Este trabalho foi financiado pelo azm Profileringsfonds (PF245).

PARTE I: identificação do patógeno 1. Preparação da Amostra Nota: O fluxo de trabalho inteiro molecular como é descrito no protocolo seguinte deve ser realizada de acordo com as recomendações para garantia da qualidade do diagnóstico molecular 3. <ol><li> Adicionar uma alíquota de 0,1 ml de cultura de sangue a 0,9 ml de NaCl 0,9% para um tubo de reacção 1,5 ml a tornar-se uma amostra 1:10 diluído. (Diluir a fim de evitar a inibição qPCR). </li><li> Centrifugar amostra a 13.400 xg durante 5 min a pelete o ADN bacteriano. </li><li> Ressuspender o sedimento bacteriano em 100 uL estéril desmineralizada H 2 O. </li><li> Armazenar a amostra de ADN a 4 ° C até uso posterior. </li></ol> 2. Ensaio de Identificação: Real-Time PCR rDNA 16s <ol><li> Preparar as misturas de reacção como se segue. O ensaio consiste em quatro reacções separadas por amostra. Cada mistura inclui 12,50 mestre ulmistura, 0,9 uM para a frente de iniciadores (5-TCCTACGGGAGGCAGCAGT-3) 7, 0,6 mM iniciador reverso (5-GGACTACCAGGGTATCTAATCCTGTT-3) 8 e um painel de sondas. A quantidade de sondas é dado para cada um dos quatro reacções separadas em baixo. </li><li> A primeira reacção inclui: <ul><li> 0,2 mM da sonda universal (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) 8 </li><li> 0,2 uM P. sonda aeruginosa (5-JOE-CCAAAACTACTGAGCTAGAGTACG-3-BHQ1) </li></ul></li><li> A segunda reacção inclui: <ul><li> 0,2 uM E. sonda coli (5-JOE-GGAGTAAAGTTAATACCTTTGCTCATT-3-BHQ1) </li><li> 0,2 uM de Pseudomonas spp. sonda (5-NED-CCTTCCTCCCAACTTAAAGTGCTT-3-MGBNFQ) </li></ul></li><li> A reacção terceira inclui: <ul><li> 0,2 mM Staphylococcus spp. sonda (5-NED-AATCTTCCGCAATGGGCGAAAGC-3-MGBNFQ) </li><li> 0,2 uM de S. sonda aureus (5-FAM-AGATGTGCACAGTTACTTACACATAT-3-BHQ1) </li><li> 0,2 uM Enterococcus spp. (5-JOE-TCCTTGTTCTTCTCTAACAACAGAG-3-BHQ1) </li></ul></li><li> A reacção quarto inclui: <ul><li> 0,2 mM da sonda universal (5-FAM-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) </li><li> 0,3 uM Streptococcus spp. sonda (5-NED-CCAGAAAGGGACSGCTAACT-3-MGBNFQ) </li><li> 0,2 uM de S. pneumoniae sonda (5-JOE-CCAAAGCCTACTATGGTTAAGCCA-3-BHQ1) </li></ul></li><li> Adicionar estéril desmineralizada H2O para atingir um volume total de 20 uL. Adicionar 20 ul de cada mistura de reacção para os poços de uma placa de PCR de 96 poços. </li><li> Adicionar 5 ul de amostra a cada poço. </li><li> Use uma película adesiva para selar a placa de 96 poços de PCR. </li><li> Execute o prato no PRISM 7900HT ABI Real Time PCR System usando as seguintes condições óptimas de ciclos térmicos: <ul><li> Pré-aquecimento a 50 ° C durante 10 min </li><li> Desnaturação inicial a 95 ° C durante 15 min </li><li> 42 ciclos de <ul type="circle"><li> Desnaturação a 95 ° C durante 15 s </li><li> Recozimento a 60 ° C durante 1 min </li></ul></li></ul></li></ol> 3. Análise dos Resultados <ol><li> Ajuste o limiar da Análise de Ct para 0,1 nas configurações de análise de tabulação. Estreitar as configurações de base para iniciar (Ciclo): 6 e End (ciclo): 15. </li><li> Registre o ciclo limiar (Ct) para todas as amostras. O valor de corte para considerar um resultado PCR positivo como pode ser definido como um Ct valor de 35. A quantidade de bactérias presentes em culturas de sangue variou de julho 10-novembro 10 CFU / ml, gerando Ct-valores abaixo de 35. </li></ol> PARTE II: TESTES susceptibilidade a antimicrobianos 4. Isolamento de bactérias a partir de hemoculturas positivas 9 <ol><li> Aspirar 5 ml de caldo de uma garrafa de sangue positivo cultura e transferi-lo para um tubo de separador de soro. </li><li> Centrifugar o tubo separador de soro a 2000 xg durante 10 min. </li><li> Descartar o sobrenadante do tubo separador de soro. </li><li> Transferência de bactérios a partir da camada de gel do tubo com uma mecha de algodão estéril em solução salina a 0,9% até 0,5 de McFarland uma suspensão padrão é obtida. </li></ol> 5. Inoculação de placas de Micro Titre <ol><li> Dilui-se a suspensão 0,5 McFarland em duplo concentrado Mueller Hinton II caldo para formar uma suspensão de 5 x 10 5 UFC / ml. </li><li> Adicionar esta suspensão para os poços de uma placa de micro titulação, contendo uma selecção de antibióticos (Tabela 1). </li><li> Incubar a placa de micro titulação, a 37 ° C durante 6 h. </li><li> Armazenar uma alíquota da suspensão a 4 ° C (como controlo de crescimento negativo). </li><li> Após 6 h de incubação, transferir o conteúdo de cada poço para um tubo de estéril, bem como a amostra de controlo negativo de crescimento que foi armazenada a 4 ° C. </li><li> Centrifugar os tubos a 16.000 xg durante 5 min. </li><li> Cuidadosamente remover o sobrenadante, sem perturbar o sedimento bacteriano. </li><li> Ressuspender o sedimento em H desmineralizada esterilizada <sub&gt; 2 O. </li><li> Diluir as amostras de 10 vezes na estéril desmineralizada H 2 O. </li></ol> 6. Real-time PCR rDNA 16s 10 <ol><li> Preparar a mistura de PCR como se segue: <ul><li> 12,50 ul iQ SYBR Green Supermix </li><li> 0,5 uM primer dianteiro 16S-1 (5-TGGAGAGTTTGATCCTGGCTCAG-3) 11 </li><li> 0,25 iM reversa 16S-2 primer (5-TACCGCGGCTGCTGGCAC-3) 11 </li><li> estéril desmineralizada H2O para um volume total de 20 uL </li></ul></li><li> Adicionar 20 ul de mistura de PCR para os poços de uma placa de PCR de 96 poços. </li><li> Adicionar 5 ul de amostra a cada poço. </li><li> Use uma película adesiva para selar a placa de 96 poços de PCR. </li><li> Execute o prato no Single-Cor MyiQ Sistema de Detecção de Real-Time PCR, utilizando os seguintes óptimas condições de ciclos térmicos: <ul><li> Desnaturação inicial a 95 ° C durante 4 min </li><li> Inicial de recozimento, a 65 ° C durante 30 s </li><li> 35 ciclos de <ul><li> Desnaturação a 95 ° C durante 15 s </li><li> Recozimento a 60 ° C durante 1 min </li></ul></li><li> Derreter análise da curva (60-95 ° C em 20 min com incrementos de 0,57 ° C) </li></ul></li></ol> 7. Análise dos Resultados <ol><li> Calcular o valor de corte Ct, utilizando uma das seguintes fórmulas (dependendo do tipo de antibiótico). <ul><li> Em geral: Cut-off Ct valor = valor Ct controle do crescimento positivo + 0,5 x (valor Ct controle do crescimento negativo - valor Ct controle de crescimento positivo) </li><li> Piperacilina, piperacilina / tazobactam e ceftazidima em bacilos Gram-negativos; amoxicilina, oxacilina e trimetoprim / sulfametoxazol em S.aureus; Amoxicilina em Enterococcus spp:. Cut-off Ct valor = valor Ct controle do crescimento positivo + 0,25 x (Ct crescimento valor negativo controle - valor Ct controle de crescimento positivo) </li></ul></li><li> Use a amostra incubada com desmineralizada estéril H 2 O, como controle de crescimento positivo.</li><li> Dependendo do microorganismo, utilizar o controlo de crescimento negativa adequada: <ul><li> Gram-negativas Amostra hastes incubadas com uma mistura de antibióticos </li><li> Enterococcus spp Amostra. Armazenado a 4 ° C </li><li> Exemplo S.aureus armazenado a 4 ° C </li></ul></li><li> Determinar a susceptibilidade (S) ou a resistência (R) da estirpe para o antibiótico testado como se segue: <ul><li> Um valor Ct superior ao valor Ct de corte indica susceptibilidade </li><li> Um valor Ct inferior ao valor Ct de corte indica resistência </li></ul></li></ol> 8. Os resultados representativos Dois organismos modelo, ou seja, uma E. Gram-negativas coli e S. uma Gram-positivos aureus, são escolhidos para visualizar o procedimento combinado para a detecção e identificação de agentes patogénicos bacterianos e de determinação do seu perfil de antimicrobiana. A primeira parte do protocolo compreende a identificação do patógeno. Spesondas específicos são concebidos para a detecção de oito microrganismos clinicamente relevantes. Em presença de um alvo incluídos no painel de bacteriana, as curvas de amplificação são gerados e valores Ct são calculados (Figura 2). O valor de corte para considerar um resultado positivo de PCR como está definido para um valor de Ct de 35. Na Figura 2A, o perfil de identificação de uma cultura de E. coli sangue infectado é mostrado. A sonda 16S universal está incluído em duas misturas de reação separadas e, conseqüentemente, gera duas curvas de amplificação (Ct de 25,20 e 25,95). O terceiro sinal é derivado da sonda específica para E. coli (Ct de 27,04). A identificação de um S. aureus infectado cultura de sangue é mostrado na Figura 2B. A sonda 16S universal tem sinais de amplificação de 33,35 e 33,71. Os dois sinais restantes são derivadas das sondas específicas para Staphylococcus spp. e S. aureus (Ct de 32,48 e 30,59). A Figura 3 é um exemplo de um antibiótico trama amplificação susceptibilidade de teste, que representa a estirpe de E. coli que foi também mostrado na Figura 2A. Cada linha representa um antibiótico que a amostra bacteriana foi incubada com. Uma amostra com um valor baixo Ct é uma amostra na qual o crescimento ocorreu na presença de uma resistência, antibiótico indicando ao antibiótico testado. Pelo contrário, um valor elevado Ct representa uma amostra na qual nenhum crescimento ocorreu por causa de o funcionamento eficaz da susceptibilidade, antibiótico indicando ao antibiótico testado. A Tabela 1 ilustra a determinação do perfil de antimicrobiana do E. coli e S. isolados aureus. Todos os valores são relatados Ct e, usando as fórmulas mencionadas no texto protocolo (7,1), dois valores de corte Ct são calculadosisoladas de distinguir entre a resistência e susceptibilidade. A estirpe é resistente ao antibiótico se o valor relatado Ct é menor do que o valor calculado Ct cut-off (e vice-versa). <img alt="A Figura 1" src="/files/ftp_upload/3254/3254fig1.jpg" /> Figura Fluxograma 1. Da identificação do patógeno e procedimento de teste de sensibilidade aos antibióticos em tempo real usando 16S rDNA PCR. <img alt="A Figura 2" src="/files/ftp_upload/3254/3254fig2.jpg" /> A Figura 2 ensaio Identificação:. Gráficos de amplificação e os valores de limiar de ciclo (valores Ct) Uma cultura de sangue positivo é detectado pelo universal 16S rDNA sonda, enquanto as sondas específicas são utilizados para a identificação do agente patogénico causal.. A. enredo ampliação da cultura de sangue contendo E. coli, B. enredo ampliação da cultura de sangue contendo S. aureus; Pseudomonas ae, Pseudomoncomo aeruginosa; uni, 16S sonda universal; Ecoli, Escherichia coli sonda; Pseudomonas sp, Pseudomonas spp. sonda; S. pneumoniae, Streptococcus pneumoniae sonda; Strep sp, Streptococcus spp. sonda; Entero, Enterococcus spp. sonda; S. aureus, Staphylococcus aureus sonda; Staph sp, Staphylococcus spp. sonda. <img alt="A Figura 3" src="/files/ftp_upload/3254/3254fig3.jpg" /> Figura enredo Amplificação 3. Dos testes de susceptibilidade a antibióticos de um E. coli isolado (amostra 1). Cada curva representa um antibiótico que a estirpe foi incubada com. Um sinal precoce é causada por uma carga elevada de bactérias, o que significa que a estirpe cresceu na presença do antibiótico testado e é, portanto, resistente ao antibiótico. Sinais tardios indicam que a estirpe não cresceu na presença do antibiótico, por outras palavras, ela é susceptível. <table border="1"><tbody&gt; <tr><td> Exemplo 1: E. coli </td><td></td><td></td><td> Exemplo 2: S. aureus </td><td></td><td></td></tr><tr><td> AST </td><td> Ct </td><td> R / S </td><td> AST </td><td> Ct </td><td> R / S </td></tr><tr><td> A amoxicilina de 8 mg / L </td><td> 16,83 </td><td> R </td><td> A amoxicilina de 0,25 mg / L </td><td> 21,03 </td><td> R </td></tr><tr><td> Amoxicilina-clavulanato mg 8/4 / L </td><td> 17,36 </td><td> R </td><td> Oxacilina 2 mg / L </td><td> 25,80 </td><td> S </td></tr><tr><td> Piperacilina 16 mg / L </td><td> 16,67 </td><td> R </td><td> A vancomicina 2 mg / L </td><td> 25,20 </td><td> S </td></tr><tr><td> Piperacilina-tazobactam 16 /4 mg / L </td><td> 24,15 </td><td> S </td><td> Gentamicina 4 mg / L </td><td> 25,86 </td><td> S </td></tr><tr><td> Ciprofloxacina 1 mg / L </td><td> 29,72 </td><td> S </td><td> Trimetoprima-sulfametoxazol mg 2/38 / L </td><td> 24,62 </td><td> S </td></tr><tr><td> A ceftazidima 1 mg / L </td><td> 24,03 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> A ceftazidima 8 mg / L </td><td> 26,58 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Gentamicina 4 mg / L </td><td> 29,83 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Trimetoprima-sulfametoxazol mg 2/38 / L </td><td> 27,60 </td><td> S </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Controlo de crescimento negativo (mistura de antibióticos) </td><td> 30,41 </td><td></td><td> Controlo de crescimento negativo (amostra armazenada a 4 ° C) </td><td> 27,42 </td><td></td></tr><tr><td> Controle de crescimento positiva </td><td> 16,90 </td><td></td><td> Controle de crescimento positiva </td><td> 20,22 </td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Cut-off valor Ct-1 * </td><td> 21,76 </td><td></td><td> Cut-off valor Ct-1 *** </td><td> 23,82 </td><td></td></tr><tr><td> Cut-off Ct-valor 2 ** </td><td> 18,75 </td><td></td><td> Cut-off Ct-valor 2 **** </td><td> 22,02 </td><td></td></tr><tr><td> * Para amoxicilina, amoxicilina-clavulanato, ciprofloxacina, gentamicna, sulfametoxazol-trimetoprim ** Para piperacilina, piperacilina-tazobactam, ceftazidima </td><td></td><td></td><td> *** Para vancomicina e gentamicina Para **** amoxicilina, oxacilina e sulfametoxazol-trimetoprim </td><td></td><td></td></tr></tbody></table> Tabela 1. Determinação dos testes de susceptibilidade aos antibióticos das duas amostras (E.coli e S.aureus). Os valores de TC do PCR-ensaio foram copiados para este ficheiro Excel, como o que pode automaticamente calcula os dois alores de corte Ct a partir do controlo de crescimento positivo e negativo, usando as fórmulas mostradas no texto de protocolo. Se um antibiótico mostra um valor Ct inferior ao valor Ct de corte, a estirpe é resistente ao antibiótico, se o valor Ct foi maior do que o ponto de corte, a estirpe é susceptível.

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Não temos nada a divulgar.

O protocolo descrito aqui permite a rápida identificação de patógenos e fornece um perfil funcional antimicrobiano que poderia conduzir a administração precoce de antibióticos adequados, melhorando o prognóstico de pacientes com infecções da corrente sanguínea. Dependendo das condições requeridas de um teste, isto é, baixo custo, com rendimento elevado, o mínimo tempo de rotação, as condições de ensaio pode ser ajustada. Todo o procedimento pode ser realizado dentro de um dia útil. Além disso, as duas partes do protocolo pode ser realizada simultaneamente, o que reduz o tempo de rotação de forma significativa. Como apresentado aqui, o painel de identificação é uma seleção das bactérias mais clinicamente relevantes em nosso hospital. Uma vez que o principal princípio tem como alvo a região do gene 16S, as sondas específicas para outros microorganismos podem ser concebidos e adicionado ao ensaio. O ensaio completo foi originalmente destinado para a análise rápida de culturas de sangue, mas também pode ser usado para o processamento de Oamostra materiais Ther. Este é também o caso para os antibióticos que foram usados ​​para os testes de susceptibilidade aos antibióticos: mais antibióticos ou outras podem ser adicionados, com base em padrões de resistência locais e orientações.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Cloreto de sódio (NaCl) </td><td> Merck Chemicals </td><td> 106404 </td><td> 0,9% em água </td></tr><tr><td> Vacutainer SST tubo separador de soro de 5 ml </td><td> BD Diagnostic Systems </td><td> 367986 </td><td></td></tr><tr><td> Mueller Hinton II caldo </td><td> BD Diagnostic Dystems </td><td> 212322 </td><td> 44 g / L em água </td></tr><tr><td> Centrifugar Rotixa 50 rs </td><td> Andreas Hettich GmbH &amp; Co. KG </td><td> 4910 </td><td></td></tr><tr><td> Centrífuga 5415 D </td><td> Eppendorf </td><td> Descontinuado </td><td></td></tr><tr><td> Primers </td> &lt;td&gt; Sigma-Aldrich </td><td> nd </td><td></td></tr><tr><td> Sondas </td><td> Sigma-Aldrich/Applied Biosystems </td><td> nd </td><td></td></tr><tr><td> TaqManEnvironmental mestre mix 2,0 </td><td> Applied Biosystems </td><td> 4396838 </td><td></td></tr><tr><td> iQ SYBRGreen Supermix </td><td> Bio-Rad Laboratories BV </td><td> 170-8880 </td><td></td></tr><tr><td> MicroAmp Optical 96-Bem Placa de Reacção </td><td> Applied Biosystems </td><td> N8010560 </td><td></td></tr><tr><td> Película adesiva MicroAmp Optical </td><td> Applied Biosystems </td><td> 4311971 </td><td></td></tr><tr><td> iQ placas de 96 poços de PCR </td><td> Bio-Rad Laboratories BV </td><td> 223-9441 </td><td></td></tr><tr><td> Microseal B Adesivo Selos </td><td> Bio-Rad Trabalhoatories BV </td><td> MSB-1001 </td><td></td></tr><tr><td> Real-time Sistema de Detecção de PCR </td><td> Applied Biosystems </td><td> ABI PRISM 7900HT </td><td></td></tr><tr><td> Real-Time PCR Detection System </td><td> Bio-Rad Laboratories BV </td><td> MyiQ Single-Cor </td><td></td></tr></tbody></table>

one-day workflow scheme for bacterial pathogen detection and antimicrobial resistance testing from blood cultures

Infecções da corrente sanguínea estão associados a altas taxas de mortalidade por causa da manifestação provável de sepse, sepse grave e choque séptico 1. Assim, a administração rápida de terapia antibiótica adequada é de grande importância no tratamento de infecções da corrente sanguínea. O elemento crítico neste processo é o tempo, fortemente dependente dos resultados da identificação bacteriana e testes de susceptibilidade aos antibióticos. Ambos estes parâmetros são rotineiramente obtido por cultura com base em testes, o que é demorado e leva em média 24-48 horas 2, 4. O objectivo do estudo foi o de desenvolver baseados em DNA ensaios para a rápida identificação de infecções da corrente sanguínea, bem como testes de susceptibilidade antimicrobiana rápida. O primeiro ensaio é um 16S eubacterianas ADNr baseado em tempo real de ensaio PCR, complementados com espécies-ou género sondas específicas 5. Usando estas sondas, bactérias gram-negativas, incluindo Pseudomonas spp., Pseudomonas aeruginosae Escherichia coli, bem como bactérias Gram-positivas, incluindo Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., e Streptococcus pneumoniae poderiam ser distinguidas. Utilizando este ensaio Multiprobe, uma primeira identificação do micro-organismo causador foi dado após 2 h. Em segundo lugar, nós desenvolvemos um ensaio semi-molecular para testes de susceptibilidade a antibióticos de S. aureus, Enterococcus spp. e (facultativo) aeróbios Gram-negativos hastes 6. Este ensaio baseou-se um estudo em que a PCR foi utilizado para medir o crescimento de bactérias 7. As bactérias colhidas directamente a partir de culturas de sangue são incubadas durante 6 h, com uma selecção de antibióticos, e seguindo um Sybr Verde baseado em tempo real ensaio de PCR determina a inibição do crescimento. A combinação destes dois métodos poderiam dirigir a escolha de uma terapia adequada antibiótico no mesmo dia (Figura 1). Em conclusão, A análise molecular de ambos identificação e antibiograma oferece uma alternativa mais rápida para a detecção de patógenos e pode melhorar o diagnóstico de infecções da corrente sanguínea.

Watch this Scientific Journal Video about One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures at JoVE.com

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

O desenho de um esquema de fluxo de trabalho simples de um dia para diagnóstico de patógenos bacterianos permite o reconhecimento rápido de infecções da corrente sanguínea. A inclusão de oito clinicamente relevantes alvos bacterianas e os seus perfis de resistência a antibióticos oferece a percepção um clínico inicial no mesmo dia, o que pode levar a uma terapia mais adequada.

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. ...

one-day-workflow-scheme-for-bacterial-pathogen-detection

Research

JoVE Journal

Immunology and Infection

25.3K visualizações.  Maastricht University Medical Center. O desenho de um esquema de fluxo de trabalho simples de um dia para diagnóstico de patógenos bacterianos permite o reconhecimento rápido de infecções da corrente sanguínea. A inclusão de oito clinicamente relevantes alvos bacterianas e os seus perfis de resistência a antibióticos oferece a percepção um clínico inicial no mesmo dia, o que pode levar a uma terapia mais adequada.

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

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 ...

Imunologia e Infecção

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 ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Alta capacidade método de detecção de vírus Influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

A saliva, glândula salivar, e Coleta de hemolinfa de carrapatos Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

Nanomecânica de interações medicamentosas-alvo e de detecção de resistência antibacteriana

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

Multiplex PCR Ensaio para a digita

Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

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

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

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 ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Isothermal amplificação (lâmpada), Ensaios mediada por loop para detecção específica da espécie os de<em&gt; Eimeria</em&gt; Que infectam Galinhas

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Usando um patógeno bacteriano para sonda para celular e organísmica-nível Host respostas

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

Um sistema de entrega e recuperação de patógenos precisos para modelos murinos de pneumonia bacteriana secundária

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...