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><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.&amp;nbsp; Soluble in DMSO.</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>216763-100ML</td><td>30 wt. % in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Stabilized.</td></tr><tr><td>Gentamicin</td><td>Gold Bio</td><td>G-400-100</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; Filter sterilize.</td></tr><tr><td>Tobramycin</td><td>Sigma-Aldrich</td><td>T4014-100MG</td><td>Soluble in H&lt;sub&gt;2&lt;/sub&gt;O.&amp;nbsp; 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 ...

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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

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

Approcci alla valutazione dello Stress ossidativo tossicologici utilizzando geneticamente codificato Fluorogenic sensori di imaging

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Ricerca

Ricerca sul cancro

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

Quantificazione di Hypopigmentation attività In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Biochimica

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Induzione farmacologica di melanina epidermica e di protezione contro le scottature in un modello di topo umanizzato

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Medicina

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

Ingegneria

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Inattivazione di agenti patogeni tramite fotolisi in luce visibile della riboflavina-5′-fosfato

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

Bioingegneria

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Induzione e analisi dello stress ossidativo nelle cellule epiteliali del pigmento retinico umano trasforato dalla bella addormentata nel bosco

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Stimolazione di nicchie di cellule staminali e rigenerazione tissutale nella pelle di topo mediante fotogenerazione commutabile protoporfirina IX-dipendente di specie reattive dell'ossigeno in situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

Biologia

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Y-27632 arricchisce la resa dei melanociti umani dai tessuti della pelle adulta

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Source: Amaro-Ortiz, A. et al. <a href="https://www.jove.com/v/50670"> Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model</a>. J. Vis. Exp. (2013)
This video describes a method to measure UV sensitivity in a humanized mouse model&#8212;a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

Saggio della dose eritematosa minima: un metodo per misurare la sensibilità ai raggi UV nei modelli murini

Source: Amaro-Ortiz, A. et al.  Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Skin Cancer

Animal model studies

A Spot Plate Assay for Evaluating Oxidative Stress Responses in Bacterial Strains

Source: Ketelboeter, L. M. et al., <a href="https://app.jove.com/v/53105">Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress.</a> J. Vis. Exp. (2015)
This video demonstrates a spot plate assay tailored to assess the oxidative stress responses of bacterial strains. Some strains produce pyomelanin, an antioxidant pigment. Conversely, strains treated with a test agent, leading to reduced pyomelanin production, exhibit increased susceptibility to oxidative stress, resulting in decreased cell viability compared to pyomelanin-producing strains.

Un test su piastra spot per la valutazione delle risposte allo stress ossidativo nei ceppi batterici

1. Preparation of Culture Media, Antibiotics, and 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)

	Make Luria-Bertani (LB) broth (1% ...

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

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Capitoli in questo video

Approcci alla valutazione dello Stress ossidativo tossicologici utilizzando geneticamente codificato Fluorogenic sensori di imaging

Quantificazione di Hypopigmentation attività In Vitro

Induzione farmacologica di melanina epidermica e di protezione contro le scottature in un modello di topo umanizzato

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Inattivazione di agenti patogeni tramite fotolisi in luce visibile della riboflavina-5′-fosfato

Induzione e analisi dello stress ossidativo nelle cellule epiteliali del pigmento retinico umano trasforato dalla bella addormentata nel bosco

Stimolazione di nicchie di cellule staminali e rigenerazione tissutale nella pelle di topo mediante fotogenerazione commutabile protoporfirina IX-dipendente di specie reattive dell'ossigeno in situ

Y-27632 arricchisce la resa dei melanociti umani dai tessuti della pelle adulta

Saggio della dose eritematosa minima: un metodo per misurare la sensibilità ai raggi UV nei modelli murini

Un test su piastra spot per la valutazione delle risposte allo stress ossidativo nei ceppi batterici