Este trabalho foi apoiado por subsídios da pesquisa nacional de chave e programa de desenvolvimento da China [2017YFD0500102]; a Fundação Nacional de ciências naturais da China [31572544]; o laboratório estadual chave de veterinária etiológico biologia [SKLVEB2016KFKT005]; a agricultura de Shanghai aplicada tecnologia desenvolvimento programa, China [G2016060201].

Os experimentos de infecção do mouse foram aprovados pelo laboratório Animal monitoramento Comité da província de Jiangsu, China e interpretados no laboratório Animal centro de Nanjing Agricultural University (de licença: SYXK (Su) 2017-0007).
1. preparação de bactérias
Nota: Cepa virulenta de S. suis sorotipo 2 P1/7 foi isolada de um porco doente com meningite19. Estirpe P1/7 foi cultivado em caldo Todd-Hewitt (THB, fórmula por litro de THB: infusão de coração, 3,1 g neopeptone, 20,0 g de dextrose, 2,0 g; cloreto de sódio, 2,0 g; fosfato dissódico, 0,4 g;, 2,5 g de carbonato de sódio) e chapeado em ágar de Todd-Hewitt (THA, fórmula por litro de THA: Infusão de coração, 3,1 g; neopeptone, 20,0 g; dextrose, 2,0 g; cloreto de sódio, 2,0 g; fosfato dissódico, 0,4 g; carbonato de sódio, 2,5 g; ágar-ágar, 15,0 g) a 37 ° C e 5% de CO2.
<ol><li>Coleção de estirpe células planctônicas P1/7<ol>
<li>Coletar 5 mL de células planctônicas de cultura fase log de mid (OD600= 0,6), centrifugar durante 3 min a 8000 × g e em seguida lavar 3 vezes com PBS.</li>
		<li>Ressuspender as células com 5 mL de glicerol de 25% no THB, alíquota para 5 tubos e em seguida, armazenar a-80 ° C.</li>
	</ol>
</li>
	<li>Coleção de estirpe P1/7 células de estado de biofilme<ol>
<li>Recolher 20 mL de uma cultura da noite e adicionar a 180 mL de THB fresco; dividir a cultura diluída em 10 placas de cultura redondo e em seguida, colocar as placas na incubadora a 37 ° C em 5% de CO2 por 24 h.</li>
		<li>Após a incubação, agitar a placa suavemente para Ressuspender as bactérias que não aderiram à placa e em seguida, descartar o sobrenadante por aspiração. Nota: Agitar a placa com cuidado e evite resuspending do sedimento.</li>
		<li>Adicione 5 mL de PBS para colher células-estado biofilme, Ressuspender o sedimento completamente e em seguida, transferir a amostra para um tubo novo.</li>
		<li>Proceda à sonicação células biofilme estado da última etapa com os seguintes parâmetros: 60 W, 4 ciclos, 5 s s no e 10 fora.</li>
		<li>Centrifugar por 3 min em 8000 × g e armazenar o sobrenadante a-80 ° C. Nota: O sobrenadante pode conter componentes de biofilme e pode ser usada para Ressuspender as bactérias do biofilme antes da infecção, conforme descrito nas experiências com animais (etapa 3).</li>
		<li>Ressuspender o sedimento com 10 mL 25% glicerol no THB e armazenar as células de estado de biofilme a-80 ° C.</li>
	</ol>
</li>
</ol>2. análise de microscopia eletrônica de varredura (MEV) de digitalização
<ol><li>Corrigir o biofilme coletadas células de estado e células planctônicas usando paraformaldeído 4% para 12-24 h a 4 ° C.</li>
	<li>Lavar as amostras rapidamente com água destilada e desidratar as amostras sequencialmente com uma crescente concentração de etanol (25%, 50%, 70%, 90% e 100%) por 30 min em cada solução.</li>
	<li>Aderir as amostras secas para titulares com fita dupla-face de metal e, finalmente, revesti-los em um evaporador com ouro e paládio.</li>
	<li>Observar as amostras por um microscópio eletrônico de varredura usando os seguintes parâmetros: EHT (Extra alta tensão) = 10 kV; WD (distância de trabalho) = 7.0 ou 8.0 mm; Ampliação = 5000 ×; A um sinal = SE1.</li>
</ol>3. animal experimentos
<ol><li>Usar o SPF 6 semanas camundongos BALB/c fêmeas neste estudo. Infectar todos os mouses através da rota intracraniana optou de infecção cujo local de injeção é localizada 3,5 mm rostral do bregma. As coordenadas estereotáxicos do local da injeção foram claramente descritas por Chiavolini et al.17.<ol>
<li>Para a análise histopatológica, dois grupos de ratos (5 ratos por grupo) de infectar células de estado usando células planctônicas ou biofilme na dose de 3 × 107 formadoras unidades (CFU), e outro 5 ratos foram injetados com PBS como um controle.</li>
		<li>Para a deteção de expressão de RNAm de TLR2 e citocinas no cérebro, infectar dois grupos adicionais de ratos (6 ratos por grupo) usando células planctônicas ou biofilme estado células na dose de 3 × 107 UFC.</li>
		<li>Determine o número de bactérias viáveis por chapeamento diluições em série para THA.</li>
		<li>Eutanásia em todos os ratos a pós-infecção de 12 h para futuras pesquisas. Nota: O glicerol em meio de estoque deve ser removido antes da infecção. Ambas as células planctônicas e células de estado de biofilme recuperadas de-80 ° C são lavadas 3 vezes com PBS. Em seguida, células planctônicas são resuspended com PBS e diluídas para a dose adequada para a infecção; biofilme estado células são resuspended com o sobrenadante armazenado (da etapa 1.2.5) e diluídas para a dose adequada para a infecção. O volume de infecção não deve ser mais de 50 µ l.</li>
	</ol>
</li>
	<li>Detectando a expressão de RNAm de TLR2 e citocinas no tecido cerebral<ol>
<li>Extrai o RNA total do tecido cerebral usando um kit de extração de RNA.<ol>
<li>Para cada rato, use tecido de cérebro inteiro para extração de RNA. Divida o tecido de cérebro inteiro em 5 tubos. Ocupam de tecido cerebral de 100mg e adicionar 1 mL de solução de Lise para cada tubo que contém a matriz lysing fornecida pelo kit.</li>
			<li>Processar o tubo em um homogeneizador para 40 s em uma configuração de 6.0 e em seguida centrifugar o tubo a 12000 × g e 4 ° C por 5 min.</li>
			<li>Após a centrifugação, transferi a fase superior para um novo tubo de microcentrifugadora.</li>
			<li>Incubar a amostra transferida durante 5 min à temperatura ambiente; Adicionar 300 µ l de clorofórmio, vórtice para 10 s e então incubar durante 5 min à temperatura ambiente.</li>
			<li>Centrifugar o tubo a 12000 × g e 4 ° C por 5 min. transferir a fase superior para um novo tubo.</li>
			<li>Adicionar 500 µ l de etanol absoluto frio para o tubo, antes invertendo por 5 vezes e em seguida armazenar a amostra a-20 ° C durante pelo menos 1 h.</li>
			<li>Centrifugar o tubo a 12000 × g e 4 ° C por 15 min e retirar o sobrenadante. Lave o pellet com 500 µ l de etanol frio de 75% em livre de RNase H2O.</li>
			<li>Remover o etanol, o ar seco a pelota durante 5 min à temperatura ambiente e ressuspender o RNA em 100 µ l de RNase-livre H2O.</li>
			<li>Incubar o RNA durante 5 min à temperatura ambiente e determinar a concentração de RNA usando um bioanalyzer RNA.</li>
		</ol>
</li>
		<li>Realize a síntese do cDNA, incluindo eliminação de DNA genômico (gDNA), usando um thermocycler com um kit de reagente de transcrição reversa (RT).<ol>
<li>Adicionar 1 µ g de RNA para um 2 µ l contendo de tubo de buffer de eliminação de x gDNA 5, 1 µ l de enzima de eliminação gDNA e livre de RNase H2O, até que o volume atinge 10 µ l. incube durante 2 min a 42 ° C.</li>
			<li>Adicionar 10 µ l de mistura de mestre (1 µ l da enzima RT Mix I, 1 µ l do Mix de Primer RT, 4 µ l de 5x Buffer 2 e 4 µ l de RNase-livre H2O) para a solução de reação da última etapa e, em seguida, misture delicadamente. Proceder imediatamente com a reação de RT: 37 ° C por 15 min e, em seguida, 85 ° C por 5 s.</li>
			<li>Realizar a análise quantitativa de PCR (RT-qPCR) em tempo real usando uma máquina de PCR em tempo real com um kit SYBR RT-qPCR. Adicione 10 µ l de 2 x enzima, 0,8 µ l de cartilha para a frente, 0,8 µ l de primer reverso, 0,4 µ l de 50 x ROX referência tintura II, 2 µ l de modelo do cDNA e livre de RNase H2O, até um volume total de 20 µ l.</li>
			<li>Executar cada amostra em triplicata, utilizando os seguintes parâmetros térmicos: 30 s a 94 ° C, seguida de 40 ciclos de 5 s a 95 ° C e 34 s a 60 ° C, com uma fase final de 15 s a 1 min a 60 ° C, 95 ° C e 15 s a 95 ° C. Primers para RT-qPCR estão listados na tabela 1. Genes de limpeza B2m e 18s rRNA foram usados como controles internos.</li>
		</ol>
</li>
		<li>Calcule a variação da dobra relativo baseada no método 2-ΔΔCt .</li>
	</ol>
</li>
	<li>
Observação de cortes histológicos
	<ol>
<li>Após a fixação de 24 h com formol a 10%, reconstrua o tecido cerebral fixa em tamanhos adequados de pedaços de tecido em 2-3 mm.</li>
		<li>Coloque os pedaços de tecido em uma gaveta de encastre e mergulhe em uma nova solução fixador para desidratação com um desidratador de vácuo automático.</li>
		<li>Retire o tecido fixo e desidratá-lo sequencialmente em concentrações crescentes de etanol (70%, 80%, 95%, 95% e 100%) por 30 min em cada solução.</li>
		<li>Transparentize a amostra com xilol durante 15 min, mergulhá-lo em parafina líquida a 60 ° C por 90 min e em seguida incorporar usando uma máquina de encastre.</li>
		<li>Corte o tecido de bloco incorporado de parafina em fatias de 3 µm, telha a fatia na água a 45 ° C e em seguida secá-lo no ar.</li>
		<li>Incubar a seção para 3h a 60 ° C, manchá-la usando a hematoxilina e eosina (HE) método e selá-lo com a goma neutra.</li>
		<li>Observe as alterações histopatológicas do cérebro com microscópio óptico. Um sistema de classificação de quatro pontos foi usado para avaliar as alterações histológicas. Utilize um teste pareado t para análise estatística. Congestão/hemorragia: ausente, Pontuação 0; pequena área de congestão/hemorragia focal, escore 1; grande área de congestão/hemorragia focal, ou vários locais de pequena área de congestão/hemorragia focal, pontuação 2; até três sites grandes congestionamentos focal/hemorragia, marcar 3; difusa de congestionamento/hemorragia, marcar 4. Necrose: ausente, Pontuação 0; necrose focal, escore 1; difundir a ocorrência de 1-10 células necróticas, pontuação 2; focos de necrose confluente, pontuação de 3; extensa necrose confluente, marcar 4. Infiltração de células inflamatórias: ausente, Pontuação 0; focais e algumas infiltrações, escore 1; infiltração multifocal ou infiltrações com formação de agregados, pontuação 2; até três agregados, marcar 3; e mais de quatro agregados, marcar 4.</li>
	</ol>
</li>
</ol>

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Os autores não têm nada para divulgar.

A rota intracraniana optou de infecção descrita aqui tem óbvias vantagens sobre outras vias de infecção. Ele permite que os investigadores a estudar a interação hospedeiro-bactéria e o efeito de componentes bacterianas em respostas imunes host diretamente no cérebro, que imitam a entrada de bactérias no sistema nervoso central. Assim, esta via de infecção pode ser estendida para investigar os mecanismos de meningite causada por outras bactérias. Além disso, ele também pode ser usado para testar a eficácia...

Streptococcus suis (S. suis) é um importante patógeno bacteriano de suínos no mundo, causando doenças graves, incluindo meningite, pneumonia, septicemia, endocardite e artrite1. É também um agente zoonótico emergente. Até agora, tem sido relatado que nove sorotipos podem causar infecção nos seres humanos, incluindo os sorotipos 2, 4, 5, 9, 14, 16, 21, 24 e 31,2,3,4. Em humanos e suínos, a meningite é um dos principais sinais clínicos de S. suis infecções. No Vietnã e Tailândia, o S. suis é a principal causa de meningite em adultos5. Biofilmes microbianos são microorganismos que aderem-se uns aos outros e se concentram em uma interface; Eles são essenciais para a virulência bacteriana, sobrevivência em diversos ambientes e a resistência aos antibióticos5. Biofilmes são tipicamente rodeados por uma matriz extracelular que geralmente contém polissacarídeos, proteínas e DNA6. O último é capaz de eliciar respostas inflamatórias do hospedeiro e a produção de citocinas7. Formação de biofilme foi relatada para ser envolvido em meningite estreptocócica em estudos anteriores. Biofilmes contribuam para meningite Streptococcus agalactiae em um modelo de peixe tilápia e formação de biofilmes foi revelada dentro de tecidos do cérebro e ao redor meníngea superfícies na vivo através de de inoculação intra-abdominal8. Durante a meningite, Streptococcus pneumoniae está em um estado de biofilme e bactérias em um biofilme estado foram mais eficazes na indução de meningite em um rato infecção modelo9. Além disso, no nosso anterior estudo, o estado de biofilme associado S. suis em cérebro de rato contribui para a virulência bacteriana por sobrevivência análise10. No entanto, a evidência direta para envolvimento de biofilme em S. suis meningite requer mais investigação.
Foram desenvolvidos modelos animais de infecção de S. suis em ratos usando o intraperitoneal (i.p.)11, intranasal (óssea)12, intravenosa (i.v.)13e as intracisternal (i.c.) rotas de infecção14, 15 , 16. no entanto, os i.p., óssea e i.v. rotas de infecção não são adequadas para estudar as funções dos componentes de superfície S. suis em meningite diretamente no cérebro. Estes incluem matriz extracelular de biofilmes. Mas a inoculação de i.c. foi usada para infecção de S. suis , o site precisa de injeção não foi descrito os jornais. Em contraste, as coordenadas estereotáxicos do local da injeção para inoculação intracraniana optou claramente tem sido descrita em um anterior estudo17. Isto permitiu o fácil reconhecimento do ponto de inoculação e mais simplista protocolo experimental. Além disso, na rota de infecção intracraniana optou imita a entrada de bactérias no sistema nervoso central dos seios do face ou do ouvido médio,17e a relação entre o ouvido médio e a meningite causada por S. suis tem sido demonstrado por Madsen et al.18. Além disso, aplicando-se a rota intracraniana optou de infecção em camundongos, demonstrámos que o S. suis pequeno RNA rss04 contribui para meningite em nosso anterior de estudo10.
No presente estudo, na rota de infecção intracraniana optou foi usada em ratos para investigar os papéis de biofilmes em meningite S. suis . Camundongos foram infectados com células planctônicas ou biofilme estado de S. suis por esta via de infecção. A análise histopatológica e aumento da expressão do mRNA de TLR2 e citocinas do tecido de cérebro de ratos injetados com células de biofilme estado claramente indicaram que o S. suis biofilme contribui para a meningite.

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="827">
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	<tbody>
		<tr>
			<td height="46">Todd Hewitt Broth(THB)</td>
			<td style="width:225px;">Becton, Dickinson and Company</td>
			<td style="width:115px;">DF0492078</td>
			<td style="width:304px;">Dissolve 30 g of the powder in 1 L of purified water. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td style="width:225px;">DSBIO</td>
			<td style="width:115px;">16C0050</td>
			<td style="width:304px;">Dissolve 15 g of the powder in 1 L of THB. Autoclave at 121&#176; for 15 min.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:184px;">Milli-Q Reference Water Purification System</td>
			<td style="width:225px;">Merck KGaA</td>
			<td style="width:115px;">Z00QSVCUS</td>
			<td style="width:304px;">Without Dnase/ Rnase</td>
		</tr>
		<tr height="21">
			<td>NaCl</td>
			<td style="width:225px;">Tianjin Kemiou Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10019318</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>Na2HPO3</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009012-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="21">
			<td>KCl</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009017-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="20">
			<td>KH2PO4</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009019-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="24">
			<td>KOH</td>
			<td style="width:225px;">Xilong Scientific Co., Ltd</td>
			<td style="width:115px;">9009014-01-09</td>
			<td style="width:304px;">Dissolve 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO3 , 0.27 g KH2PO4 in 1 L of purified water. Autoclave at 121&#176; for 15 min. Use KOH to adjust pH to 7.4.</td>
		</tr>
		<tr height="42">
			<td>Glycerol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10010618</td>
			<td style="width:304px;">Diluted with equal volumu of purified water, autoclave at 121&#176; for 15 min</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">4% paraformaldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">80096675</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">25% Glutaraldehyde</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">30092436</td>
			<td style="width:304px;">10-fold diluted with purified water for fixation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ethanol</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10009218</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td style="width:225px;">Sionpharm Chemical Reagent Co., Ltd</td>
			<td style="width:115px;">10006818</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spctrophotometre</td>
			<td style="width:225px;">DeNovix Inc.</td>
			<td style="width:115px;">DS-11+</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Ultrasound cell crusher</td>
			<td style="width:225px;">NingBo Scientz Biotechnology Co.,Ltd</td>
			<td style="width:115px;">JY96-IIN</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Centrifuge</td>
			<td style="width:225px;">Hitachi Koki Co., Ltd</td>
			<td style="width:115px;">CT15RE</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Refrigerator</td>
			<td style="width:225px;">Aucma Co., Ltd</td>
			<td style="width:115px;">DW-86L500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Scanning electron microscope</td>
			<td>Zeiss</td>
			<td style="width:115px;">EVO-LS10</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastRNA Pro Green Kit</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">#6045-050</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPrep-24 Instrument</td>
			<td>MP Biomedicals</td>
			<td style="width:115px;">116005500</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Instrument for PCR</td>
			<td style="width:225px;">SensoQuest GmbH</td>
			<td style="width:115px;">1124310110</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantStudio 6 Flex</td>
			<td style="width:225px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">4485689</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">SYBR Premix Ex Taq II</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR820A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">PrimeScript RT reagent kit with gDNA Eraser</td>
			<td style="width:225px;">Takara Biomedical Technology (Beijing) Co., Ltd</td>
			<td style="width:115px;">RR047A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Fully Enclosed Tissue Processor</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ASP200S</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Heated Paraffin Embedding Module</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">EG1150H</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Semi-Automated Rotary Microtome</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">RM2245</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Water bath for paraffin sections</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">HI1210</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:184px;">Autostainer XL</td>
			<td style="width:225px;">Leica Biosystems Nussloch GmbH</td>
			<td style="width:115px;">ST5010</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Agilent 2100</td>
			<td style="width:225px;">Agilent Technologies</td>
			<td style="width:115px;">G2939A</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:184px;">Optical microscope</td>
			<td style="width:225px;">Olympus</td>
			<td style="width:115px;">BX51</td>
			<td style="width:304px;"></td>
		</tr>
	</tbody>
</table>

intracranial subarachnoidal route of infection for investigating roles of streptococcus suis biofilms in meningitis in a mouse infection model

Streptococcus suis é não só um importante patógeno bacteriano de suínos no mundo, mas também um agente zoonótico emergente. Em humanos e suínos, a meningite é uma grande manifestação de S. suis infecções. Um modelo apropriado de infecção é uma ferramenta essencial para compreender os mecanismos das doenças causadas por agentes patogénicos. Várias rotas de infecção em camundongos foram desenvolvidas para estudar a patogênese da infecção por S. suis . No entanto, as rotas intraperitoneal, intranasais e intravenosa de infecção não são adequadas para estudar as funções dos componentes de superfície S. suis em meningite diretamente no cérebro, tais como a matriz extracelular de biofilmes. Embora intracisternal inoculação tem sido usada para infecção de S. suis , o site precisa de injeção não foi descrito. Aqui, na rota de infecção intracraniana optou foi descrita em um modelo do rato para investigar os papéis de biofilmes em S. suis meningite. S. suis planctônicos nas células ou biofilme estado foram diretamente injetado no espaço subaracnoide dos ratos através do site de injeção localizado 3,5 mm rostral do bregma. A análise histopatológica e aumento da expressão do mRNA de TLR2 e citocinas do tecido cerebral dos ratos injetados com células de biofilme estado claramente indicaram que o S. suis biofilme tem um papel definitivo na meningite S. suis . Esta rota de infecção tem óbvias vantagens sobre outras vias de infecção, permitindo o estudo da interação hospedeiro-bactéria. Além disso, ele permite que o efeito de componentes bacterianas no anfitrião respostas imunes diretamente no cérebro para ser avaliado e imita a entrada de bactérias no sistema nervoso central. Esta via de infecção pode ser estendida para investigar os mecanismos de meningite causada por outras bactérias. Além disso, ele também pode ser usado para testar a eficácia das drogas contra meningite bacteriana.

SEM análise foi realizada para examinar a formação de biofilmes nas condições experimentais. Como mostrado na Figura 1, há uma diferença significativa na formação de biofilme entre células planctônicas (Figura 1A) e células de estado de biofilme (Figura 1B). SEM a análise mostrou que as bactérias do biofilme foram aos montes e múltiplas camadas e eles ...

Watch this Scientific Journal Video about Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model at JoVE.com

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Aqui, descrevemos a rota intracraniana optou de infecção em camundongos para estudar funções de biofilmes de Streptococcus suis meningite. Este modelo de infecção também é adequado para estudar a patogênese das outra meningite bacteriana e a eficácia de novas drogas contra meningite bacteriana.

Intracraniana optou rota de infecção para investigar papéis de biofilmes de Streptococcus suis em meningite em um modelo de infecção do rato

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

intracranial-subarachnoidal-route-infection-for-investigating-roles

Research

JoVE Journal

Immunology and Infection

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

7.2K visualizações.  Nanjing Agricultural University. Aqui, descrevemos a rota intracraniana optou de infecção em camundongos para estudar funções de biofilmes de Streptococcus suis meningite. Este modelo de infecção também é adequado para estudar a patogênese das outra meningite bacteriana e a eficácia de novas drogas contra meningite bacteriana.

Vídeo: Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Inferência genotípica do HIV-1 Tropismo Usando de base populacional de Seqüenciamento V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Imunologia e Infecção

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

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

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Análise de uma única célula de Bacillus subtilis Os biofilmes utilizando microscopia de fluorescência e citometria de fluxo

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extração de proteínas e 2-D métodos Eletroforese em Gel de<em&gt; Burkholderia</em&gt; Espécies

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Acompanhamento da evolução das Membrane Polaridade, integridade de membrana e intracelulares Ion concentrações no<em&gt; Streptococcus pneumoniae</em&gt; Usando corantes fluorescentes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsular sorotipagem de<em&gt; Streptococcus pneumoniae</em&gt; Por aglutinação em látex

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagem não invasivo do imunitário inatas Response em um modelo de peixe-zebra larval de<em&gt; Streptococcus iniae</em&gt; Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Estudos do Vírus Influenza A em um Modelo de Infecção de Camundongos

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

Construindo Mutantes na Cepa 519/43 do Streptococcus pneumoniae do Serotipo 1

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...