Ringraziamo Pr. Bernard La Scola per utili consigli tecnici e interessante discussione sulla co-coltura amoebal e di arricchimento amoebal. Ringraziamo anche il Dott. Vincent Thomas per il suo aiuto nella realizzazione tecnica nel nostro laboratorio.

1. Amoebal coculture 1.1 Preparazione del campione <ol><li> Campione ambientale <ol><li> I campioni di acqua Filtrare il campione d&#39;acqua (500 ml a 1 L) attraverso una membrana dimensione dei pori di 0,22 micron. Poi, scuotere la membrana in ameba saline medie PAS della pagina (120 mg di NaCl, 4 mg di MgSO4 • 7H 2 O, 4 mg di CaCl 2 • 2H 2 O, 142 mg di Na 2 HPO 4, e 136 mg di KH 2 PO 4 in 1 L di acqua distillata). </li><li> Campioni solidi Risospendere campioni solidi come campioni di terreno o sabbia e campioni semi-solidi come fanghi attivi in ​​acqua distillata o PBS e filtrare attraverso una membrana dimensione dei pori 0,22 micron. Poi, scuotere la membrana in PAS. </li><li> I campioni altamente contaminati da amebe endogena e protozoi Prima sedimentare i campioni mediante centrifugazione a bassa velocità (180 xg) for 10 min o filtrare attraverso una membrana dimensione dei pori 5 micron. Ulteriori elaborare il surnatante (rispettivamente filtrato), come in 1.1.1. Nota: Altre tecniche di decontaminazione potrebbero essere utilizzati per decontaminare ulteriori campioni ambientali: Riscaldare il campione a 50 ° C per 30 minuti o trattare con soluzioni acide o basiche 14. </li></ol></li><li> Campione clinico Elaborare campioni clinici seconda delle loro proprietà chimico-fisiche. Filtrare o centrifugare liquidi per rimuovere grosse impurità. Risospendere campioni solidi. Per utilizzare tessuti, macinare, ad esempio utilizzando un homogeneiser Dounce o perline di vetro, e lisare le cellule per liberare i batteri intracellulari. </li></ol> 1.2 Amebe preparazione <ol><li> Preparato per brodo Preparare i seguenti mezzi: un ricco supporto contenente peptone, estratto di lievito e glucosio (PYG, 100 g di peptone proteoso, 10 g di estratto di lievito, 4,9 g di MgSO4 • 7H 2O, 5 g di sodio citrato • 2H 2 O, 0,1 g di Fe (NH 4) 2 (SO 4) 2 • 6H 2 O, 1,7 g di KH 2 PO 4, 1,97 g di Na 2 HPO 4 • 7H 2 O , 45 g di glucosio, e 0,295 g di CaCl 2 a 5 L di acqua distillata) e un mezzo non nutritivo come PAS per specie Acanthamoeba. </li><li> Cultura amoebal <ol><li> Coltivare amebe (Acanthamoeba preferenzialmente castellanii ATCC 30010 o A. polyphaga Linc-Ap1) a 25 ° C in cella fiasche di coltura contenente 30 ml di PYG mezzo. </li><li> Raccogliere il amebe forte agitazione del pallone e centrifugare la sospensione cellulare per 10 minuti a 1500 x g. Lavare il pellet due volte con mezzo di PAS. Contare le cellule in una diapositiva Kova e regolare il volume per ottenere una sospensione di 5 x 10 5 cellule per ml. </li><li> Trasferire la sospensione amoebal in micropiastre. Utilizzare 1 ml per pozzetto per 12 - e 24-wpiastre ell, 500 microlitri per piastre 48 pozzetti e 300 microlitri per piastre da 96 pozzetti. Incubare la micropiastra per almeno 2 ore a 25 ° C. Questo permette sedimentazione e il fissaggio amebe al fondo di ciascun pozzetto. </li></ol></li></ol> 1.3 coculture <ol><li> Esempio di inoculazione <ol><li> Seminare la piastra da 2.3.3 con diluizioni seriali del campione da 1.1 o 1.2, di solito con serie di diluizioni di 10 volte, partendo con 100 ml di campione non diluito. </li><li> Centrifugare la micropiastra a 1.800 xg per 10 min a sedimenti sul prato amoebal microrganismi potenzialmente presenti nel campione. Questo aumenta il contatto e la fagocitosi dei microrganismi da amebe. </li><li> Incubare le piastre per 45 minuti a 25 ° C e lavate tre volte con PAS sostituendo il materiale con PAS fresco. Aggiungere 1 ml per pozzetto di PAS con o senza aggiunta di antibiotici (streptomicina, penicillina, gentamicina e / o vancomicina), a seconda delle batteriologicheAL specie che sono cercato. </li><li> Incubare la micropiastra a 32 ° C in atmosfera umidificata per evitare incistamento del amebe. Osservare ogni bene ogni giorno con un obiettivo 20X per rilevare la presenza di batteri invasori e lisi amebe. </li><li> Nel caso di lisi, eseguire una subcultura su amebe fresco inoculando 100 pl di co-colture di un monostrato di circa 10 5 amebe / cm 2. Per isolare in particolare una determinata specie batteriche, inoculare anche agar specifico progettato per questi batteri (cioè BCYE agar per Legionella spp.). </li><li> In assenza di lisi, prendere 100 ml di co-colture quattro a sette giorni dopo la prima inoculazione e inoculare una coltura fresca amoebal di 900 microlitri in una piastra da 24 pozzetti. Se rapida lisi delle amebe si osserva senza proliferazione di batteri, virus potrebbe essere presente. In questo caso, filtrare il supernatante a 0,22 micron e usare la sospensione filtrata per infettare amebe fresco. </li></ol&gt; </li></ol> 1.4 isolamento batterica e la caratterizzazione <ol><li> Colorazione batterica Eseguire co-colture direttamente su vetrini a 24 pozzetti. Rimuovere il supporto ed eseguire la colorazione o immunofluorescenza. <ol><li> Modificato colorazione Romanowsky <ol><li> Lasciare asciugare il vetrino. Immergere il vetrino cinque volte nella soluzione fissativo (2 mg / L veloce verde in metanolo). </li><li> Immergere il coprioggetto cinque volte nella soluzione colorante I (1,22 g / L Eosina G in tampone fosfato pH 6,6) </li><li> Infine, immergere il vetrino 5 volte nella soluzione colorante II (1.1 g / L tiazina in tampone fosfato pH 6,6). </li><li> Sciacquare il campione con acqua distillata. Lasciate asciugare e osservare al microscopio. </li></ol></li><li> Ziehl-Neelsen <ol><li> Lasciare asciugare il vetrino. Coprire il campione con Ziehl fucsina. Riscaldare il colorante con una fiamma fino all&#39;apparizione vapori. </li><li> Raffreddare a temperatura ambiente per almeno 5 mine risciacquare con acqua distillata. Coprire il campione con una soluzione di acido cloridrico al 3% in isopropanolo per 2 minuti e risciacquare con acqua distillata. </li><li> Copertina per 30 sec con blu di metilene e risciacquare con acqua distillata. Lasciate asciugare e osservare al microscopio 22. </li></ol></li><li> Gimenez colorazione <ol><li> Preparare una soluzione stock fucsina base miscelando 100 ml di 10% fucsina basica (10 g di fucsina basica in 100 ml di etanolo al 95%), 250 ml di 4% fenolo acquoso e 650 ml di acqua distillata. Incubare a 37 ° C per 48 h prima dell&#39;uso. </li><li> Lasciate asciugare il vetrino e fissare mediante passaggio su fiamma. Coprire il campione con fucsina basica appena filtrata (4 ml di fucsina magazzino soluzione di base in 10 ml di 0,1 M tampone fosfato di sodio, pH 7,45) per 2 minuti. </li><li> Risciacquare il campione con acqua e incubare in verde malachite (0,8% in acqua distillata) per 10 sec. Sciacquare nuovamente con acqua e ripetere la colorazione verde malachite. Sciacquare nuovamente con acqua. Lasciate che il vetrino asciutto, mount, e osservare al microscopio 23. </li></ol></li><li> Immunofluorescenza <ol><li> Fissare il vetrino incubando in metanolo per 5 min o con paraformaldeide al 4% per 10 min. </li><li> Lavare tre volte con PBS e incubare per 2 ore in una soluzione bloccante (5% BSA, 0.1% saponina in PBS) a temperatura ambiente. </li><li> Incubare il vetrino per 1 ora in una soluzione bloccante contenente anticorpi contro il microrganismo di interesse. </li><li> Lavare nuovamente per tre volte in PBS e incubare per 1 ora con un anticorpo secondario diretto contro l&#39;anticorpo primario e collegato a un fluoroforo. Lavare tre volte con PBS, montare il coprioggetto ed osservare al microscopio a fluorescenza. </li></ol></li></ol></li><li> Rilevazione del DNA mediante PCR Estrarre il DNA da 100 a 200 ml di co-coltura amoebal. Rileva microrganismi con primer universali rivolti al gene 16S rRNA o primer specifici per le specie di interesse come i micobatteri 24, Legionella 25 o membri del Chlamydiales 26. </li></ol> 2. Arricchimento amoebal 2.1. Preparazione del campione Risospendere campioni solidi e semi-solidi in PAS vortex. Centrifugare la sospensione a bassa velocità (180 xg) per 10 min. Questo permette di arricchimento di amebe a vita libera nel pellet. Il supernatante può essere utilizzato per coculture amoebal e il pellet di arricchimento amoebal 21. 2.2. Preparazione medio <ol><li> Aggiungere 1,5 g di agar per 100 ml di PAS e sterilizzare in autoclave il mezzo 15 min a 121 ° C. Versare il terreno caldo in piastre di Petri e lasciare solidificare a temperatura ambiente. </li><li> Grow Escherichia coli (ATCC 25922) in brodo LB o tioglicolato notte a 37 ° C. Lavare i batteri due volte con PBS e risospendere in mezzo PAS. Diluire 10x in PAS e diffondere 2-3 ml di questa diluizione su una piastra di PAS agar e lasciare asciugare. </li></ol> Aggiungere una goccia di campione (o un pezzo di filtro) su un lato della piastra e lasciare scorrere sulla piastra di Petri per formare una linea al centro del piatto. 2.4. La crescita amoebal e sottocultura amoebal <ol><li> Osservare la piastra di Petri quotidiano. Se viene rilevato un fronte di migrazione amoebal, tagliare un piccolo pezzo di agar al fronte di migrazione e inoculare un piatto fresco NNA Petri ricoperta da un prato di E. coli. </li><li> Ripetere la reinoculazione più volte a seconda della purezza del campione, in modo da avere una coltura pura di un dato ceppo amoebal. </li></ol> 2.5. Amebe e batteri caratterizzazione <ol><li> Raschiare le cellule e risospendere in PAS. </li><li> Estrarre il DNA e determinare l&#39;identità di amebe e / o endosimbionti batterici mediante PCR e sequenziamento (16S rRNA di amplificazione per i batteri, risp. 18S rRNA per la amebe e sequenziamento, per esempio). </li> &lt;li&gt; Utilizzare queste cellule raschiate in PAS per inoculare amebe fresco ed eseguire una co-coltura amoebal per rilevare i batteri eventualmente presenti nel campione. </li></ol>

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	<li>Fraser, D. 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=Legionnaires'+disease:+description+of+an+epidemic+of+pneumonia.">Legionnaires' disease: description of an epidemic of pneumonia.</a> New Engl. J. Med. 297, 1189-1197 (1977).</li><li>McDade, J. E., 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=Legionnaires'+disease:+isolation+of+a+bacterium+and+demonstration+of+its+role+in+other+respiratory+disease.">Legionnaires' disease: isolation of a bacterium and demonstration of its role in other respiratory disease.</a> New Engl. J. Med. 297, 1197-1203 (1977).</li><li>Rowbotham, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+report+on+the+pathogenicity+of+Legionella+pneumophila+for+freshwater+and+soil+amoebae.">Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae.</a> J. Clin. Pathol. 33, 1179-1183 (1980).</li><li>Rodriguez-Zaragoza, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+free-living+amoebae.">Ecology of free-living amoebae.</a> Crit. Rev. Microbiol. 20, 225-241 (1994).</li><li>Booton, G. C., Visvesvara, G. S., Byers, T. J., Kelly, D. J., Fuerst, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+distribution+of+Acanthamoeba+species+genotypes+associated+with+nonkeratitis+infections.">Identification and distribution of Acanthamoeba species genotypes associated with nonkeratitis infections.</a> J Clin. Microbiol. 43, 1689-1693 (2005).</li><li>Brussow, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+between+protists+and+phages:+from+antipredation+strategies+to+the+evolution+of+pathogenicity.">Bacteria between protists and phages: from antipredation strategies to the evolution of pathogenicity.</a> Molecular microbiology. 65, 583-589 (2007).</li><li>Greub, G., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microorganisms+resistant+to+free-living+amoebae.">Microorganisms resistant to free-living amoebae.</a> Clin. Microbiol. Rev. 17, 413-433 (2004).</li><li>Thomas, V., McDonnell, G., Denyer, S. P., Maillard, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-living+amoebae+and+their+intracellular+pathogenic+microorganisms:+risks+for+water+quality.">Free-living amoebae and their intracellular pathogenic microorganisms: risks for water quality.</a> FEMS Microbiol Rev. 34, 231-259 (2010).</li><li>Birtles, R. J., Rowbotham, T. J., Storey, C., Marrie, T. J., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydia-like+obligate+parasite+of+free-living+amoebae.">Chlamydia-like obligate parasite of free-living amoebae.</a> Lancet. 349, 925-926 (1997).</li><li>Amann, R., 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=Obligate+intracellular+bacterial+parasites+of+acanthamoebae+related+to+Chlamydia+spp.">Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp.</a> Appl. Environ. Microbiol. 63, 115-121 (1997).</li><li>Birtles, R. J., 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=Candidatus+Odyssella+thessalonicensis'+gen.+nov.,+sp.+nov.,+an+obligate+intracellular+parasite+of+Acanthamoeba+species.">Candidatus Odyssella thessalonicensis' gen. nov., sp. nov., an obligate intracellular parasite of Acanthamoeba species.</a> Int. J. Syst. Evol. Microbiol. 50, 63-72 (2000).</li><li>Thomas, V., Casson, N., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Criblamydia+sequanensis,+a+new+intracellular+Chlamydiales+isolated+from+Seine+river+water+using+amoebal+co-culture.">Criblamydia sequanensis, a new intracellular Chlamydiales isolated from Seine river water using amoebal co-culture.</a> Environ. Microbiol. 8, 2125-2135 (2006).</li><li>Pagnier, I., Raoult, D., La Scola, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+amoeba-resisting+bacteria+from+water+in+human+environment+by+using+an+Acanthamoeba+polyphaga+co-culture+procedure.">Isolation and identification of amoeba-resisting bacteria from water in human environment by using an Acanthamoeba polyphaga co-culture procedure.</a> Environ. Microbiol. 10, 1135-1144 (2008).</li><li>Thomas, V., Loret, J. F., Jousset, M., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiversity+of+amoebae+and+amoebae-resisting+bacteria+in+a+drinking+water+treatment+plant.">Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant.</a> Environ. Microbiol. 10, 2728-2745 (2008).</li><li>Corsaro, D., 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=Novel+Chlamydiales+strains+isolated+from+a+water+treatment+plant.">Novel Chlamydiales strains isolated from a water treatment plant.</a> Environ. Microbiol. 11, 188-200 (2009).</li><li>Goy, G., Croxatto, A., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waddlia+chondrophila+enters+and+multiplies+within+human+macrophages.">Waddlia chondrophila enters and multiplies within human macrophages.</a> Microbes Infect. 10, 556-562 (2008).</li><li>Kebbi-Beghdadi, C., Cisse, O., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permissivity+of+Vero+cells,+human+pneumocytes+and+human+endometrial+cells+to+Waddlia+chondrophila.">Permissivity of Vero cells, human pneumocytes and human endometrial cells to Waddlia chondrophila.</a> Microbes Infect. 13, 566-574 (2011).</li><li>Kebbi-Beghdadi, C., Batista, C., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permissivity+of+fish+cell+lines+to+three+Chlamydia-related+bacteria:+Waddlia+chondrophila,+Estrella+lausannensis+and+Parachlamydia+acanthamoebae.">Permissivity of fish cell lines to three Chlamydia-related bacteria: Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae.</a> FEMS Immunol. Med. Microbiol. 63, 339-345 (2011).</li><li>Fry, N. K., Rowbotham, T. J., Saunders, N. A., Embley, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+amplification+and+sequencing+of+the+16S+ribosomal+DNA+of+an+intracellular+Legionella+species+recovered+by+amoebal+enrichment+from+the+sputum+of+a+patient+with+pneumonia.">Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia.</a> FEMS Microbiol. Lett. 67, 165-168 (1991).</li><li>Rowbotham, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Legionella+pneumophila+serogroup+1+from+human+feces+with+use+of+amebic+cocultures.">Isolation of Legionella pneumophila serogroup 1 from human feces with use of amebic cocultures.</a> Clin. Infect. Dis. 26, 502-503 (1998).</li><li>Greub, G., La Scola, B., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoebae-resisting+bacteria+isolated+from+human+nasal+swabs+by+amoebal+coculture.">Amoebae-resisting bacteria isolated from human nasal swabs by amoebal coculture.</a> Emerging Infect. Dis. 10, 470-477 (2004).</li><li>Isenberg, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical microbiology procedures handbook. , (1992).</li><li>Gimenez, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+Rickettsiae+in+Yolk-Sac+Cultures.">Staining Rickettsiae in Yolk-Sac Cultures.</a> Stain Technol. 39, 135-140 (1964).</li><li>Thomas, V., Herrera-Rimann, K., Blanc, D. S., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiversity+of+amoebae+and+amoeba-resisting+bacteria+in+a+hospital+water+network.">Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network.</a> Appl. Environ. Microbiol. 72, 2428-2438 (2006).</li><li>Miyamoto, 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=Development+of+a+new+seminested+PCR+method+for+detection+of+Legionella+species+and+its+application+to+surveillance+of+legionellae+in+hospital+cooling+tower+water.">Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water.</a> Appl. Environ. Microbiol. 63, 2489-2494 (1997).</li><li>Lienard, J., 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=Development+of+a+new+chlamydiales-specific+real-time+PCR+and+its+application+to+respiratory+clinical+samples.">Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples.</a> J. Clin. Microbiol. 49, 2637-2642 (2011).</li><li>Wang, Y., Ogawa, M., Fukuda, K., Miyamoto, H., Taniguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+mycobacteria+from+soils+at+an+illegal+dumping+site+and+landfills+in+Japan.">Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan.</a> Microbiol. Immunol. 50, 513-524 (2006).</li><li>Corsaro, D., Pages, G. S., Catalan, V., Loret, J. F., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiversity+of+amoebae+and+amoeba-associated+bacteria+in+water+treatment+plants.">Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants.</a> Int. J. Hygiene Environ. Health. 213, 158-166 (2010).</li><li>La Scola, B., 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=Legionella+drancourtii+sp.+nov.,+a+strictly+intracellular+amoebal+pathogen.">Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen.</a> Int. J. Syst. Evol. Microbiol. 54, 699-703 (2004).</li><li>Thomas, V., Casson, N., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Afipia+and+Bosea+strains+isolated+from+various+water+sources+by+amoebal+co-culture.">New Afipia and Bosea strains isolated from various water sources by amoebal co-culture.</a> Syst. Appl. Microbiol. 30, 572-579 (2007).</li><li>La Scola, B., 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=Amoeba-resisting+bacteria+and+ventilator-associated+pneumonia.">Amoeba-resisting bacteria and ventilator-associated pneumonia.</a> Emerging Infect. Dis. 9, 815-821 (2003).</li><li>Collingro, A., 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=Recovery+of+an+environmental+Chlamydia+strain+from+activated+sludge+by+co-cultivation+with+Acanthamoeba+sp.">Recovery of an environmental Chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp.</a> Microbiology. 151, 301-309 (2005).</li><li>Lienard, J., Croxatto, A., Prod'hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrella+lausannensis,+a+new+star+in+the+Chlamydiales+order.">Estrella lausannensis, a new star in the Chlamydiales order.</a> Microbes Infect. 13, 1232-1241 (2011).</li><li>La Scola, B., 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+giant+virus+in+amoebae.">A giant virus in amoebae.</a> Science. 299, 2033 (2003).</li><li>Boyer, 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=Giant+Marseillevirus+highlights+the+role+of+amoebae+as+a+melting+pot+in+emergence+of+chimeric+microorganisms.">Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 21848-21853 (2009).</li><li>Thomas, V., 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=Lausannevirus,+a+giant+amoebal+virus+encoding+histone+doublets.">Lausannevirus, a giant amoebal virus encoding histone doublets.</a> Environ. Microbiol. 13, 1454-1466 (2011).</li><li>Raoult, D., Renesto, P., Brouqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+infection+of+a+technician+by+mimivirus.">Laboratory infection of a technician by mimivirus.</a> Ann. Internal Med. 144, 702-703 (2006).</li><li>Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parachlamydia+acanthamoebae,+an+emerging+agent+of+pneumonia.">Parachlamydia acanthamoebae, an emerging agent of pneumonia.</a> Clin. Microbiol. Infect. 15, 18-28 (2009).</li><li>Lamoth, F., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoebal+pathogens+as+emerging+causal+agents+of+pneumonia.">Amoebal pathogens as emerging causal agents of pneumonia.</a> FEMS Microbiol. Rev. 34, 260-280 (2010).</li><li>Lienard, J. G., Ashbolt, K., Sen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+6.">Ch. 6.</a> Environmental microbiology, current technology and water applications. , 143-162 (2011).</li><li>Boughalmi, 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=High-throughput+isolation+of+giant+viruses+of+the+Mimiviridae+and+Marseilleviridae+families+in+the+Tunisian+environment.">High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.</a> Environ. Microbiol. , (2012).</li><li>Ovrutsky, A. R., 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=Cooccurrence+of+Free-Living+Amoebae+and+Nontuberculous+Mycobacteria+in+Hospital+Water+Networks,+and+Preferential+Growth+of+Mycobacterium+avium+in+Acanthamoeba+lenticulata.">Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata.</a> Appl. Environ. Microbiol. 79, 3185-3192 (2013).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Coculture amoebal e l&#39;arricchimento amoebal sono metodi efficaci che hanno permesso l&#39;isolamento di molte nuove specie batteriche e amoebal. I risultati ottenuti con questi metodi confermano la presenza ubiquitaria di entrambi amebe e batteri-ameba resistere nell&#39;ambiente, e la più interessante delle reti idriche artificiali che sono considerati essere controllato da trattamenti chimici come la clorazione e ozonizzazione. Coculture amoebal e l&#39;arricchimento amoebal sono strumenti essenziali per isolare ...

Prima dell&#39;avvento di diagnosi molecolare, microrganismi presenti in nicchie ambientali o in campioni clinici sono stati spesso individuati da loro coltivazione su diversi terreni selettivi, soprattutto su agar in piastre di Petri. Il fenotipo delle colonie batteriche e la loro attività metabolica quindi lasciata classificazione batterica a livello di specie. Brodo può essere anche usato per aumentare la sensibilità del rilevamento. Tuttavia, entrambe le tecniche non consentono il recupero di batteri che crescono lentamente o per niente su questi terreni. Questo è il motivo per cui gli approcci molecolari sono così ampiamente utilizzati al giorno d&#39;oggi. Tuttavia, il rilevamento del DNA fornisce alcun indizio sulla vitalità dei batteri. Inoltre, contrariamente alla cultura, approcci molecolari non comportino un ceppo che può essere ulteriormente caratterizzato. Studiare gli agenti patogeni che crescono male su terreni solidi o che le cellule hanno bisogno per crescere è complicato. La maggior parte di questi &quot;difficile da coltivare&quot; i batteri sono intr esigentibatteri acellulare, spesso scoperti e caratterizzati seguente grandi epidemie come era il caso di Legionella pneumophila. Questo batterio è stato caratterizzato in seguito ad un&#39;epidemia che si è verificato nel corso di un convegno American Legion. Ben 182 persone sono state infettate e 29 morti a causa di una grave polmonite 1,2. E &#39;stato poi dimostrato che le amebe erano i padroni di casa naturali di questo batterio e che la loro presenza nelle reti albergo di sistema di aria condizionata e acqua è stata all&#39;origine dello scoppio della malattia, la cosiddetta del legionario 3. Amebe sono presenti in tutto il mondo e sono stati isolati dal suolo, l&#39;aria, l&#39;acqua e la mucosa nasale di volontari umani (recensiti 4). Queste amebe &quot;vita libera&quot; sono generalmente dividendo autonomamente nell&#39;ambiente, ma può occasionalmente invadere padroni di casa permissive 5. Mangimi amebe su vari microrganismi attraverso la fagocitosi e la successiva digestione lisosomiale da hydrolases 6. Molti batteri intracellulari facoltativi o di obbligare sono in grado di resistere digestione e infettare e dividere in amebe come per esempio batteri o micobatteri Legionella, Chlamydia correlati (recensito in 7 e 8) così. Amebe a vita libera probabilmente rappresentano un importante bacino potenziale di batteri intracellulari che non sono ancora stati scoperti. Ciò ha portato il nostro gruppo ad attuare a Losanna due tecniche principali, chiamati co-coltura amoebal e arricchimento amoebal, che ha permesso di isolare i diversi gruppi diversi nuovi microrganismi intracellulari obbligati da vari campioni ambientali 9-15. Poiché amebe sono fagociti professionali pascolo su batteri, un batterio in grado di resistere fagocitosi e di crescere all&#39;interno di questi protisti potrebbe anche colonizzare fagociti umani ed essere patogeni verso gli esseri umani. Questo è stato parzialmente dimostrata per alcuni batteri Chlamydia-correlati, come Waddlia chondrophila. W. chondrophila può crescere non solo in amebe ma anche in diversi tipi cellulari quali cellule di mammifero epiteliali, macrofagi e linee cellulari di pesce 16-18. La co-coltura amoebal appare rilevante anche per la rilevazione di batteri intracellulari in campioni clinici 19,20, compresi sgabelli che sono fortemente contaminati da diverse specie batteriche 21. Qui si descrivono le fasi principali di co-coltura amoebal e arricchimento amoebal, tra cui (a) il trattamento di campioni ambientali o cliniche; (b) la crescita di amebe su supporti axeniche e su un prato batterica di Escherichia coli e (c) la selezione e la caratterizzazione di batteri intracellulari.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>108342</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m pore size membrane</td>
			<td>Merck Millipore, Darmstadt, Germany</td>
			<td>SCVPU11RE</td>
			<td></td>
		</tr>
		<tr>
			<td>proteose peptone</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>Kova slide</td>
			<td>Hycor, Indianapolis, IN</td>
			<td>87144</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture microplates</td>
			<td>Corning Inc, Corning, NY</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>Diff-Quik staining kit</td>
			<td>Siemens Healthcare diagn., Munich, Germany</td>
			<td>130832</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziehl fuchsin</td>
			<td>Fluka, St-Louis, MI</td>
			<td>21820</td>
			<td></td>
		</tr>
		<tr>
			<td>basic fuchsin</td>
			<td>Sigma, St-Louis, MI</td>
			<td>857843</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma, St-Louis, MI</td>
			<td>P1037</td>
			<td>Corrosive and mutagenic</td>
		</tr>
		<tr>
			<td>malachite green oxalate</td>
			<td>Fluka, St-Louis, MI</td>
			<td>63160</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution</td>
			<td>Electron Microscopy Sciences, Hatfield, PA</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma, St-Louis, MI</td>
			<td>84510</td>
			<td></td>
		</tr>
	</tbody>
</table>

discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches

Patogeni intracellulari come la legionella, micobatteri e organismi Chlamydia simili sono difficili da isolare perché spesso crescono poco o per niente su terreni selettivi che di solito vengono utilizzati per coltivare i batteri. Per questo motivo, molti di questi agenti patogeni sono state scoperte solo recentemente o seguente focolai importanti. Questi agenti patogeni sono spesso associati con amebe, che servono come cellula ospite e consentire la sopravvivenza e la crescita dei batteri. Intendiamo qui per fornire una dimostrazione di due tecniche che permettono l&#39;isolamento e la caratterizzazione dei patogeni intracellulari presenti in campioni clinici e ambientali: la co-coltura amoebal e l&#39;arricchimento amoebal. Coculture amoebal permette il recupero di batteri intracellulari inoculando campione indagato su un prato amoebal che può essere infettato e lisate dai batteri intracellulari presenti nel campione. Arricchimento amoebal permette il recupero di amebe presente in un campione clinico o ambientale. Thsi può portare alla scoperta di nuove specie amoebal ma anche di nuovi batteri intracellulari che crescono in particolare in queste amebe. Insieme, queste due tecniche aiutano a scoprire nuovi batteri intracellulari in grado di crescere in amebe. A causa della loro capacità di infettare amebe e resistere fagocitosi, questi batteri intracellulari potrebbero anche sfuggire fagocitosi da parte dei macrofagi e quindi, per essere patogeni eucarioti superiori.

Utilizzo di co-coltura amoebal e l&#39;arricchimento amoebal, tutta una serie di batteri ambientali e / o patogeni sono stati scoperti (Tabella 1). Coculture amoebal è stato utilizzato dal nostro gruppo ed altri per analizzare campioni ambientali, impianti di trattamento delle acque e sistemi di distribuzione dell&#39;acqua. Una vasta gamma di microrganismi potrebbe essere isolato con questa tecnica. I più comuni batteri isolati da co-coltura amoebal sono membri del genere...

Watch this Scientific Journal Video about Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches at JoVE.com

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Coculture amoebal è un sistema di coltura cellulare usando amebe aderente a crescere selettivamente patogeni intracellulari in grado di resistere fagociti come amebe e macrofagi. Rappresenta quindi uno strumento chiave per scoprire nuovi agenti infettivi. Arricchimento amoebal permette la scoperta di nuove specie amoebal e dei loro specifici batteri intracellulari.

Scoperta di nuovi agenti patogeni intracellulari da amoebal co-coltura e approcci amoebal arricchimento

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

discovery-new-intracellular-pathogens-amoebal-coculture-amoebal

Research

JoVE Journal

Immunology and Infection

14.4K Views.  University Hospital Center and University of Lausanne.  Coculture amoebal è un sistema di coltura cellulare usando amebe aderente a crescere selettivamente patogeni intracellulari in grado di resistere fagociti come amebe e macrofagi. Rappresenta quindi uno strumento chiave per scoprire nuovi agenti infettivi. Arricchimento amoebal permette la scoperta di nuove specie amoebal e dei loro specifici batteri intracellulari. 

Video: Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

L&#39;infezione di embrioni di zebrafish con batteri patogeni intracellulari

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Ricerca

Immunologia e infezioni

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

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.

Monitoraggio dei cambiamenti di polarità membrana, membrana integrità e intracellulare di ioni concentrazioni in<em&gt; Streptococcus pneumoniae</em&gt; Utilizzo di tinture fluorescenti

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

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Applicazione di nanoparticelle fluorescenti per studiare rimodellamento del sistema Endo-lisosomiale da batteri intracellulari

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Trasformazione Biolistic di una fluorescente Tagged Gene nel Opportunistic Fungal patogeni<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

Identificazione di batteri patogeni rari da 16S rRNA sequenziamento del gene e MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Detection High-throughput respiratorie patogeni nei campioni animali da Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Visualizing intracellulare rullante traffico da fluorescenza Lifetime Imaging microscopia

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Virus di Zika specifico diagnostico dell'epitopo Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Monostrati Colonoid umana per studiare le interazioni tra gli agenti patogeni, Commensals ed epitelio intestinale Host

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...