Somos gratos pelo apoio financeiro da Fundação de Ciências Naturais da Província de Shaanxi (número de concessão 2021SF-118) e da Fundação Nacional de Ciências Naturais da China (números de concessão 81973409, 82204631).

Os animais experimentais, camundongos BALB/c machos (12-16 g), foram adquiridos no Laboratory Animal Center, Xi'an Jiaotong University Health Science Center. Todos os procedimentos foram aprovados pelo Comitê Institucional de Ética Animal da Universidade Xi'an Jiaotong com o número de licença SCXK (Shaanxi) 2021-103.
1. Preparação do tampão e do equipamento
<ol><li>Transfect cepas de C. albicans com plasmídeo de alta expressão pCaExp.<ol><li>Compre C. albicans (SC5314) da ATCC. Obter a cepa fluorescente de alta expressãoEGFP 22 transfectando C. albicans com um plasmídeo de alta expressão pCaExp contendo o quadro completo de leitura aberta do gene EGFP (o mapa do plasmídeo é mostrado na Figura 1) e usá-lo para experimentos subsequentes.</li>
	</ol></li>
	<li>Cultura de cepas transfectadas de C. albicans .<ol><li>Selecionar colônias monoclonais de cepa fluorescente de C. albicans a partir da placa de meio peptona dextrose (YPD) do extrato de levedura e cultura durante a noite (30 °C e 220 rpm) em 5 mL de meio líquido YPD (YPD + 50 μg/mL carbenicilina).</li>
		<li>Ressuspender C . albicans em solução salina normal após centrifugação a 400 x g por 5 min no TR.</li>
		<li>Ajustar a concentração da suspensão de C. albicans para 1 x 108 células/mL comparando a turbidez ao padrão de McFarland de 0,5.</li>
	</ol></li>
	<li>Preparar os instrumentos cirúrgicos.<ol><li>Certifique-se de autoclavar todos os instrumentos cirúrgicos (tesoura, pinça, pinça hemostática, porta-agulhas, agulhas de sutura) a 121°C por 30 min. São utilizados cateteres estéreis de polietileno (diâmetro interno: 0,28 mm; diâmetro externo: 0,63 mm). OBS: Os cateteres utilizados neste estudo foram esterilizados com gás óxido de etileno e a embalagem foi aberta em mesa ultralimpa exposta aos raios UV por mais de 30 min. Antes do implante em camundongos, os cateteres foram imersos em etanol 75% para evitar contaminação.</li>
	</ol></li>
</ol>2. Estabelecimento de um modelo de CRI de rato
OBS: O procedimento cirúrgico é mostrado na Figura 2.
<ol><li>Aclimatar 30 camundongos BALB/c (12-16 g, machos) em condições específicas livres de patógenos (FPS) com livre acesso a água e alimento e 12 h-12 h alternando ciclo claro e escuro.</li>
	<li>Divida aleatoriamente 30 camundongos BALB/c em três grupos (n = 10 camundongos/grupo): (A) grupo controle normal; (B) grupo cateter (cateteres implantados sem C. albicans); (C) Grupo modelo (cateteres implantados com C. albicans).</li>
	<li>Anestesiar os camundongos com isoflurano a 1-4% e colocá-los em uma mesa cirúrgica em decúbito ventral. A perda do reflexo de retificação e a ausência de resposta à estimulação dos pododáctilos confirmam o sucesso da anestesia. Remover os cabelos e esterilizar o sítio cirúrgico com três rodadas alternadas de iodóforo ou clorexidina e esfoliação com álcool.</li>
	<li>Deixe os ratos no grupo de controle normal sem qualquer tratamento e forneça acesso livre a comida e água.</li>
	<li>Para os camundongos dos grupos cateter e modelo, manter a anestesia com isoflurano a 3%. Confirmar a profundidade anestésica adequada pela ausência de resposta ao pinçamento dos dedos e ajustar a concentração de isoflurano conforme necessário.</li>
	<li>Para os camundongos do grupo cateter, insira por via intradérmica uma agulha de seringa estéril de 1 mL na área depilada de trás para fazer um furo. Insira um cateter (cerca de 1 cm de comprimento) no orifício depois de remover a agulha da seringa.</li>
	<li>Para os camundongos do grupo modelo, pipetar 20 μL de suspensão de C. albicans na área de retrodepilação para simular o comensal de C. albicans na pele.</li>
	<li>Após a absorção da solução pela pele, inserir um cateter na área retrodepilada com os mesmos procedimentos descritos no passo 2.5.</li>
	<li>Pipetar mais 20 μL de suspensão de C. albicans ao longo do cateter para o tecido para simular C. albicans no meio externo.</li>
	<li>Fixar os cateteres com fita adesiva e gaze e retornar os camundongos às gaiolas para alimentação. Ao final do tratamento, injetar meloxicam (4 mg/kg) por via subcutânea como analgesia por três dias consecutivos. NOTA: Após a cirurgia, os camundongos foram cuidadosamente alimentados com água e comida. Os camundongos foram monitorados duas vezes ao dia. Os camundongos foram eutanasiados por um método aprovado pela IACUC se apresentassem dificuldades alimentares, perda de peso significativa (10-20%) e hipotermia.</li>
</ol>3. Avaliação do modelo CRI
<ol><li>Após 3 dias, anestesiar os camundongos com isoflurano a 3% e sacrificá-los por deslocamento cervical. Coletar os cateteres e amostras de tecido cutâneo da parte de trás dos camundongos.</li>
	<li>Observar C. albicans e biofilmes no cateter por microscopia eletrônica de varredura.<ol><li>Imergir os cateteres em solução de glutaraldeído a 2,5% a 4 °C por 48 horas. Enxaguar os cateteres com PBS estéril três vezes.</li>
		<li>Fixar os cateteres com ácido ósmico a 1% por 3 h e enxaguar com PBS estéril três vezes.</li>
		<li>Desidratar as células dos cateteres em solução de etanol gradiente com concentrações crescentes (50%, 70%, 80%, 90% e 100%, 15 min/gradiente).</li>
		<li>Imergir os cateteres em álcool terc-butílico três vezes (30 min cada vez).</li>
		<li>Congele rapidamente os cateteres em nitrogênio líquido e liofilize a amostra em um liofilizador de acordo com as instruções do fabricante.</li>
		<li>Revestir as amostras do cateter com ouro de 10 nm por deposição de feixe de íons.</li>
		<li>Observar a presença de C. albicans e seu biofilme na superfície do cateter de cada grupo em microscópio eletrônico de varredura (em condições de alto vácuo, 1,5 kV) e registrar as imagens em cada grupo.</li>
	</ol></li>
	<li>Observar C. albicans no cateter por microscopia de fluorescência.<ol><li>Imergir os cateteres em solução de paraformaldeído a 4% para fixação a 4 °C por 48 h.</li>
		<li>Observar a presença de C. albicans e seu biofilme na superfície do cateter de cada grupo por meio de um microscópio de fluorescência sob excitação de 484 nm e registrar as imagens em cada grupo. NOTA: A ampliação é de 400x. A fluorescência de Candida albicans pode ser observada com excitação a 490 nm e emissão a 510 nm.</li>
	</ol></li>
	<li>Observar C. albicans residindo na pele de camundongos.<ol><li>Imergir os tecidos cutâneos dorsais de camundongos em solução de paraformaldeído a 4% para fixação a 4 °C por 48 h.</li>
		<li>Desidratar os tecidos dorsais da pele em solução de etanol gradiente com concentrações crescentes (50%, 70%, 80%, 90% e 100%, 15 min/gradiente).</li>
		<li>Incorporar os tecidos cutâneos dorsais desidratados em parafina a 55-60 °C. Preste atenção à temperatura para evitar tecidos quebradiços. Para remover o maior número possível de impurezas, repita este passo três vezes (30 min cada).</li>
		<li>Seccionar os tecidos dorsais da pele (espessura = 5 μm) com micrótomo.</li>
		<li>Desencenar as seções de parafina mergulhando as lâminas em xileno duas vezes por 20 min.</li>
		<li>Reidratar os trechos por eluição com etanol gradiente (etanol absoluto, etanol 90%, etanol 75%, água) por 5 min de cada vez.</li>
		<li>Manchar as secções com ácido periódico imergindo a secção na solução de ácido periódico durante 15 minutos antes de lavar com água corrente uma vez e água destilada duas vezes.</li>
		<li>Manchar as secções com uma solução corante de divisas (de acordo com as instruções do fabricante) durante 30 minutos no escuro e enxaguar as secções em água corrente durante 5 minutos.</li>
		<li>Imergir os cortes em solução de hematoxilina por 3-5 min antes de lavar com água corrente (2-3 min), solução diferenciada (5-10 min) e água corrente, respectivamente.</li>
		<li>Imergir as seções em etanol três vezes (5 min cada) e xileno duas vezes (5 min cada) antes de selar a seção com goma neutra.</li>
		<li>Observe as imagens do espécime com um microscópio e analise os resíduos de C. albicans na pele de camundongos. OBS: A ampliação é de 10x para a ocular e 4x ou 10x para a objetiva.</li>
	</ol></li>
	<li>Observar as alterações histopatológicas nos tecidos cutâneos dorsais.<ol><li>Imergir os tecidos cutâneos dorsais em solução de paraformaldeído a 4% para fixação a 4 °C durante 48 horas. Desidratar os tecidos dorsais da pele em solução de etanol gradiente com concentrações crescentes (50%, 70%, 80%, 90% e 100%, 15 min/gradiente).</li>
		<li>Incorporar os tecidos cutâneos dorsais desidratados em parafina, conforme descrito no passo 3.4.3.</li>
		<li>Seccionar os tecidos dorsais da pele (espessura = 5 mm) com micrótomo.</li>
		<li>Desencenar as seções de parafina mergulhando as lâminas em xileno duas vezes por 20 min.</li>
		<li>Reidratar os trechos por eluição com etanol gradiente (etanol absoluto, etanol 90%, etanol 75%, água) por 5 min de cada vez.</li>
		<li>Manchar os cortes com hematoxilina por 4 min antes de enxaguar com água da torneira para remover a cor da boia.</li>
		<li>Diferenciar o espécime com solução de etanol de ácido clorídrico a 1% antes de enxaguar as lâminas com água corrente.</li>
		<li>Imergir o espécime em etanol 85% e 95% por 5 min e corá-los com solução de eosina por 3 min.</li>
		<li>Desidratar o espécime mergulhando-o em etanol gradiente (70%, 90%, 95% e 100%) e xileno por 2 min cada.</li>
		<li>Selar o espécime com resina neutra.</li>
		<li>Observe as imagens do espécime com um microscópio e analise as alterações patológicas.</li>
	</ol></li>
</ol>

Estabelecendo um modelo de camundongo para infecção relacionada a cateter associada a Candida albicans

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenging+diagnostics+of+biofilm+associated+periprosthetic+infection+in+immunocompromised+patient:+A+clinical+case.">Challenging diagnostics of biofilm associated periprosthetic infection in immunocompromised patient: A clinical case.</a> Open Access Macedonian Journal of Medical Sciences. 7 (5), 786-790 (2019).</li><li>Cai, Y., 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=Metagenomic+next+generation+sequencing+improves+diagnosis+of+prosthetic+joint+infection+by+detecting+the+presence+of+bacteria+in+periprosthetic+tissues.">Metagenomic next generation sequencing improves diagnosis of prosthetic joint infection by detecting the presence of bacteria in periprosthetic tissues.</a> International Journal of Infectious Diseases. 96, 573-578 (2020).</li><li>Samanipour, A., Dashti-Khavidaki, S., Abbasi, M. R., Abdollahi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+patterns+of+microorganisms+isolated+from+nephrology+and+kidney+transplant+wards+of+a+referral+academic+hospital.">Antibiotic resistance patterns of microorganisms isolated from nephrology and kidney transplant wards of a referral academic hospital.</a> Journal of Research in Pharmacy Practice. 5 (1), 43-51 (2016).</li><li>Huang, G., Huang, Q., Wei, Y., Wang, Y., Du, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+and+diverse+regulation+of+the+Ras/cAMP/protein+kinase+A+pathway+in+Candida+albicans.">Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans.</a> Molecular Microbiology. 111 (1), 6-16 (2019).</li><li>Garlito-D&#237;az, H., 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+new+antifungal-loaded+sol-gel+can+prevent+candida+albicans+prosthetic+joint+infection.">A new antifungal-loaded sol-gel can prevent candida albicans prosthetic joint infection.</a> Antibiotics (Basel). 10 (6), 711 (2021).</li></ol>

Os autores declaram não ter interesses financeiros ou relações pessoais concorrentes conhecidos que possam ter influenciado o trabalho relatado neste artigo.

A IRC é uma das infecções hospitalares mais comuns na práticaclínica23. Patógenos nos anexos cutâneos, como epiderme, glândulas sebáceas e folículos pilosos, são possíveis causas de IRC23,24. A Candida é o terceiro maior patógeno causador da IRC, sendo a Candida albicans o tipo mais comum de infecção por biofilme25,26. Portanto, nosso objetivo foi ...

Nos últimos anos, com o desenvolvimento e aplicação de materiais biomédicos, infecções relacionadas a implantes vêm emergindo como problemas clínicos difíceis 1,2. Com a ampla aplicação de cateteres médicos em clínicas, o número de infecções e mortes relacionadas é enorme a cada ano 3,4. As vias de infecção comuns de uma infecção relacionada ao cateter (IRC) incluem: (1) patógenos na superfície da pele infiltram-se no corpo e aderem à superfície externa do cateter 5,6,7; (2) patógenos derivados de operações assépticas inadequadas invadem, aderem e colonizam o cateter; (3) patógenos na circulação sanguínea aderem e colonizam no cateter; (4) drogas contaminadas por microrganismos patogênicos.
A Candida é a terceira causa mais comum de IRC 8,9. É muito provável que cause infecção da corrente sanguínea e outras candidíase invasiva com risco de vida depois que biofilmes são formados na superfície do implante. O prognóstico é ruim e a taxa de mortalidade é alta2. Relata-se que biofilmes são formados na superfície do cateter dentro de 2 semanas após a inserção venosa central e no lúmen do cateter algumas semanas depois10,11.
Biofilmes de Candida albicans (C. albicans) formados em cateteres médicos exibem uma rede de dupla camada composta por levedura, estroma e micélio12,13. A formação de biofilmes de C. albicans não é apenas uma chave para a resistência a drogas e evasão imune13, mas também vital para a produção de esporos disseminados, o que leva a uma maior infecção hematogênica 2,12 e resulta em consequências mais graves e até fatais. A IRC associada a C. albicans é uma importante causade infecções fúngicas clínicas da corrente sanguínea7,14, e mais de 40% dos pacientes com infecção por C. albicans no cateter venoso central evoluirão para bacteremia15.
De acordo com a Infectious Disease Society of America, o tratamento recomendado da Candida CRI inclui (1) remoção do cateter infectado; (2) submeter os pacientes a uma terapia antifúngica sistêmica de 14 dias8; (3) reimplante de novo cateter4. No entanto, em aplicações clínicas, os cateteres não podem ser totalmente removidos às vezes. Alguns pacientes só podem ser tratados com antibióticos sistêmicos e terapia antimicrobiana de bloqueio, acompanhados de fortes efeitos colaterais16,17.
Os modelos animais existentes de C. albicans, como o modelo de candidíase orofaríngea, o modelo de candidíase vaginal e o modelo de infecção sistêmica invasiva causada por candidíase18,19, não podem se correlacionar bem com a IRC clínica. Portanto, neste estudo, um modelo de IRC associado a C. albicans em camundongos foi estabelecido. Cateteres de polietileno de uso clínico comum foram utilizados como implantes subcutâneos20,21, e C. albicans foi inoculado na superfície da pele para simular a adesão de C. albicans aos cateteres médicos e a formação de biofilmes.
Esse modelo tem sido utilizado com sucesso em nosso laboratório para rastrear o efeito anti-biofilme de diferentesterapêuticas22. Além disso, devido à detecção de defasagem de C. albicans após infecção por cateter, uma cepa de C. albicans contendo proteína fluorescente verde reforçada (EGFP) foi construída e inoculada em camundongos para facilitar a observação intuitiva das colônias e biofilmes de C. albicans no cateter implantado.

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			<td>MLS-3750</td>
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			<td>Tianjin Chemio Reagent Co., Ltd</td>
			<td>200-889-7</td>
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			<td>Carbenicillin</td>
			<td>Amresco</td>
			<td>C0885</td>
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			<td>Eclipse Ci Nikon upright optical microscope&#160;</td>
			<td>Nikon</td>
			<td>Eclipse Ts2-FL</td>
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			<td>Glucose</td>
			<td>Macklin&#160;</td>
			<td>D823520</td>
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			<td>Inoculation ring</td>
			<td>Thermo Scientific</td>
			<td>251586</td>
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			<td>Isoflurane</td>
			<td>RWD</td>
			<td>20210103</td>
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			<td>Paraformaldehyde</td>
			<td>Beyotime Biotechnology</td>
			<td>P0099</td>
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			<td>PAS dye kit</td>
			<td>Servicebio</td>
			<td>G1285</td>
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			<td>Peptone</td>
			<td>Beijing Aoboxing Bio-tech Co., Ltd</td>
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			<td>Polyethylene catheter</td>
			<td>Shining Plastic Mall</td>
			<td>PE100</td>
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			<td>RWD R550 multi-channel small animal anesthesia machine&#160;</td>
			<td>RWD</td>
			<td>R550</td>
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			<td>SEM</td>
			<td>Hitachi</td>
			<td>TM-1000</td>
			<td></td>
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			<td>Temperature incubator</td>
			<td>Shanghai Zhichu Instrument Co., Ltd</td>
			<td>ZQTY-50N</td>
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			<td>Ultrapure water water generator</td>
			<td>Heal Force</td>
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			<td>DS10260D</td>
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			<td>Xylene</td>
			<td>Sinopharm&#160; Chemical Reagent Co., Ltd</td>
			<td>10023428</td>
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			<td>Yeast extract</td>
			<td>Thermo Scientific&#160;Oxoid</td>
			<td>LP0021B</td>
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a catheter-related candida albicans infection model in mouse

A infecção relacionada ao cateter (IRC) é uma infecção nosocomial comum causada por candida albicans durante o implante do cateter. Tipicamente, biofilmes são formados na superfície externa do cateter e levam a infecções disseminadas, que são fatais para os pacientes. Não há prevenção e manejo de tratamento efetivos nas clínicas. Portanto, é urgente o estabelecimento de um modelo animal de IRC para a triagem pré-clínica de novas estratégias para sua prevenção e tratamento. Neste estudo, um cateter de polietileno, um cateter médico amplamente utilizado, foi inserido no dorso dos camundongos BALB/c após a depilação. Candida albicans ATCC MYA-2876 (SC5314) expressando proteína fluorescente verde reforçada foi subsequentemente inoculado na superfície da pele ao longo do cateter. Intensa fluorescência foi observada na superfície do cateter sob microscópio fluorescente 3 dias depois. Biofilmes maduros e espessos foram encontrados na superfície do cateter por microscopia eletrônica de varredura. Esses resultados indicaram a adesão, colonização e formação de biofilme de candida albicans na superfície do cateter. A hiperplasia da epiderme e a infiltração de células inflamatórias nos espécimes cutâneos indicaram as alterações histopatológicas da pele associada à IRC. Em resumo, um modelo de CRI de camundongo foi estabelecido com sucesso. Espera-se que esse modelo seja útil na pesquisa e desenvolvimento do manejo terapêutico da IRC associada a candida albicans .

A C. albicans e os biofilmes nos cateteres puderam ser observados pela MEV. Como mostrado na Figura 322, a superfície dos cateteres de polietileno do grupo de cateteres era lisa e nenhum microrganismo patogênico aderido foi observado. Entretanto, biofilmes maduros e densos de C. albicans foram visíveis na superfície dos cateteres de polietileno do grupo modelo, indicando que C. albicans foi capaz de colonizar e formar biofilmes na superf...

Watch this Scientific Journal Video about Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging at JoVE.com

Autor em destaque: Aprimorando a detecção de Candida albicans em infecções por cateter usando marcação de proteína fluorescente 

Estabelecemos um modelo murino de infecção relacionada ao cateter (IRC) associada a C.albicans, no qual o biofilme se forma no cateter, e a interação entre C.albicans e hospedeiro correlaciona-se bem com a IRC clínica. Esse modelo ajuda a selecionar terapias para IRC associada ao biofilme de C.albicans , estabelecendo uma base para a transformação clínica.

Modelo de Infecção por Candida albicans Relacionada a Cateter em Camundongos

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

Nossa pesquisa visa estabelecer um modelo murino de infecção associada ao cateter de Candida albicans, na qual se forma biofilme no cateter. Esse modelo é útil para correlacionar com a IRC clínica e a interação entre Candida albicans e hospedeiro. Os modelos animais existentes de Candida albicans, como o modelo de candidíase orofaríngea, o modelo de candidíase vaginal e o modelo de infecção sistêmica invasiva causada por candidíase, não podem se correlacionar bem com a IRC clínica.Para melhorar a detecção de Candida albicans em infecções por cateter, nosso protocolo empregou uma coloração com proteína fluoresceína verde reforçada. Isso permitiu uma visualização clara da colônia e biofilmes de Candida albicans em cateteres em camundongos e na detecção oportuna.

a-catheter-related-candida-albicans-infection-model-in-mouse

Research

JoVE Journal

Immunology and Infection

A Catheter-Related Candida albicans Infection Model in Mouse

991 visualizações.  Xi’an Jiaotong University. Nossa pesquisa visa estabelecer um modelo murino de infecção associada ao cateter de Candida albicans, na qual se forma biofilme no cateter. Esse modelo é útil para correlacionar com a IRC clínica e a interação entre Candida albicans e hospedeiro. Os modelos animais existentes de Candida albicans, como o modelo de candidíase orofaríngea, o modelo de candidíase vaginal e o modelo de infecção sistêmica invasiva causada por candidíase, não podem se correlacionar bem com a IRC clí...

Vídeo: Autor em destaque: Aprimorando a detecção de Candida albicans em infecções por cateter usando marcação de proteína fluorescente 

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

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

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

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

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

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

Imunologia e Infecção

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Estudar comunidades microbianas<em&gt; In Vivo</em&gt;: Um modelo de interação mediada por anfitrião entre<em&gt; Candida Albicans</em&gt; E<em&gt; Pseudomonas aeruginosa</em&gt; Nas Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Visualização da formação de biofilmes de Candida albicans , usando um dispositivo Microfluidic automatizada

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Profunda dérmica injeção como um modelo de Candida albicans de infecção da pele para análise histológica

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Diária fototerapia com luz vermelha para regular crescimento de biofilme de Candida albicans

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualização de candida albicans no trato gastrointestinal murina usando fluorescente in situ hibridização

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

Esclarecendo e Imagem Candida albicans Biofilmes

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Um ensaio ex vivo para estudar candida albicans Hórgênese Háfal no Trato Gastrointestinal

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Terapia fotodinâmica mediada por Rose Bengala para inibir a candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

Uso de imagens in vivo para rastrear fenótipos de morfogênese em cepas mutantes de Candida albicans durante a infecção ativa em um hospedeiro mamífero

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...