Reconhecemos o Chi-Chung Chen do departamento de pesquisa médica, Chi Mei Medical Center (Tainan, Taiwan) pela ajuda no mouse, infecção e o apoio do centro de animais de laboratório da Universidade Nacional de Cheng Kung. Este trabalho é apoiado pelo Ministro da ciência e tecnologia (a maioria) concede (mais 104-2321-B-006-019, 105-2321-B-006-011,-005 and106-2321-B-006) para CC.

Cuidado: EHEC O157: H7 é um nível de biossegurança 2 (BSL-2) patógeno de acordo com o Centers for Disease Control and Prevention (CDC) instrução de biossegurança (https://www.cdc.gov/). Portanto, todos os procedimentos experimentais envolvendo EHEC devem ser executados em uma facilidade BSL-2. Use luvas e jalecos de laboratório durante a realização do experimento. Trabalho em uma gabinete de biossegurança certificada (BSC). Desinfete a bancada experimental, antes e após o procedimento experimental com etanol a 70%. Todos os instrumentos ou equipamentos que EHEC contato (ou potencialmente contato) deve ser desinfectado com etanol 70% ou água sanitária. Resíduos contaminados (ou potencialmente contaminados) devem ser selado e esterilizado com cuidado. Use uma máscara, óculos de proteção, luvas duplo ou um macacão, se necessário. Os ratos fêmeas C57BL/6 de 6 semanas de idade foram comprados e mantidos pela Universidade de laboratório Animal centro de Nacional Cheng Kung (NCKU). As experiências com animais foram aprovadas pelo cuidado de Animal institucional e Comitê de uso de NCKU (número de aprovação 104-039).
1. geração de bactéria EHEC Bioluciferase-etiquetado
<ol><li>Mix 50 ng o Ácido desoxirribonucleico (DNA), 1 μL de cada 10 μm para a frente e reverter as primeiras demão, desoxinucleótidos de 2,5 mM 2 μL (dNTPs), 2 μL de 10x buffer, 0,2 µ l de DNA polimerase e água de bidestilada estéril (ddH2O) para um volume final de 20 μL para amplificar o antigaveta canamicina, de gene nptII 24. O modelo de DNA é de pBSL18024, que é comprado de projeto nacional de BioResource (NBRP). As condições PCR são listadas na tabela 1 e a sequência de cartilha está disponível na tabela 2.</li>
	<li>Ligam os produtos PCR para um vetor de clonagem comercial seguindo o kit protocolo (consulte Tabela de materiais).</li>
	<li>Excisar o fragmento do gene nptII do vetor clonagem por NsiI e SmaI e clonar em pBBR1MCS4, que é digerido pela ScaI e NsiI pAKlux29 -criar o plasmídeo canamicina resistente, pWF27813.</li>
	<li>Excisar o operon luxCDABE , do luciferase expressando de shRNA9, pAKlux2, pela SpeI-HF e ScaI e clone SpeI-HF e SmaI digerido pWF278 para gerar canamicina resistente e luciferase expressando de shRNA, pWF27913. Nota: Para as extrações do plasmídeo e purificações o fragmento extirpado, use a extração comercial do plasmídeo e o kit de extração do gel, respectivamente. Para a digestão enzimática, misturar 100 ng DNA, 1 μL de buffer x 10, 1 μL de cada enzima de restrição selecionada e ddH estéril2O para um volume total de 10 μL e incubar a 37 ° C para 2,5-3 h.</li>
	<li>Transforme o plasmídeo pWF279 em células competentes de Escherichia coli O157: H7 EDL933 por eletroporação com 2.500 V para ms 4.</li>
	<li>Incube as células de bactérias transformadas em 1 mL de meio de Luria-Bertani (LB) a 37 ° C por 1h.</li>
	<li>As bactérias em uma placa de ágar LB suplementado com 50 µ g/mL de canamicina a 37 ° C por 16-18 h da placa.</li>
	<li>Verificar o sinal bioluminescente da placa por um na vivo de imagem máquina no dia seguinte. Escolher uma única colônia da placa e a cultura em 3 mL LB suplementado com 50 canamicina µ g/mL a 37 ° C por 16-18 h.</li>
	<li>Prepare o suporte de ações bacteriana diluindo 100% glicerol em ddH estéril2O tornar a solução de glicerol de 30%.</li>
	<li>Congelar a canamicina resistente bactéria de e. coli O157: H7 EDL933 abrigando o plasmídeo luciferase como um estoque bacteriano em uma tampa de rosca cryovial como uma proporção de 1:1 de solução de glicerol cultura e 30% bactérias a-80 ° C. A concentração final de glicerol é 15%.</li>
</ol>2. preparação de bactéria EHEC bioluminescentes para inoculação Oral
Nota: O fluxograma cronograma dos procedimentos experimentais para a preparação de EHEC e gavagem oral rato é apresentado na Figura 1 para auxiliar na preparação experimental.
<ol><li>Raia e. coli O157: H7 EDL933 as bactérias abrigando plasmídeo luciferase em uma placa de ágar LB com 50 canamicina µ g/mL de estoque-80 ° C. Crescem as bactérias para 16-18 h a 37 ° C.</li>
	<li>Escolha uma única colônia da placa durante a noite e cultura no meio de 3 mL LB com 50 canamicina µ g/mL para 16-18 h em uma incubadora de 37 ° C a 220 rpm.</li>
	<li>Subcultura as bactérias (diluição de 1: 100) em canamicina (50 µ g/mL) contendo caldo LB para 2,5-3 h em uma incubadora de 37 ° C a 200 rpm. (Por exemplo, adicione 2 mL a noite cultivadas bactérias para 200 mL LB suplementado com canamicina (50 µ g/mL)).</li>
	<li>Incubar as bactérias para 2,5-3 h e medir o valor de densidade óptica em 600 nm (OD600) até o valor é entre 0,9 a 1. O número de células bacterianas é em uma densidade de cerca de 108 colônia formando unidades (CFU) / mL.</li>
	<li>Centrifugar as bactérias re-cultivadas em 8.000 x g durante 30 minutos, 4 ° C.</li>
	<li>Desprezar o sobrenadante sem agitar o sedimento de bactérias e lave a pelota com 100 mL 0,9% solução salina normal estéril por agitação suave.</li>
	<li>Repita os passos de 2.5 e 2.6 uma vez.</li>
	<li>Após a lavagem, centrifugar a cultura bacteriana a 8.000 x g por 30 min, 4 ° C e descartar o sobrenadante suavemente.</li>
	<li>Condensado a pelota em 100 vezes com solução salina normal estéril 0,9%. Por exemplo, se são centrifugadas bactérias de 200 mL, adicione salina 2 mL para suspender o sedimento.</li>
	<li>Confirme as bactérias CFU (deve ser aproximadamente 109 UFC/100 μL após a condensação em solução salina normal) com chapeamento concentradas bactérias através de um de 10 vezes de diluição serial25.</li>
</ol>3. Mouse gavagem Oral de EHEC
<ol><li>Trate de camundongos C57BL/6 fêmeas de 6 semanas com água de estreptomicina (5 g/L) por 24 h.</li>
	<li>Após 24h, mude para regular água potável para outro 24h antes de gavage. Após o tratamento com água normal para 24h, os ratos estão prontos para gavagem oral de EHEC.</li>
	<li>Encha a seringa com bactérias EHEC 100 μL, puxando o êmbolo para trás. Não garantir nenhuma bolha de ar na seringa. Remova as bolhas, encaixe a seringa com os dedos.</li>
	<li>Levante o animal delicadamente e coloque-o no topo da gaiola com cuidado.</li>
	<li>Segure o mouse pela cauda cuidadosamente, e o animal vai agarrar o topo da gaiola e tentar te afastar de mim.</li>
	<li>Contenha suavemente o mouse segurando a pele solta do pescoço e costas o animal com o polegar e o indicador para evitar que a cabeça do rato se mova.</li>
	<li>Segure o mouse na posição vertical para inserir a agulha de gavagem e garantir que a cabeça de rato é imobilizado e vertical.</li>
	<li>Insira a agulha de gavagem para a boca do rato após o céu da boca e mover para baixo no esôfago e em direção ao estômago.</li>
	<li>Quando a agulha inserida é metade ou o comprimento de dois terços no mouse, injete 100 bactéria EHEC μL, que contêm aproximadamente 109 células CFU.</li>
</ol>4. visualização
<ol><li>Após a intubação oral, detecta os sinais bioluminescentes em 1 a 2 dias.</li>
	<li>Antes de examinar os sinais bioluminescentes de animais, anestesia, colocando-os em uma câmara de isoflurano em 2,5% 1,5 L/min de oxigênio.</li>
	<li>Espere por 2-5 min até que todos os ratos se tornar inconsciente e parada em movimento. Animais estão prontos para a deteção em vivo de bioluminescência.</li>
	<li>Detectar e imagem bioluminescentes sinais dos animais por um na vivo sistema de imagem. Por favor, veja a seção 5 "Aquisição de dados" para operação de software. Durante o processo de geração de imagens, todos os animais estão sob um fornecimento contínuo de isoflurano em 2,5% 1,5 L/min de oxigênio.<ol>
<li>Clique em arquivo > ao vivo e manualmente o foco do mouse.</li>
		<li>Selecione a intensidade de laser e tempo de exposição.</li>
		<li>Clique em adquirir > capturar.</li>
	</ol>
</li>
	<li>Para a imagem latente ex vivo , eutanásia os ratos por deslocamento cervical e retire o intestino inteiro de ratos infectados. Coloque os intestinos em uma placa de Petri-9cm e imagem pelo na vivo sistema de imagem. A configuração é o mesmo que na vivo imagens exceto o campo de visão é definida como A/B. Por favor consulte a seção 5 "Aquisição de dados" para obter detalhes. Nota: Método por deslocamento cervical: conter os ratos no topo da gaiola agarrando a cauda com uma mão, para que os animais agarrar a gaiola. Coloca uma caneta ou o polegar e o primeiro dedo da outra mão contra o pescoço na base do crânio. Rapidamente empurre para a frente com a mão ou objeto, enquanto a cabeça de restrição e puxar para trás com a mão segurando a cauda. Olhe com cuidado para confirmar respiratória e sem pulsação.</li>
</ol>5. aquisição de dados
Nota: O software utilizado para aquisição de dados é listado na Tabela de materiais.
<ol><li>Para aquisição de imagem, abra o painel de controle de aquisição do software (Figura 2).</li>
	<li>Selecione "Luminescentes", "fotografia" e "Sobreposição".</li>
	<li>Definir o tempo de exposição, como "Auto". Definir Binning como "Meio".</li>
	<li>Luminescentes e 8 estabelecem ƒ/parar como 1 para fotografia. Ƒ/parar controles a quantidade de luz recebida pelo detector CCD.</li>
	<li>Defina o campo de visão, com base no campo de imagens que são de interesse para adquirir. Opção "D" pode caber cinco ratos de 6 semanas de idade e imagem-los todos em um tempo. "C" pode imagem três ratos de 6 semanas de idade em um campo.</li>
	<li>Uma vez que os ratos/amostras estão prontas para tratamento de imagens, clique em "Adquirir" para aquisição de imagens.</li>
	<li>Abra os dados de imagem que foi adquiridos.</li>
	<li>Abra o painel de paleta de ferramentas (Figura 3).</li>
	<li>Selecione Ferramentas ROI. Recomendamos o círculo (o um mais à esquerda) para variar a área bioluminescente em imagens (Figura 4).</li>
	<li>Clique em "Medida ROIs" (ícone de lápis) para medir a intensidade de superfície bioluminescente (Figura 3). O painel de medições de ROI e os valores de quantificação aparecem (Figura 5).</li>
	<li>Use a medida de configurar no canto esquerdo do painel de medições de ROI para selecionar as valores/informações necessárias (Figura 6), caso contrário clique em "Exportar" para exportar esta tabela de dados e salvar como arquivo. csv (Figura 5).</li>
	<li>Use os valores da coluna "Fluxo Total (p/s)" como a quantificação de intensidade bioluminescentes no arquivo. csv.</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+green+fluorescence.">Improved green fluorescence.</a> Nature. 373 (6516), 663-664 (1995).</li><li>Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clearer+vision+for+in+vivo+imaging.">A clearer vision for in vivo imaging.</a> Nat Biotechnol. 19 (4), 316-317 (2001).</li><li>Frangioni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+near-infrared+fluorescence+imaging.">In vivo near-infrared fluorescence imaging.</a> Current Opinion in Chemical Biology. 7 (5), 626-634 (2003).</li><li>Collins, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrobacter+rodentium:+infection,+inflammation+and+the+microbiota.">Citrobacter rodentium: infection, inflammation and the microbiota.</a> Nat Rev Microbiol. 12 (9), 612-623 (2014).</li><li>Mallick, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+murine+infection+model+for+Shiga+toxin-producing+Escherichia+coli.">A novel murine infection model for Shiga toxin-producing Escherichia coli.</a> J Clin Invest. 122 (11), 4012-4024 (2012).</li><li>Petty, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Citrobacter+rodentium+genome+sequence+reveals+convergent+evolution+with+human+pathogenic+Escherichia+coli.">The Citrobacter rodentium genome sequence reveals convergent evolution with human pathogenic Escherichia coli.</a> J Bacteriol. 192 (2), 525-538 (2010).</li><li>Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson , G. T., Farris, M. A., Roop, R. M., Peterson, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+new+derivatives+of+the+broad-host-range+cloning+vector+pBBR1MCS,+carrying+different+antibiotic-resistance+cassettes.">Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.</a> Gene. 166 (1), 175-176 (1995).</li><li>Galen, J. E., Nair, J., Wang , J. Y., Wasserman, S. S., Tanner, M. K., Sztein , M. B., Levine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Plasmid+Maintenance+in+the+Attenuated+Live+Vector+Vaccine+Strain+Salmonella+typhiCVD+908-htrA.">Optimization of Plasmid Maintenance in the Attenuated Live Vector Vaccine Strain Salmonella typhiCVD 908-htrA.</a> Infect Immun. 67 (12), 6424-6433 (1999).</li><li>Francis, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+pneumococcal+infections+in+the+lungs+of+live+mice+using+bioluminescent+Streptococcus+pneumoniae+transformed+with+a+novel+gram-positive+lux+transposon.">Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.</a> Infect Immun. 69 (5), 3350-3358 (2001).</li><li>Goldwater, P. N., Bettelheim, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+enterohemorrhagic+Escherichia+coli+(EHEC)+infection+and+hemolytic+uremic+syndrome+(HUS).">Treatment of enterohemorrhagic Escherichia coli (EHEC) infection and hemolytic uremic syndrome (HUS).</a> BMC Med. 10, (2012).</li></ol>

Os autores não têm nada para divulgar.

Tem sido relatado que EHEC transformada com o plasmídeo luciferase tem sido utilizada para examinar sua localização em hosts ou expressão de gene em vivo8,11,12. O modelo murino demonstrado aqui também tem sido relatado para detectar o sincronismo de EHEC colonizado e localização no anfitrião murino8. Não obstante, nós fornecemos o protocolo de detalhe de como administrar a inoculação...

EHEC O157: H7 é um patógeno que causa diarreia1, HS2, HUS3e insuficiência renal aguda mesmo4 através de água ou alimentos contaminados. EHEC é um enterobacterium patogênico e coloniza para o trato gastrointestinal dos seres humanos1. Quando EHEC primeiro aderir ao epitélio intestinal do hospedeiro, eles injetam os fatores de colonização em células hospedeiras através do sistema de secreção de III do tipo (T3SS) que funciona como uma seringa molecular induzindo um anexar e apagar (A/E) lesão posteriormente para impor adesão (colonização)5. Estes genes envolvidos na formação da lesão de A/E são codificados pelo locus do enterócito (LEE) de apagamento de ilha de patogenicidade5.
Bioluminescência é uma reação química produzindo a luz, na qual luciferase catalisa sua Luciferina de substrato para gerar luz visível6. Este processo enzimático, muitas vezes, requer a presença de oxigênio ou trifosfato de adenosina (ATP)6. Bioluminescência (BLI) de imagem permite que os pesquisadores a visualização e a quantização das interações patógeno-hospedeiro em animais vivos7. BLI pode caracterizar o ciclo de infecção bacteriana em animais vivos, seguindo as bactérias bioluminescentes que migrar para e invadir tecidos diferentes7; Isto revela uma dinâmica progressão da infecção. Além disso, a carga bacteriana nos animais está relacionada com o sinal bioluminescentes8; assim, é um conveniente indicador para estimar as condições patológicas de animais experimentais em uma maneira simples e direta.
O plasmídeo utilizado aqui contidos o operon do luciferase, luxCDABE, que é a partir da bactéria Photorhabdus luminescens que codifica seu próprio substrato de luciferase7,9. Transformando este plasmídeo luciferase-expressando em bactérias, os processos de colonização e infecção podem ser monitorados, observando estas bactérias bioluminescentes em animais vivos. Em geral, BLI e rotulado de bioluminescência bactérias permitem aos investigadores monitorar os números bacterianos e localização, viabilidade bacteriana com antibióticos/terapia tratamento e expressão de gene bacteriano em infecção/colonização6, 7. numerosas bactérias patogênicas têm sido relatadas que expressam o operon luxCDABE para examinar a sua expressão de ciclo e/ou gene infecção na infecção. Estas bactérias, incluindo uropathogenic e. coli10, EHEC8,11,12,13, enteropatogênica Escherichia coli (EPEC)8, Citrobacter rodentium14,15, Salmonella typhimurium16, Listeria monocytogenes17, Yersinia enterocolitica18,19, e Vibrio cholerae20, foram documentados.
Vários modelos experimentais foram desenvolvidos para facilitar o estudo da EHEC colonização in vitro e in vivo21,22,23. No entanto, há uma falta de modelos animais apropriados para estudar o EHEC colonização na vivoe, portanto, uma escassez resultante de detalhes. Para facilitar o estudo da EHEC colonização mecanismo na vivo, que é valioso para construir modelos animais para observar e quantificar a colonização de EHEC em animais vivos em um método não-invasivo.
Este manuscrito descreve um modelo de colonização de rato-EHEC que usa um sistema expressando bioluminescente para monitorar a colonização de EHEC ao longo do tempo em hospedeiros vivos. Ratos intragastrically são inoculados com bioluminescência-rotulado EHEC e o sinal bioluminescente é detectado em ratos com um não-invasivo na vivo de imagem de sistema13. Camundongos infectados com bioluminescência-rotulado EHEC mostrou sinais bioluminescentes significativos em seu intestino, depois de 2 dias pós infecção, que sugeriu que essas bactérias colonizaram no intestino hospedeiro após 2 dias pós infecção. Dados de imagem ex vivo mostram que esta colonização é especificamente no ceco e cólon de ratos. Usando este modelo de rato-EHEC, a colonização de EHEC bioluminescente pode ser detectada no hospedeiro vivo por um na vivo de imagem de sistema, para estudar os mecanismos detalhados da colonização de bactérias entéricas, que pode promover uma compreensão mais em EHEC-induzido por alterações fisiológicas e patológicas.

<table><tbody><tr><td>Shaker incubator</td><td>YIH DER</td><td>LM-570R</td><td>bacteria incubation&amp;nbsp;</td></tr><tr><td>Orbital shaking incubator</td><td>FIRSTEK</td><td>S300</td><td>bacteria incubation&amp;nbsp;</td></tr><tr><td>pBSL180</td><td></td><td></td><td>source of nptII gene</td></tr><tr><td>pAKlux2</td><td></td><td></td><td>source of luxCDABE operon</td></tr><tr><td>T&amp;amp;A Cloning Kit</td><td>Yeastern Biotech</td><td>FYC001-20P</td><td>use for TA cloning&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Nsi&lt;/em&gt; I</td><td>NEB</td><td>R0127S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Sca&lt;/em&gt; I</td><td>NEB</td><td>R0122S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Spe&lt;/em&gt; I-HF</td><td>NEB</td><td>R0133S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Sma&lt;/em&gt; I&amp;nbsp;</td><td>NEB</td><td>R0141S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>T4 ligase</td><td>NEB</td><td>M0202S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>Ex Taq</td><td>TaKaRa</td><td>RR001A</td><td>use for PCR amplification</td></tr><tr><td>10X Ex Taq Buffer</td><td>TaKaRa</td><td>RR001A</td><td>use for PCR amplification</td></tr><tr><td>dNTP Mixture&amp;nbsp;</td><td>TaKaRa</td><td>RR001A</td><td>use for PCR amplification</td></tr><tr><td>PCR machine</td><td>applied Biosystem&amp;nbsp;</td><td>2720 thermal cycler&amp;nbsp;</td><td>&amp;nbsp;for PCR amplification</td></tr><tr><td>Glycerol</td><td>SIGMA</td><td>G5516-1L</td><td>use for bacteria stocking solution</td></tr><tr><td>NaCl</td><td>Sigma</td><td>31434-5KG-R</td><td>chemical for making LB medium, 10 g/L</td></tr><tr><td>Tryptone</td><td>CONDA pronadisa</td><td>Cat 1612.00</td><td>chemical for making LB medium, 10 g/L</td></tr><tr><td>Yeast Extract powder</td><td>Affymetrix</td><td>23547-1 KG</td><td>chemical for making LB medium, 5 g/L</td></tr><tr><td>Agar</td><td>CONDA pronadisa</td><td>Cat 1802.00</td><td>chemical for making LB agar</td></tr><tr><td>kanamycin&amp;nbsp;</td><td>Sigma</td><td>K4000-5G</td><td>antibiotics, use for seleciton</td></tr><tr><td>streptomycin&amp;nbsp;</td><td>Sigma</td><td>S6501-100G</td><td>antibiotics, eliminate the microbiota in mice</td></tr><tr><td>EDL933 competent cell</td><td>Homemade</td><td></td><td>method is on supplemental document&amp;nbsp;</td></tr><tr><td>Electroporator</td><td>MicroPulser</td><td></td><td>for electroporation</td></tr><tr><td>Electroporation Cuvettes</td><td>Gene Pulser/MicroPulser</td><td>1652086</td><td>for electroporation</td></tr><tr><td>High-speed centrifuge</td><td>Beckman Coulter</td><td>Avanti, J-26S XP</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>Fixed-Angle Rotor</td><td>Beckman Coulter</td><td>JA25.5</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>Fixed-Angle Rotor</td><td>Beckman Coulter</td><td>JLA10.5</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>centrifuge bottles</td><td>Beckman Coulter</td><td>REF357003</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>centrifuge bottles</td><td>Thermo Fisher scientific</td><td>3141-0500</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>eppendorf biophotometer plus&amp;nbsp;</td><td>eppendorf</td><td>AG 22331 hamburg</td><td>for measuring the OD600 value of bacteria</td></tr><tr><td>C57BL/6 mice&amp;nbsp;</td><td>Laboratory Animal Center of NCKU</td><td></td><td></td></tr><tr><td>lab coat, gloves</td><td></td><td></td><td>for personnel protection&amp;nbsp;</td></tr><tr><td>isoflurane&amp;nbsp;</td><td>Panion &amp;amp; BF Biotech Inc.</td><td>G-8669</td><td>for mice anesthesia, pharmaceutical grade</td></tr><tr><td>1ml syringe&amp;nbsp;</td><td></td><td></td><td>use for oral gavage of mice</td></tr><tr><td>Reusable 22 G ball-tipped feeding needle</td><td></td><td>&amp;phi;0.9 mm X L 50 mm</td><td>use for oral gavage of mice</td></tr><tr><td>surgical&amp;nbsp; scissors&amp;nbsp;</td><td></td><td></td><td>use for mice experiment</td></tr><tr><td>Xenogen IVIS 200 imaging system</td><td>Perkin Elmer</td><td>IVIS spectrum</td><td>use for bioluminescent image capture&amp;nbsp;</td></tr><tr><td>Living Image Software</td><td>Perkin Elmer</td><td>version 4.1</td><td>use for quantifying the image data</td></tr></tbody></table>

detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system

Enterohemorrhagic Escherichia coli (EHEC) O157: H7, que é um patógeno de origem alimentar que causesdiarrhea, (HS), a colite hemorrágica e Síndrome hemolítico-urêmica (HUS), colonizar para o trato intestinal de seres humanos. Para estudar o mecanismo detalhado da colonização de EHEC in vivo, é essencial ter modelos animais para monitorar e quantificar a colonização de EHEC. Demonstramos aqui um modelo de colonização de rato-EHEC, transformando o plasmídeo expressando bioluminescente de EHEC para monitorar e quantificar a colonização de EHEC em hospedeiros vivos. Animais inoculados com bioluminescência-rotulado EHEC mostram sinais bioluminescentes intensos em camundongos por detecção com um não-invasivo na vivo sistema de imagem. Após 1 a 2 dias pós infecção, sinais bioluminescentes poderiam ainda ser detectados em animais infectados, o que sugere que o EHEC colonizar em hospedeiros pelo menos 2 dias. Também demonstramos que estes EHEC bioluminescentes localizar de intestino de rato, especificamente no ceco e cólon, de imagens ex vivo . Este modelo de colonização de rato-EHEC pode servir como uma ferramenta para fazer avançar o conhecimento atual do mecanismo de colonização EHEC.

Nós administramos bioluminescência-rotulado EHEC (~ 109 células bacterianas) para 6 semana de idade camundongos C57BL/6 fêmeas, por gavagem oral. Após a inoculação oral de EHEC aos ratos dentro de 1h, os animais foram examinados pelo sinal bioluminescente, na vivo sistema de imagem como mostrado na Figura 7. Os resultados mostraram um forte sinal bioluminescente em camundongos gavage com bioluminescência-rotulado EHEC. Examinamos o...

Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Um protocolo detalhado de um modelo de mouse para enterohemorrhagic Escherichia coli (EHEC) colonização usando bactérias bioluminescência-etiquetado é apresentado. A detecção destas bactérias bioluminescentes por um não-invasivo na vivo sistema de animais vivos de imagem pode avançar nossa compreensão atual da colonização de EHEC.

Deteção de colonização Enterohemorrhagic Escherichia Coli murino Host pelo sistema não-invasivo na Vivo bioluminescência

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

detection-enterohemorrhagic-escherichia-coli-colonization-murine-host

Research

JoVE Journal

Immunology and Infection

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

9.4K visualizações.  National Cheng Kung University. Um protocolo detalhado de um modelo de mouse para enterohemorrhagic Escherichia coli (EHEC) colonização usando bactérias bioluminescência-etiquetado é apresentado. A detecção destas bactérias bioluminescentes por um não-invasivo na vivo sistema de animais vivos de imagem pode avançar nossa compreensão atual da colonização de EHEC.

Vídeo: Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Usando a luciferase Infecções bacterianas Imagem em camundongos

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Imunologia e Infecção

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

In vitro e in vivo de imagens Gene Reporter bioluminescência de células estaminais embrionárias humanas

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Biologia

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

In Situ Medição e correlação da densidade celular e emissão de luz de bactérias bioluminescentes

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Bioquímica

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Imagem bacteriana Bioluminescent<em&gt; Em Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

In Vivo Imaging Systems Detecção (IVIS) de um vírus de Neuro-invasiva encefalítico

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D Multimodalidade de imagens de<em&gt; Citrobacter rodentium</em&gt; Infecções em Ratos

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Monitoramento do crescimento do câncer de mama e formação de colônia metastática em camundongos usando bioluminescência

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Pesquisa do câncer

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combinado<em&gt; In vivo</em&gt; Optical e μCT imagem para monitorar infecções, inflamações, and Bone Anatomy em uma infecção Orthopaedic Implant em Ratos

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Medicina

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

Usando avançada proteína de fluorescência verde-expressando Escherichia Coli para avaliar a fagocitose de macrófagos peritoneais de Mouse

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

Acompanhamento longitudinal de infecções do trato urinário e seu tratamento em camundongos usando bioluminescência imagem

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

Deteção de colonização Enterohemorrhagic Escherichia Coli murino Host pelo sistema não-invasivo na Vivo bioluminescência

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