The authors thank Dara Frank and Carrie Harwood for their generous contribution of strains. University of Wisconsin Milwaukee Research Foundation holds patent no. 8,354,451; with claims broadly directed to treating or inhibiting the progression of infection of a microorganism in a patient by administering a 4-hydroxyphenylpyruvate dioxygenase-inhibiting compound such as 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). Inventors are Graham Moran and Pang He. This research was supported by the National Institutes of Health (R00-GM083147). The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507.

1. Preparazione della Cultura Media, antibiotici, e 2- [2-nitro-4- (trifluorometil) benzoile] -1,3-cicloesandione (NTBC) <ol><li> Rendere brodo LB (1% triptone, estratto di lievito 0,5%, 0,25% di NaCl in H 2 O) e aliquota in volumi appropriati. Sterilizzare in autoclave. Conservare a temperatura ambiente. </li><li> Rendere 100 ml LB agar (1% triptone, estratto di lievito 0,5%, 0,25% NaCl, 1,5% agar in H 2 O) in 250 ml beute. Sterilizzare in autoclave e conservare a temperatura ambiente. Assicurarsi che l&#39;agar viene fuso prima di versare in piastre. NOTA: Boccette contenenti 100 ml di LB agar produrrà 4 piatti. La quantità di LB agar può essere modificato per corrispondere al numero di lastre necessarie per il dosaggio. </li><li> Fai la PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4, 2 mM KH 2 PO 4) 16. Sterilizzare in autoclave o filtrazione e conservare a temperatura ambiente. </li><li> Preparare le soluzioni madre di antibiotici per gentamicina, kanamicina, und tobramicina. <ol><li> Preparare opportune concentrazioni azionari antibiotici per P. aeruginosa ceppi contenenti 100 mg / ml gentamicina, 30 mg / ml di kanamicina, e 10 mg / ml tobramicina. Sciogliere gli antibiotici in acqua, sterilizzare il filtro (0,2 micron), e conservare a 4 ° C. Modificare gli antibiotici e le concentrazioni seconda batterio studiata. </li></ol></li><li> Preparare le soluzioni madre NTBC. Disciogliere 10 mg di NTBC in 400 ml di DMSO. Questo produce una concentrazione di 75,9 NTBC mM. Store Solutions NTBC azionari a -20 ° C. Soluzioni scongelare a temperatura ambiente, se necessario. NOTA: diverse fonti di NTBC hanno differenze di solubilità. Determinare il veicolo appropriato per sciogliere il NTBC in base alle raccomandazioni del costruttore e regolare Passo 1.5 di conseguenza. </li></ol> 2. NTBC titolazioni di Ceppi batterici <ol><li> Impostare le culture durante la notte dei ceppi da testare. Aggiungere 2 ml di brodo LB a 16 x 150 mm provettas (uno per ceppo) e inoculare con 1 isolato colonia da ogni ceppo. Incubare per una notte a 37 ° C con aerazione su un rotatore coltura tissutale in un incubatore dell&#39;aria. </li><li> Il giorno seguente, preparare titolazioni di NTBC in LB brodo. Utilizzare una prima serie 0-900 micron NTBC da ceppi diversi hanno differenze nella sensibilità al NTBC. <ol><li> Aggiungere 1 ml di brodo LB per 4 a 5 provette (16 x 150 mm) per ceppo. </li><li> Aggiungere la soluzione di riserva NTBC (75,9 mm) per le provette (16 x 150 mm) in un intervallo di concentrazioni. Vedi Tabella 1 per le concentrazioni di NTBC e corrispondenti volumi delle scorte di aggiungere ad 1 ml di brodo LB. </li></ol></li><li> Misurare il diametro esterno 600 delle culture durante la notte. Lavare le culture prima di OD 600 letture per eliminare pyomelanin presente nei media. <ol><li> Lavare le culture centrifugazione 1 ml di coltura in una microcentrifuga a 16.000 xg per 2 min. Rimuovere il surnatante e tutte le cellule senza bloccare in pellet conun micropipettatore e risospendere il pellet cellulare solido in 1 ml LB. </li></ol></li><li> Inoculare tubi di titolazione a OD 600 0.05. Calcolare la quantità di coltura lavato necessari per inoculare le provette. NOTA: Utilizzare le culture lavati per vaccinazioni da pyomelanin non dovrebbe essere presente. </li><li> Incubare le provette titolazione per circa 24 ore a 37 ° C con aerazione usando un tessuto coltura rotatore in un incubatore dell&#39;aria. </li><li> Fotografa i tubi di titolazione e confrontare produzione di pigmenti all&#39;interno e tra i ceppi per determinare la quantità di NTBC da utilizzare per MIC e lo stress ossidativo saggi. Utilizzare OD 600 letture per determinare la quantità di pyomelanin in cella libera supernatante di coltura e per determinare la densità cellulare. NOTA: Il rapporto di OD 600 pyomelanin nella cultura surnatante in cellule può essere calcolato per quantificare le differenze nella produzione pyomelanin dopo il trattamento con NTBC. </li></ol> 3. antibiotico minima inibizioneConcentrazione tory (MIC) Assay in piastre con 96 pozzetti <ol><li> Impostare le culture durante la notte dei ceppi da testare in LB con e senza NTBC. NOTA: Questo protocollo è descritta utilizzando il livello rappresentativo di 300 micron NTBC. Il livello adeguato di NTBC da utilizzare è determinato nel passo 2.6. <ol><li> Aggiungere 300 micron NTBC a 2 ml LB. Aggiungere un volume equivalente di veicolo (DMSO) a 2 ml LB per la condizione non NTBC. </li><li> Utilizzando stuzzicadenti sterili, inoculare provette con uno isolato colonia di batteri. Ci sarà una cultura con NTBC e una cultura senza NTBC per ogni ceppo. Incubare per una notte a 37 ° C con aerazione usando un tessuto coltura rotatore in un incubatore dell&#39;aria. </li></ol></li><li> Il giorno seguente, fare LB + NTBC e soluzioni maestri LB + DMSO per l&#39;impostazione del test MIC. Aggiungere NTBC ad una concentrazione di 600 mM come questa saranno diluiti due volte quando si aggiunge inoculo, ottenendo una concentrazione finale di 300 mM. <ol><li> Per provare uno antibiotic per un ceppo, aggiungere 600 micron NTBC a 2 ml di LB e mescolare per rendere la soluzione principale NTBC. Aggiungere un volume equivalente di veicolo (DMSO) a 2 ml LB e mescolare per rendere la soluzione maestro non NTBC. Utilizzare queste soluzioni per la creazione di soluzioni madre di antibiotici, nonché per la creazione di una serie di diluizioni in piastre da 96 pozzetti. NOTA: Le formulazioni in soluzione maestri produrrà soluzione in più per tenere conto di errori di pipettamento. Le soluzioni master può essere aumentato o giù come richiesto a seconda del numero di antibiotici e ceppi testati. </li></ol></li><li> Preparare le soluzioni antibiotiche nelle soluzioni maestri LB + NTBC o LB + DMSO. NOTA: La concentrazione di antibiotico in queste soluzioni dovrebbero essere il doppio della concentrazione finale desiderata. Soluzione abbastanza dovrebbe essere fatto per trasferire 100 ul di quattro pozzi in una piastra da 96 pozzetti. <ol><li> Preparare gentamicina +/- soluzione madre NTBC a 64 mg / ml. Per rendere questa soluzione, aggiungere 0,288 ml di gentamicina magazzino (100 mg / ml) per 450ul LB + NTBC o soluzione maestro LB + DMSO. NOTA: la concentrazione massima di gentamicina per P. aeruginosa PAO1 è 32 mg / ml. </li><li> Rendere la kanamicina +/- soluzione di riserva NTBC a 256 mg / ml. Per rendere questa soluzione, aggiungere 3,84 ml di kanamicina magazzino (30 mg / ml) per 450 microlitri soluzioni maestri LB + NTBC o LB + DMSO. NOTA: la concentrazione massima di kanamicina per P. aeruginosa PAO1 è di 128 mg / ml. </li><li> Preparare tobramicina +/- soluzione madre NTBC a 8 mg / ml. Per rendere questa soluzione, aggiungere 0,36 ml di tobramicina magazzino (10 mg / ml) per 450 microlitri soluzioni maestri LB + NTBC o LB + DMSO. NOTA: Per P. aeruginosa PAO1, la massima concentrazione di tobramicina è 4 mg / ml. NOTA: Gli antibiotici e le concentrazioni possono essere regolati per i batteri da testare. </li></ol></li><li> Aggiungere 100 ml di ogni soluzione antibiotica 2x a quattro pozzi in una piastra da 96 pozzetti. Mettere queste soluzioni in fila A. Per example, gentamicina deve essere posto in A1 a A4, kanamicina deve essere posto in A5 attraverso A8 e tobramicina deve essere posto in A9 attraverso A12. Vedere Figura 1A per un diagramma di una piastra a 96 pozzetti impostato. NOTA: più antibiotici possono essere testati in un piatto, ma solo ceppo devono essere testati per piastra di eliminare il rischio di contaminazione incrociata da altri ceppi. </li><li> Aggiungere 50 ml di soluzione di maestro LB + NTBC o LB + DMSO alle righe B attraverso H della piastra a 96 pozzetti. Assicurarsi che un piatto è LB + NTBC e un piatto è LB + DMSO. Vedere la Figura 1A. <ol><li> Utilizzare LB + NTBC per gli antibiotici in LB + NTBC. Utilizzare LB + DMSO per gli antibiotici in LB + DMSO. </li></ol></li><li> Utilizzando un micropipettatore eseguire due diluizioni seriali piega degli antibiotici trasferendo 50 ml di soluzione dalla fila A alla fila B. Mix la soluzione, modificare i puntali, e trasferire 50 ml di soluzione da riga B a remare C. Ripetere l&#39;operazione per il remainirighe ng. Dopo diluizione fila G, rimuovere 50 ml di soluzione da quella riga e scarti. Utilizzare fila H come nessun controllo antibiotico per la crescita batterica. Vedere la Figura 1B. NOTA: ogni bene nelle righe da A a G ora contiene 50 ml di antibiotici in LB + NTBC o LB + DMSO a 2X la concentrazione finale desiderata. Fila H contiene LB + NTBC o LB + DMSO con antibiotici. </li><li> Misurare il diametro esterno 600 delle culture durante la notte. Lavare tutte le culture prima di OD 600 letture per eliminare pyomelanin presente nei media. <ol><li> Lavare le culture centrifugazione 1 ml di coltura in una microcentrifuga a 16.000 xg per 2 min. Rimuovere il surnatante con un micropipettatore e risospendere il pellet cellulare in 1 ml LB. </li></ol></li><li> Diluire le culture durante la notte per 2.75x10 5 CFU / ml in LB. NOTA: Si supponga che una OD 600 unità è l&#39;equivalente di 1x10 9 CFU / ml per P. aeruginosa. OD di CFU / ml conversioni può essere different in altri batteri. </li><li> Aggiungere 50 ml di coltura batterica diluito nel pozzetto. NOTA: Le culture coltivati ​​in NTBC dovrebbero essere aggiunti ai pozzetti contenenti NTBC e culture cresciute in DMSO dovrebbero essere aggiunti ai pozzetti contenenti DMSO. Vedere la Figura 1B. <ol><li> Aggiungi ai batteri di tre pozzi per ogni ceppo e la concentrazione di antibiotico. Aggiungere 50 ml di LB alla quarta e di agire come un controllo per la contaminazione batterica. Vedere la Figura 1B. </li><li> Utilizzare un micropipettatore multicanale per inoculare i pozzi. Assicurarsi che pipetta suggerimenti sono vicino al fondo dei pozzetti quando si aggiunge inoculo per prevenire la contaminazione dei pozzi vicini. NOTA: L&#39;aggiunta di coltura batterica ai pozzetti diluire le concentrazioni di antibiotici e NTBC due volte. </li></ol></li><li> Coprire le piastre a 96 pozzetti con parafilm e incubare circa 24 ore a 37 ° C. Incubare le piastre a 96 pozzetti staticamente, in un incubatore dell&#39;aria. </li><li> Esaminarele piastre per la crescita batterica nei pozzetti. La MIC è la più bassa concentrazione di antibiotico in cui nessuna crescita batterica è visibile per tutte tre repliche di ciascun ceppo. <ol><li> Esaminare visivamente la piastra per la crescita o leggere utilizzando un lettore di set piatto di OD 600. </li></ol></li></ol> 4. Spot Assay Piastra per Stress ossidativo risposta <ol><li> Impostare le culture durante la notte dei ceppi da testare in LB con e senza NTBC come descritto al punto 3.1. </li><li> Il giorno successivo, preparare piastre di agar LB contenenti H 2 O 2 come fattore di stress ossidativo. Una gamma di H 2 O 2 concentrazioni da 0 a 1 mm è un buon punto di partenza. <ol><li> Sciogliere i palloni LB agar. Supporti Raffreddare a circa 50 ° C a temperatura ambiente. </li><li> Aggiungere H 2 O 2 direttamente ai media raffreddati alle concentrazioni desiderate. Fiaschi Swirl per mescolare. Vedere la Tabella 2 per le concentrazioni di H 2 O 2e volumi di H 2 O 2 concentrato da aggiungere. Questi valori sono basati su 100 ml di LB agar. </li><li> Versare in piastre subito dopo l&#39;aggiunta H 2 O 2 e fiamme la superficie per rimuovere bolle. La resa è di 4 piastre per 100 ml di LB agar. Segnare i piatti con la concentrazione di H 2 O 2. </li><li> Posizionare le piastre scoperto in una cappa a flusso biologico con il ventilatore acceso per 30 minuti per rimuovere l&#39;umidità in eccesso dalle piastre. NOTA: Utilizzare le lastre lo stesso giorno in cui vengono preparati. In caso contrario si potrebbe causare dati incoerenti. NOTA: fattori di stress ossidativo, come il paraquat può essere sostituito per H 2 O 2 in questo saggio. Le concentrazioni utilizzate per altri fattori di stress ossidativi possono variare rispetto a quelli utilizzati per H 2 O 2. </li></ol></li><li> Lavare e misurare il diametro esterno 600 delle culture durante la notte come descritto al punto 3.7. </li><li> Normalizzare la OD 600 di tutta la cu durante la notteltures al valore più basso per il set di ceppi testati. P. aeruginosa ha generalmente un diametro esterno 600 di circa 2,5 quando cresciuto durante la notte in LB + NTBC o LB + DMSO. <ol><li> Determinare il volume della cultura necessaria per diluire la coltura al OD più basso 600 in un volume totale di 1 ml. Ad esempio, se una coltura ha un diametro esterno 600 del 3 e l&#39;OD più basso 600 per il set di ceppi è 2.5, eseguire il seguente calcolo: (2,5) (1 ml) = (3) (x). x = 0,833 ml. 0,833 ml di coltura saranno collocati in una provetta per microcentrifuga. </li><li> Calcolare la quantità di LB + NTBC (300 micron) o LB + DMSO necessaria a portare il volume cultura di 1 ml. Per l&#39;esempio nel passaggio 4.4.1, la quantità di LB + NTBC o LB + DMSO aggiunto alla cultura sarebbe 0.167 ml (1 ml di volume totale - 0.833 ml cultura). Fai soluzioni stock di LB + NTBC e LB + DMSO da utilizzare per queste diluizioni in base al volume necessario per diluire tutti i ceppi. </li><li> Mescolare la cultura e LB + NTBC o LB + DMSO nel vortex. </li></ol></li><li> Per mantenere le culture in una concentrazione costante di NTBC o DMSO, eseguire dieci diluizioni seriali di piegatura delle culture durante la notte normalizzati in PBS + NTBC o PBS + DMSO. <ol><li> Fai soluzioni stock di PBS + NTBC e PBS + DMSO. Per una serie di diluizioni di un ceppo, mescolare 300 pM NTBC o un volume equivalente di DMSO con PBS per ottenere un volume totale di 720 microlitri. Scala questi stock alto o in basso a seconda di quante ceppi vengono testati. </li><li> Tubi Label microcentrifuga per 10 -1 attraverso diluizioni 10 -7 seriali. Aggiungere 90 ml di PBS + NTBC o PBS + DMSO ai tubi appropriati. Usa PBS + NTBC per i ceppi cresciuti in LB + NTBC e utilizzare PBS + DMSO per i ceppi cresciuti in LB + DMSO. </li><li> Aggiungere 10 ml di cultura alla competente 10 -1 provetta di diluizione. Mescolare nel vortex e trasferire 10 ml di diluizione 10 -1 al tubo di diluizione 10 -2. Ripetere l&#39;operazione fino a quando tutte le diluizioni sono stati perfoconfermate. Cambiare puntali tra diluizioni. </li></ol></li><li> Spot 5 ml di 10 -3 attraverso 10 -7 diluizioni su LB + H 2 O 2 piastre in duplicato per ogni ceppo. Utilizzare una punta della pipetta se i punti sono placcati da più diluito al meno diluito (10 -7 a 10 -3). Non ribaltare la piastra fino a quando il liquido si è asciugata nella piastra. </li><li> Incubare le piastre per 24-48 ore a 37 ° C (incubatore aria), a seconda del ceppo. NOTA: P. aeruginosa PAO1 avrà buone colonie dimensioni su LB dopo 24 ore di incubazione. Incubare ceppi fino colonie hanno approssimativamente la stessa dimensione PAO1. </li><li> Fotografare i piatti utilizzando una fotocamera CCD sopra un transluminator. Facoltativamente, modificare le foto per il contrasto e la coltivazione alla stessa dimensione. Contare il numero di colonie in ogni posto per determinare le variazioni di sensibilità allo stress ossidativo. </li></ol>

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D., Dankai, W., Vanittanakom, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanogenesis+in+dermatophyte+species+in+vitro++during+infection.">Melanogenesis in dermatophyte species in vitro during infection.</a> Microbiology. 157, 2348-2356 (2011).</li><li>Khajo, 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=Protection+of+Melanized+Cryptococcus+neoformans+Lethal+Dose+Gamma+Irradiation+Involves+Changes+in+Melanin's+Chemical+Structure+and+Paramagnetism.">Protection of Melanized Cryptococcus neoformans Lethal Dose Gamma Irradiation Involves Changes in Melanin's Chemical Structure and Paramagnetism.</a> PLoS ONE. 6, e25092 (2011).</li><li>Hancock, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hancock Laboratory Methods. , (2015).</li></ol>

The authors declare that they have no competing financial interests.

The NTBC titration method described in this protocol will allow the user to determine if NTBC can reduce or eliminate pyomelanin production in bacteria, and determine the concentration of NTBC required. The most critical step in the NTBC titration assay is determining the range of NTBC concentrations to use in the assay. Different strains of P. aeruginosa have different sensitivities to NTBC, and laboratory strains may be more sensitive to NTBC than clinical isolates12 (Figure 2). The...

Pseudomonas aeruginosa is a Gram negative bacterium that produces a variety of pigments including pyomelanin, a red-brown pigment that helps provide protection from oxidative stress1-4 and binds a variety of compounds, including aminoglycoside antibiotics5-7. Pyomelanin production is caused by a defect in the tyrosine catabolism pathway4,8, either through deletions or mutations of the gene encoding homogentisate 1,2-dioxygenase (HmgA)1,9 or through imbalances in the various enzymes in the pathway10. Homogentisate accumulates due to inactivation of HmgA, and is secreted and oxidized to form pyomelanin11. Production of pyomelanin can be abolished or reduced in a dose dependent manner through treatment with the herbicide 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)12, which inhibits 4-hydroxyphenylpyruvate dioxygenase (Hpd) in the tyrosine catabolism pathway13. Hpd is required for the formation of homogentisate, and therefore pyomelanin11. 
We describe in detail three techniques that were important in our studies of NTBC treatment of pyomelanin producing strains of P. aeruginosa. These techniques include titration of NTBC to determine the concentrations that will abolish or reduce pyomelanin production in laboratory and clinical pyomelanin producing strains, determination of the minimum inhibitory concentration (MIC) of antibiotics when bacteria are treated with NTBC, and the resulting sensitivity to oxidative stress with NTBC treatment.
The titration assay we developed serves two purposes. First, the assay will allow the user to determine if NTBC can abolish or reduce pyomelanin production in the bacterium being studied and at which concentrations. This will allow the user to determine sensitivity to NTBC, since different strains of bacteria may have different sensitivities to this compound, as observed in P. aeruginosa12. Second, the NTBC titration assay will allow the user to determine the appropriate concentration of NTBC to use in subsequent assays, such as antibiotic MIC and oxidative stress response assays, if the goal is to abolish or reduce pyomelanin production and determine the effects of pigment reduction. 
The titration assay works because a visible difference in pyomelanin production can be seen in strains treated with NTBC and the differences in pyomelanin production are dose dependent12. Additionally, this technique can be applied to the study of other compounds that may eliminate or enhance pigment production in bacteria.
Antibiotic MICs are used to determine the sensitivity of bacteria to antibiotics. There are several methods to determine MICs, including agar dilution plates and broth dilutions14. Broth dilutions can be performed in small test tubes or in a 96-well microtiter plate. The microtiter plate method of MIC determination described herein will allow the user to test a wide range of antibiotics using a minimum of resources. The assay provides reproducibility as well as flexibility in the number of antibiotics and strains tested by this method. Additionally, with the incorporation of NTBC in the assay, the user can determine if elimination or reduction of pyomelanin production alters antibiotic sensitivity in bacteria that produce pyomelanin.
Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time1, or oxidative stress disc diffusion assays15. These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates. The viable count assay also tends to use more agar plates than the other methods. The spot plate assay we describe uses low concentrations of H2O2 and allows the user to test the oxidative stress response of multiple strains using a minimum of plates. The assay is also consistently reproducible between technical and biological replicates. As pyomelanin is involved in resistance to oxidative stress, the incorporation of NTBC in the assay allows the user to determine the effects of elimination of pyomelanin production on oxidative stress resistance.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)</td>
			<td>Sigma-Aldrich</td>
			<td>SML0269-50mg</td>
			<td>Also called nitisinone.&#160; Soluble in DMSO.</td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td>30 wt. % in H2O.&#160; Stabilized.</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gold Bio</td>
			<td>G-400-100</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014-100MG</td>
			<td>Soluble in H2O.&#160; Filter sterilize.</td>
		</tr>
	</tbody>
</table>

methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Titolazioni NTBC Le titolazioni NTBC sono stati utilizzati per determinare se NTBC è stata in grado di ridurre la produzione pyomelanin in P. aeruginosa, e anche identificare la concentrazione di NTBC che elimina o riduce pyomelanin produzione per uso in test aggiuntivi. Ci possono essere variazioni nei livelli di pyomelanin prodotto in diverse repliche, ma tendenze generali rimanere costante. Il saggio di titolazione NTBC potrebbe anche essere modificato per testare altri...

Watch this Scientific Journal Video about Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress at JoVE.com

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Metodi Per inibire batterica Pyomelanin produzione e determinare il corrispondente aumento di sensibilità allo stress ossidativo

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

methods-to-inhibit-bacterial-pyomelanin-production-determine

Research

JoVE Journal

Immunology and Infection

8.1K Views.  University of Wisconsin Milwaukee. Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Video: Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

L&#39;introduzione di shear stress nello studio di adesione batterica

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

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Immunologia e infezioni

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Utilizzando il Overlay saggio misurare Qualitativamente produzione batterica di sensibilità e di pneumococcico batteriocine

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

I metodi per aumentare la sensibilità di High Resolution Melting polimorfismo a singolo nucleotide genotipizzazione a malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modello per studiare l&#39;adesione batterica alla parete del serbatoio in condizioni di flusso

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrofili Isolamento e analisi per determinare il loro ruolo nel linfoma a cellule sensibilità agli agenti terapeutici

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Un metodo rapido per la lavorazione del sangue per aumentare la resa dei livelli plasmatici del peptide nel sangue umano

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Sovrapressione e purificazione dell&#39;uomo<em&gt; Cis</em&gt; -preniltransferasi in<emEscherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation.
We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P)+ and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase&#39;s activity. The formazan&#39;s absorbance is therefore a direct measure of the dehydrogenase&#39;s activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays.
Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Here, we present a protocol that can be used to microscopically visualize and quantify activity of dehydrogenases in cells and tissues and its kinetics, function and subcellular localization.

Mappatura metabolica: Enzima quantitativa citochimica e istochimica per determinare l'attività della deidrogenasi in cellule e tessuti

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells ...