida de um sedimento de bactérias hemocultura tem um impacto significativo sobre o tempo de identificação e AST reviravolta e, portanto, sobre o sucesso de pacientes que sofrem de infecções da corrente sanguínea.

No estudo realizado por Prod&#39;hom et al. 15, peletes bacterianas obtidas por lise de cloreto de amónio a centrifugação de 122 cultura sanguínea positiva a partir de 78 doentes foram analisadas por MALDI-TOF MS. Fora de 122 hemocultura positiva, 95 (77,9%) foram corretamente identificados ao nível da espécie e um (0,8%) no nível de gênero. Os restantes 26 (21,3%) pelotas de hemocultura não deu nenhuma identificação confiável por MALDI-TOF. Entre esses, 21 eram bactérias gram positivas,...

Watch this Scientific Journal Video about Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing at JoVE.com

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Uma preparação sedimento bacteriano rápida de uma cultura de sangue positiva pode ser utilizado como uma amostra para aplicações tais como a identificação por MALDI-TOF, a coloração de Gram, teste de susceptibilidade aos antibióticos e ensaios baseados em PCR. Os resultados podem ser rapidamente comunicados aos médicos para melhorar a evolução dos pacientes que sofrem de infecções da corrente sanguínea.

Preparação de uma Cultura Pellet sangue para rápida identificação bacteriana e Antibiótico Teste à Susceptibilidade

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

preparation-blood-culture-pellet-for-rapid-bacterial-identification

Research

JoVE Journal

Immunology and Infection

35.4K visualizações.  University Hospital Center and University of Lausanne.  Uma preparação sedimento bacteriano rápida de uma cultura de sangue positiva pode ser utilizado como uma amostra para aplicações tais como a identificação por MALDI-TOF, a coloração de Gram, teste de susceptibilidade aos antibióticos e ensaios baseados em PCR. Os resultados podem ser rapidamente comunicados aos médicos para melhorar a evolução dos pacientes que sofrem de infecções da corrente sanguínea. 

Vídeo: Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

A 96 Bem microtitulação Método da Placa com base para a Formação Monitoramento e Teste de susceptibilidade antifúngica de Candida albicans Biofilmes

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Imunologia e Infecção

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocolo para vacinas recombinantes RBD baseado SARS: Preparação de proteínas, Vacinação Animal e Detecção de Neutralização

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Um Procedimento de 1,5 horas para a Identificação de Enterococcus Espécies Diretamente de hemoculturas

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Teste de susceptibilidade antimicrobiana do complexo Mycobacterium tuberculosis da Droga primeira e segunda linha de diluição em caldo em um formato de placa de microtitulação

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

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

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

Um dia-Esquema de fluxo de trabalho para detecção de patógenos de bactérias e de resistência antimicrobiana de hemoculturas

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Rápida identificação de bactérias gram-negativas de Sangue Cultura Caldo Usando MALDI-TOF Espectrometria de Massa

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Um método de microdiluição em caldo de referência para Dalbavancin<em&gt; In Vitro</em&gt; Teste à Susceptibilidade de bactérias que crescem aeròbia

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Um método rápido para processamento de sangue para aumentar o rendimento dos níveis plasmáticos de peptídeos no sangue humano

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Cultura de macrófagos macrófagos Colony estimulante fator diferenciado Humano monócitos derivados

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...