Os autores agradecem Mattias Gyllborg para squid apoio instalações e dos comentários deste manuscrito, Michael Hadfield eo Kewalo Laboratório Marinho de assistência durante a coleta de campo, e os membros do Laboratório de Ruby e McFall-Ngai para contribuições para este protocolo. Trabalho no Laboratório de Mandel é suportado pela NSF IOS-0843633.

1. Preparação de inóculos <ol><li> Dia 0 Dois dias antes da inoculação lula, o prato de cepas bacterianas relevantes sobre LBS 13 ágar. </li><li> Incubar as bactérias em 25-28 ° C durante a noite. </li><li> Dia 1 Inocular 3 ml de meio LBS em um tubo de vidro com uma cultura de cada colônia V. fischeri linhagem de infecção. Prepare duplicar tubos como backup. </li><li> Dia 2 (Coordenar passos bacterianas 1,4-1,6 com passos de lula 3.7-3.10) 1 h antes da inoculação, as bactérias subcultura 1:80 (37,5 ul) em 3 lbs ml num tubo de cultura de vidro e crescer durante 1 h, com arejamento. </li><li> Medir a OD 600 da amostra antes da inoculação. Medições típicas são 0,3-0,6, dependendo da estirpe. </li><li> Para um inoculo alvo de 3-5 x 10 3 UFC / ml calcular o volume de inoculo como se segue: Inoculo volume (ul) = 1,25 / OD 600 (por exemplo, para OD 600 = 0,5, o volume de inóculo calculado = 1.25/0.5 = 2,5 ul). Este montante é adicionado diretamente à água do mar contendo lula no Passo 4.1. Este cálculo pode necessitar de ser ajustada para diferentes estirpes de V. fischeri ou para inoculação em níveis mais baixos ou mais elevado do que aqueles aqui especificado. </li></ol> 2. Preparação de placas de ágar para contagem dos inóculos <ol><li> Para cada tratamento, as placas de etiquetas lbs (2 por tratamento) para amostras de placa do inoculo no Passo 4,1. </li><li> Adicionar 5 gotas de revestimento estéreis por placa. </li></ol> 3. Colecção de Jovens Squid <ol><li> Medir a salinidade do oceano instantânea utilizando o refratômetro e ajustar a 35 ‰. </li><li> Filtrar 1 L de Instant Ocean usando a unidade de filtração e uma linha de vácuo anexado ou bomba de vácuo, para gerar esterilizada por filtração instantânea Marinho (FSIO). Água oxigenar o agitando vigorosamente antes de cada distribuição. The unidade de filtro pode ser reutilizado durante 2 dias. </li><li> Alíquota 40-50 ml de FSIO em cada um de dois (2) taças de amostra descartáveis. Um rótulo como earlies e um como timelies. </li><li> Preparar um excesso de pipetas de transferência de plástico para a aquisição de lula juvenil, cortando a pipeta cerca de 1 cm a partir da ponta, acima as mais baixas cristas (ver Figura 3). Isso facilita uma zona mais ampla através do qual o lula pode passar após a coleta. Descartar quaisquer pipetas de transferência em que há uma superfície áspera exposta. </li><li> Usando pipetas de transferência preparados, recolher E. scolopes que nasceram durante a noite e transfira para a tigela earlies de FSIO. Hatchlings primeiros foram no sistema de ovo por mais de 1 hora e são suscetíveis à colonização por meio da contaminação V. fischeri no sistema de ovo. Não use earlies para experimentos de colonização sensíveis. </li><li> Verifique todos os tanques de ovos min 30-45 para filhotes novos. Assegurar que todos os filhotes são apagadas durante cada cParreira. Remover filhotes com uma pipeta de transferência e depósito no vaso timelies de FSIO. Animais coletados em tempo hábil estão disponíveis para experimentos de colonização. </li><li> Quando a coleta foi concluída (~ 45 min depois do anoitecer), transferir a lula para o laboratório principal. Empiricamente, é vantajoso para colonizar os animais em condições de laboratório ininterruptas de luz para as inoculações 3 h. </li><li> Para cada tratamento, preparar uma tigela com 40 ml FSIO. Adicionar lula para as tigelas para o ensaio (no máximo n = 40 por tigela). </li><li> Prepare uma tigela adicional como um controle (negativo) aposymbiotic. </li><li> Prepara-se uma pipeta de transferência dedicado para cada tratamento. </li><li> Euthanize lula extra em etanol a 2%. </li></ol> 4. Colonização Squid <ol><li> Dia 2 - Usando um Pipetman P10, dispensar a alíquota calculada de bactérias (Passo 1,5) em cada taça lula (Passo 3,8) para cada tratamento. Iniciar uma 3 h temporizador imediatamente apósa primeira inoculação. </li><li> Para cada tratamento, criar um &quot;vórtice&quot; na tigela com a pipeta de transferência dedicado, colocando a pipeta perto da borda da taça e pipetagem cima e para baixo repetidamente para misturar a água e lulas por cerca de 10 seg. Uma mistura completa é crítica. </li><li> UL placa 50 de cada recipiente para uma placa de agar a partir do Passo LBS 2,2 (para técnica repetições, placa duas placas 50 ul por tratamento). Incubar a 25-28 ° C durante a noite. </li><li> Preparar taças de lavagem (100 ml FSIO / EA) para cada tratamento. </li><li> Preparar Drosophila frascos (4 ml FSIO / EA) para cada lula. </li><li> Depois de exatamente 3 horas, a lula para transferir suas tigelas de lavagem respectivos (completa para todos os tratamentos). Isso interrompe a inoculação. </li><li> Vá para transferir cada lula indivíduo à sua própria Drosophila frasco com FSIO. Usar uma pipeta de transferência designado para cada tratamento. </li><li> Mover bandejas de Drosophila frascos à facilidade de lula para retornar ao dia / nciclo de luz ight os animais experimentaram durante a embriogênese. </li><li> Dia 3 - Prepare Drosophila frascos (4 ml FSIO / ea) para cada lula. </li><li> Antes de anoitecer em 22-24 h pós-inoculação, transferir cada lula para um frasco de Drosophila novo. Usar uma pipeta de transferência designado para cada tratamento. </li><li> Dia 4 - Prepare rotulado tubos de 1,5 ml de microcentrífuga (1/squid). </li><li> Antes de anoitecer, às 46-48 h pós-inoculação, medir e registrar a luminescência de cada lula no frasco Drosophila (conjunto luminômetro de integração 6 s e auto-ler sobre fechamento da tampa). </li><li> Como um controlo negativo para a luminescência de fundo, medir um frasco com FSIO que não contém qualquer lula. </li><li> Transfira cada lulas em um volume de aproximadamente 700 ul a um tubo de microcentrífuga de 1,5 ml a partir do Passo 4,12. Mover-se para uma caixa de papelão freezer. Uma vez que a tampa é colocada na caixa, não removê-lo como as pistas de luz para a expulsão bactérias não são well-entendido. </li><li> Congelar tubos de microcentrífuga a -80 ° C durante a noite. </li></ol> 5. Determinação dos níveis de colonização <ol><li> Para cada lula, preparar dois tubos (2) microcentrífuga, cada um com 475 FSIO ul (ou autoclavados 70% Instant Ocean). </li><li> Preparar pilões usando primeiro uma Kimwipe para limpar o pilão e remover detritos bruto e / ou tecido. </li><li> O local de pilões ponta para baixo em um copo de 50 ml contendo etanol a 95%. Etanol deve ser adicionado a uma altura de cerca de 3 cm. </li><li> Para cada pilão, retire do copo e limpe a ponta com uma Kimwipe. </li><li> Mergulhar o pilão de volta para o banho de etanol, remover e inserir (ponta para cima) em um suporte de tubos de microcentrífuga e deixa-se secar ao ar completamente durante aproximadamente 15 minutos. </li><li> Lulas descongelar em um suporte de tubos de microcentrífuga (no máximo n = 8). </li><li> Se necessário, ajustar o volume para 700 ul. </li><li> Usando um pilão a partir do Passo 5,5, perturbar o tecido animal até que a tinta saco rupestruturas (a água vai virar uma cor cinza escuro). </li><li> Remover o pilão e assegurar todo o tecido permanece no tubo. </li><li> Vortex brevemente o tecido por exatamente 10 segundos (use um timer). </li><li> Permitir que o tecido a descansar durante 10 min. O tecido vai assentar e as bactérias e de tinta permanecer na solução. Para os cálculos que se seguem, as bactérias / solução de tinta é a [A] de diluição (isto é, a E. scolopes homogenato órgão de luz em 700 uL). Diluições 1:20 em série ([B] [C]) são descritos abaixo. </li><li> Para a [B] diluição, adicionar 25 uL [A] a um dos tubos de microcentrífuga preparado no passo 5.1. Vortex. </li><li> Para a [C] diluição, adicionar 25 uL [B] a um dos tubos de microcentrífuga preparado no passo 5.1. Vortex. </li><li> UL placa 50 de cada diluição em agar LBS, 2 repetições por tratamento. </li><li> Incubar as placas a 25-28 ° C durante a noite. </li></ol> 6. Análise de Dados <ol><li> Para calcular o UFC / órgão luz (LO), paísescolónias t sobre a placa para cada tratamento em que 10-400 colónias estão presentes, e utilizar a fórmula apropriado: UFC / LO = (colônias em [A] placa) x 14, ou UFC / LO = (colônias em [B] placa) x 280, ou UFC / LO = (colônias em [C] placa) x 5600. </li><li> Traçar pontos de dados individuais e medianas em uma escala logarítmica. </li><li> Os dados não são frequentemente normalmente distribuído, com desvios diferentes, e os valores extremos podem conter biologicamente significativa de informação. Portanto, não paramétricos testes fornecem um método útil para determinar se os tratamentos diferem de forma significativa. </li><li> Use o software GraphPad Prism para análise estatística. Por dois tratamentos, use o teste de Wilcoxon Rank Sum. Para comparações entre mais de dois tratamentos, utilize o teste Kruskal-Wallis com pós-testes apropriados. </li></ol> 7. Os resultados representativos Os resultados de um ensaio de colonização da amostra são mostrados na Figura 4. Duas cepas de <em&gt; V. fischeri que apresentam diferentes níveis relativos de luminescência foram inoculadas em seis lula, juntamente com seis lula que serviu aposymbiotic (colonizada) controles. O E. scolopes simbionte, ES114 14, e mais brilhante Sepiola robusta simbionte, SR5 15,16. Níveis de inóculo similares (Fig. 4A) levar a colonização 100% dentro de 3 h. Às 48 h, os níveis de luminescência (Fig. 4B) e as contagens de CFU (Fig. 4C) foram determinados para avaliar a proficiência colonização da estirpe. Determinação da luminescência específico (Fig. 4D; bactéria per) permite a determinação do brilho de cada estirpe bacteriana durante o simbiose. <img alt="A Figura 1" src="/files/ftp_upload/3758/3758fig1.jpg" /> Figura Fluxograma 1. Do procedimento de colonização. As bactérias e as lulas são colhidas separadamente e depois misturado ao inóculo especificado. Lulas foram lavadas e, em seguida transferida para a água de novo em 3 h, 24 h, e 4<html> 8 após a inoculação h. Aos 48 h, a luminescência é medida e os animais são congelados, que serve para a superfície a esterilizar os animais. Luz órgão-colonizado bactérias permanecem viáveis ​​a -80 ° C através de um descongelamento (sem adicionais de congelação-descongelação ciclos). <img alt="A Figura 2" src="/files/ftp_upload/3758/3758fig2.jpg" /> Figura Fluxograma 2. Ilustrando homogeneização e diluição em placas de bactérias. Serial 20-diluições fornecer a gama dinâmica adequado para enumeração de bactérias colonizadas. <img alt="A Figura 3" src="/files/ftp_upload/3758/3758fig3.jpg" /> Figura 3. Transferir pipetas com uma conicidade eixo adequadamente estreita para um furo estreito (A), que prejudicaria lula juvenil. O pré-tratamento, cortando o mais estreita secção com uma tesoura ou uma lâmina de barbear produz uma ferramenta apropriada (B) para a transferência de lula juvenil. s/ftp_upload/3758/3758fig4.jpg &quot;/&gt; Figura 4. Dados de amostra para um ensaio de colonização. (A) Os níveis de bactéria nas bacias de inóculo. (B) luminescência de lula individual. (C) contagem de colônias de lula individual. (D) luminescência específico de lula individual. Apo, Aposymbiotic (não colonizadas controle negativo).

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O ensaio de colonização descrito permite a análise de um processo natural simbiótica num ambiente controlado de laboratório. Como tal, pode ser usado para avaliar a colonização por estirpes mutantes, por diferentes isolados naturais, e sob regimes químicas diferentes. Variações sobre os experimentos descritos são comumente usados ​​para avaliar diferentes aspectos da simbiose. A cinética da colonização pode ser medido por análise de luminescência durante as primeiras 24 h, o que pode ser detectado au...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> Tubos de Vidro Cultura, 16 mm de diâmetro </td><td> VWR </td><td> 47729-580 </td><td></td></tr><tr><td> Tampas para tubos de cultura de vidro </td><td> Pescador </td><td> NC9807998 </td><td></td></tr><tr><td> Espectrofotómetro visível para a determinação de OD 600 </td><td> Biowave </td><td> CO8000 </td><td> Qualquer espectrofotómetro capaz de medir OD 600 irá funcionar. Esta unidade pode medir a OD 600 de líquido directamente nos tubos de cultura de vidro. Alguns ajustamento do cálculo inoculo pode ser necessário, dependendo do instrumento utilizado. </td></tr><tr><td> GloMax 20/20 Luminometer Único-Tube </td><td> Promega </td><td> E5311 </td><td> Equivalente ao BioSystems Turner 20/20n Luminometer. Inclui o suporte do tubo de microcentrífuga. </td></tr><tr><td> GloMax 20/20 Luz padrão </td><td> Promega </td><td> E5341 </td><td> Para a calibração luminómetro. </td></tr><tr><td> Refratômetro, Handheld </td><td> Foster e Smith Aquatics </td><td> CD-14.035 </td><td> Calibrar antes de cada uso com água deionizada. Lavar depois de cada uso com água deionizada para evitar acúmulo de sal. </td></tr><tr><td> Instantânea Oceano (concentrado de água do mar artificial) </td><td> Foster &amp; Smith Aquatics </td><td> CD-16.881 </td><td> Preparar a 35 ‰ em água desionizada, utilizando o refractómetro, seguida de filtração através de um filtro de 0,2 uM SFCA. </td></tr><tr><td> Unidade de filtração </td><td> Nalgene </td><td> 158-0020 </td><td> Surfactante livre de membrana de acetato de celulose (SFCA), 0,2 m. Temos observado resultados variáveis ​​com alguns filtros surfactante contendo PES. </td></tr><tr><td> Pipetas de transferência </td><td> Pescador </td><td> 13-711 -09:00 </td><td> Usando uma tesoura ou lâmina de barbear, cortar a ponta de forma limpa acima do rebordo primeiro para aumentar o diâmetro da ponta da pipeta e evitar espremer os filhotes de lula. </td></tr><tr><td> Descartáveis ​​Bowls exemplo (copos de plástico) </td><td> Cometa </td><td> T9 (9 oz.) </td><td> Taças para a inoculação, com diâmetro superior 3 ¼ &quot;, menor diâmetro 2 ¼&quot;, altura 3 &quot;. Bowls criar um ambiente homogêneo, pois não possuem borda inferior, em que lula pode ficar preso em um nicho de baixo oxigênio. O tamanho é otimizado para 40 ml de inóculo. Disponível em webstaurantstore.com, # 619PI9. </td></tr><tr><td> Drosophila frascos </td><td> VWR </td><td> 89092-720 </td><td> Vial diâmetro corresponde a abertura na PMT luminómetro. </td></tr><tr><td> Tubos de microcentrífuga de 1,5 ml </td><td> ISC Bioexpress </td><td> C-3217-1CS </td><td> Os tubos devem caber a forma dos pilões. </td></tr><tr><td> Etanol, 200 Prova </td><td> Pescador </td><td> BP2818-100 </td><td></td></tr><tr><td> Pilões </td><td> Kimble Chase / Kontes </td><td> 749521-1500 </td><td></td></tr><tr><td> Contas do chapeamento, 5 mm de diâmetro </td><td> Kimble perseguição </td><td> 13500 5 </td><td> Preparar 5 por tubo e em autoclave. </td></tr></tbody></table>

colonization of euprymna scolopes squid by vibrio fischeri

Bactérias específicas são encontrados em associação com tecidos animais 1-5. Tais host-bacterianas associações (simbiose) pode ser prejudicial (patogênicos), não tem conseqüência fitness (comensal), ou ser benéfica (mutualismo). Enquanto muita atenção tem sido dada às interações patogênicas, pouco se sabe sobre os processos que determinam a aquisição reprodutível de benéficos / comensal bactérias do ambiente. O mutualismo luz de órgãos entre a bactéria Gram-negativa marinha V. fischeri eo havaiano bobtail lula, E. scolopes, representa uma interação altamente específica em que um host (E. scolopes) estabelece uma relação simbiótica com apenas uma espécie bacteriana (V. fischeri) durante todo o curso da sua vida 6,7. A bioluminescência produzido por V. fischeri durante essa interação fornece um benefício anti-predatória de E. scolopes durante atividades noturnas 8,9, enquantoo tecido hospedeiro rico em nutrientes fornece V. fischeri com um nicho protegido 10. Durante cada geração de acolhimento, esta relação é recapitulada, representando assim um processo previsível que pode ser avaliado em detalhe em vários estágios de desenvolvimento simbiótico. No laboratório, a juvenil lula escotilha aposymbiotically (não colonizada), e, se recolhida dentro dos primeiros 30-60 minutos e transferida para simbionte livre de água, não podem ser colonizados excepto por o inoculo experimental 6. Esta interacção proporciona assim um sistema modelo útil no qual se pode avaliar os passos individuais que levam à aquisição específica de um micróbio simbiótica a partir do ambiente 11,12. Aqui nós descrevemos um método para avaliar o grau de colonização que ocorre quando recém-eclodidos E. aposymbiotic scolopes são expostos a (artificial) a água do mar que contêm V. fischeri. Este ensaio simples descreve a inoculação, a infecção natural, e recuperaçãodo simbionte bacteriano do órgão luz nascente da E. scolopes Care. é levado para proporcionar um ambiente consistente para os animais durante o desenvolvimento simbiótico, especialmente no que diz respeito à qualidade da água e sinais de luz. Métodos para caracterizar a população simbiótico descrito incluem (1) medição da bioluminescência bactérias derivados, e (2) colônia contagem direta de simbiontes recuperados.

Watch this Scientific Journal Video about Colonization of Euprymna scolopes Squid by Vibrio fischeri at JoVE.com

Colonization of Euprymna scolopes Squid by Vibrio fischeri

O método descreve o procedimento pelo qual o squid bobtail havaiana,<em&gt; Euprymna scolopes</em&gt; E seu simbionte bacteriano,<em&gt; Vibrio fischeri</em&gt;, São levantadas separadamente e, em seguida, introduzido para permitir a colonização específica do órgão luz lula pelas bactérias. Detecção de colonização por bactérias luminescência e derivados por meio de contagem direta colônia são descritas.

Colonização de Euprymna scolopes Squid por Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

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Research

JoVE Journal

Immunology and Infection

Colonization of Euprymna scolopes Squid by Vibrio fischeri

19.8K visualizações.  Northwestern University. O método descreve o procedimento pelo qual o squid bobtail havaiana, Euprymna scolopes E seu simbionte bacteriano, Vibrio fischeri, São levantadas separadamente e, em seguida, introduzido para permitir a colonização específica do órgão luz lula pelas bactérias. Detecção de colonização por bactérias luminescência e derivados por meio de contagem direta colônia são descritas.

Vídeo: Colonization of Euprymna scolopes Squid by Vibrio fischeri

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

Avaliar as alterações metabólicas hepáticas Durante a colonização progressiva de Germ-free Mouse 1 H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Imunologia e Infecção

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

Quantificação da colonização fúngica, esporogênese, e produção de micotoxinas Utilizando Bioensaios Kernel

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

TransFLP - um método para modificar geneticamente<em&gt; Vibrio cholerae</em&gt; Com base Transformação Natural e recombinação FLP-

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caracterização de Respostas Inflamatórias Durante Colonização Intranasal com Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Investigando os efeitos dos probióticos sobre pneumocócica Colonização Usando um<em&gt; In Vitro</em&gt; Adesão Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Avaliação da invasão bacteriana de células cardíacas na cultura e colonização cardíaca em camundongos infectados usando listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Desenvolvimento de uma mais sensível e específica Cromogênico Agar para a detecção de<em&gt; Vibrio parahaemolyticus</em&gt; E outro<em&gt; Vibrio</em&gt; Espécies

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Um modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonização Vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Técnicas de laboratório usadas para manter e diferenciar biótipos de<em&gt; Vibrio cholerae</em&gt; Isolados clínicos e ambientais

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

Indução de diferenciação celular e imagens de células<em&gt; Vibrio parahaemolyticus</em&gt; Células Swimmer e Swarmer

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...