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. Preparação de Meios de Cultura, antibióticos e 2- [2-nitro-4- (trifluorometil) benzoil] -1,3-ciclo-hexanodiona (NTBC) <ol><li> Adicione caldo LB (1% de triptona, 0,5% de extracto de levedura, 0,25% de NaCl em H 2 O) e alíquota em volumes apropriados. Esterilizar em autoclave. Guarde-o em temperatura ambiente. </li><li> Adicione 100 ml de agar LB (1% de triptona, 0,5% de extracto de levedura, 0,25% de NaCl, 1,5% de agar em H2O) em 250 ml de frascos. Esterilizar em autoclave e armazenar à temperatura ambiente. Certifique-se de que o agar é derretido antes de verter em placas. NOTA: Os frascos contendo 100 ml de LB agar irá originar 4 placas. A quantidade de agar LB pode ser alterada para corresponder ao número de placas necessárias para o ensaio. </li><li> Adicione PBS (NaCl 137 mM, KCl 2,7 mM, 10 mM de Na 2 HPO 4, KH 2 mM 2 PO 4) 16. Esterilizar em autoclave ou filtração e armazenamento à temperatura ambiente. </li><li> Preparar as soluções de antibióticos para a gentamicina, a canamicina, umad tobramicina. <ol><li> Prepare concentrações banco de antibióticos apropriados para P. aeruginosa estirpes contendo 100 mg / ml de gentamicina, 30 mg / ml de canamicina, e 10 mg / ml de tobramicina. Dissolver os antibióticos em água, Esterilizar por filtração (0,2 mm), e armazenar a 4 ° C. Alterar os antibióticos e concentrações, dependendo da bactéria em estudo. </li></ol></li><li> Prepare as soluções estoque NTBC. Dissolve-se 10 mg de NTBC em 400 ul de DMSO. Isto produz uma concentração de 75,9 mM NTBC. Loja soluções estoque NTBC a -20 ° C. Soluções descongelar à temperatura ambiente, conforme necessário. NOTA: Diferentes fontes de NTBC têm diferenças de solubilidade. Determinar o veículo apropriado em que para dissolver o NTBC com base nas recomendações do fabricante e ajustar Passo 1.5 em conformidade. </li></ol> 2. NTBC Titulações de Estirpes Bacterianas <ol><li> Defina-se culturas nocturnas das estirpes a serem testadas. Adicionar 2 ml de caldo LB a 16 x 150 mm tubo de ensaios (um por cepa) e inocular com uma colônia isolada de cada estirpe. Incubar durante a noite a 37 ° C com arejamento num agitador rotativo de cultura de tecidos em uma incubadora de ar. </li><li> No dia seguinte, preparar titulações de NTBC em caldo LB. Use uma primeira série 0-900 mM NTBC uma vez que diferentes cepas têm diferenças na sensibilidade à NTBC. <ol><li> Adicionar 1 ml de caldo LB a 4 para 5 tubos de ensaio (16 x 150 mm) por estirpe. </li><li> Adicionar a solução estoque NTBC (75,9 mM), para os tubos de ensaio (16 x 150 mm) numa gama de concentrações. Ver Tabela 1 para as concentrações e volumes NTBC imagens correspondentes a adicionar a 1 mL de caldo de LB. </li></ol></li><li> Medir a OD 600 das culturas durante a noite. Lave as culturas antes de tomar OD 600 leituras para eliminar pyomelanin presente na mídia. <ol><li> Lavam-se as culturas por centrifugação de 1 ml de cultura em uma microcentrífuga a 16000 xg durante 2 min. Remover o sobrenadante e as células peletizadas com frouxamenteuma micropipeta e ressuspender o pellet celular sólido em 1 ml de LB. </li></ol></li><li> Inocular tubos de titulação em OD 600 de 0,05. Calcula-se a quantidade necessária da cultura lavou-se para inocular os tubos. NOTA: Use as culturas lavadas para inoculações desde pyomelanin não deve estar presente. </li><li> Incubar os tubos de titulação durante aproximadamente 24 horas a 37 ° C com arejamento usando um tecido rotador cultura numa incubadora de ar. </li><li> Fotografar os tubos de titulação e comparar a produção de pigmento dentro e entre as cepas para determinar a quantidade de NTBC de usar para MIC e estresse oxidativo ensaios. Use OD 600 leituras para determinar a quantidade de pyomelanin na célula livre sobrenadante da cultura e para determinar a densidade celular. Observação: A razão OD 600 de pyomelanin no sobrenadante da cultura de células pode ser calculada para quantificar as diferenças na produção pyomelanin após o tratamento com NTBC. </li></ol> 3. Antibiótico Inhibi mínimoConcentração (MIC) Ensaio tory em placas de 96 poços <ol><li> Configure culturas durante a noite das estirpes a ser testado em LB com e sem NTBC. NOTA: Este protocolo é descrito usando o nível representante de 300? M NTBC. O nível apropriado de NTBC a ser utilizado é determinado no passo 2.6. <ol><li> Adicionar 300 ^ M a 2 ml NTBC LB. Adicionar um volume equivalente de veículo (DMSO) a 2 ml de LB para a condição não NTBC. </li><li> Usando palitos estéreis, inocule os tubos com uma colônia de bactérias isoladas. Haverá uma cultura com NTBC e uma cultura sem NTBC para cada estirpe. Incubar durante a noite a 37 ° C com arejamento usando um tecido rotador cultura numa incubadora de ar. </li></ol></li><li> No dia seguinte, fazer LB + NTBC e soluções mestre LB + DMSO para a criação do ensaio MIC. Adicionar NTBC a uma concentração de 600 uM como este será diluído duas vezes quando inoculo é adicionado, dando origem a uma concentração final de 300 uM. <ol><li> Para testar um antibiótica para uma estirpe, adicionar 600 | iM NTBC a 2 ml de LB e misturar para tornar a solução NTBC mestre. Adicionar um volume equivalente de veículo (DMSO) a 2 ml de LB e misturar para fazer a solução não NTBC mestre. Usar estas soluções para criar soluções-mãe de antibióticos, bem como para a criação de séries de diluição em placas de 96 poços. NOTA: As formulações de solução mestre trará solução extra para contabilizar erros de pipetagem. As soluções mestre pode ser escalada para cima ou para baixo, conforme necessário, dependendo do número de antibióticos e estirpes testadas. </li></ol></li><li> Preparar as soluções de antibióticos nas soluções mestre LB + NTBC ou LB + DMSO. NOTA: A concentração do antibiótico na Estas soluções devem ser o dobro da concentração final desejada. Deve ser feita solução suficiente para transferir 100 ul de quatro poços de uma placa de 96 poços. <ol><li> Prepare gentamicina +/- solução estoque NTBC a 64 ug / ml. Para tornar esta solução, adicionar 0,288 ul de estoque de gentamicina (100 mg / mL) a 450jil de LB + NTBC ou mestre solução de LB + DMSO. NOTA: A concentração máxima de gentamicina para P. aeruginosa PAO1 é de 32 ug / ml. </li><li> Faça a canamicina +/- solução estoque NTBC a 256 ug / ml. Para tornar esta solução, adicionar 3,84 l de estoque canamicina (30 mg / ml) a 450 ul soluções mestre LB + NTBC ou LB + DMSO. NOTA: A concentração máxima de canamicina para P. aeruginosa PAO1 é de 128 ug / ml. </li><li> Prepare tobramicina +/- solução estoque NTBC a 8 ug / ml. Para tornar esta solução, adicionar 0,36 l de estoque tobramicina (10 mg / ml) a 450 ul soluções mestre LB + NTBC ou LB + DMSO. NOTA: Para P. aeruginosa PAO1, a concentração máxima de tobramicina é de 4 ug / ml. NOTA: As concentrações de antibióticos e pode ser regulado para as bactérias a serem testadas. </li></ol></li><li> Adiciona-se 100 ul de cada solução de antibiótico 2x para poços quatro numa placa de 96 poços. Coloque essas soluções em linha A. Para example, a gentamicina, deve ser colocado em A1 a A4, canamicina devem ser colocados em A5 a A8 e tobramicina deve ser colocado em meio A9 A12. Ver Figura 1A um diagrama de uma placa de 96 poços configurado. NOTA: antibióticos múltiplos pode ser testado em um prato, mas apenas uma estirpe deve ser testado por placa para eliminar o potencial de contaminação cruzada de outras estirpes. </li><li> Adicionar 50 ul da solução de mestre de LB + NTBC ou LB + DMSO a linha B a H da placa de 96 poços. Certifique-se que uma placa é LB + NTBC e um prato é LB + DMSO. Veja a Figura 1A. <ol><li> Use de LB + NTBC para os antibióticos em LB + NTBC. Use de LB + DMSO para os antibióticos em LB + DMSO. </li></ol></li><li> Usando uma micropipeta realizar diluições em série duas vezes dos antibióticos através da transferência de 50 mL da solução de linha A para a linha B. Misturar a solução, mudar as pontas de pipetas, e transferir 50 ul da solução a partir da linha B para a linha C para Repetir o remainilinhas GN. Depois de se diluir linha L, remover 50 ul da solução a partir dessa linha e descarte. Utilize linha H como um antibiótico sem controle para o crescimento bacteriano. Ver Figura 1B. NOTA: Cada poço nas filas A a G contém agora 50 uL de antibiótico em LB + NTBC ou LB + DMSO a 2X a concentração final desejada. Fila H contém LB + NTBC ou LB + DMSO sem antibióticos. </li><li> Medir a OD 600 das culturas durante a noite. Lave todas as culturas antes de tomar OD 600 leituras para eliminar pyomelanin presente na mídia. <ol><li> Lavam-se as culturas por centrifugação de 1 ml de cultura em uma microcentrífuga a 16000 xg durante 2 min. Remover o sobrenadante com uma micropipeta e ressuspender o pellet celular em 1 ml de LB. </li></ol></li><li> Dilui-se as culturas durante a noite para 2.75x10 5 ufc / mL em LB. NOTA: Assume-se que uma unidade DO600 é o equivalente de 1x10 9 CFU / ml para P. aeruginosa. OD para as conversões de UFC / mL podem ser different em outras bactérias. </li><li> Adicionar 50 ul da cultura bacteriana diluído ao poço adequado. NOTA: As culturas são crescidas em NTBC deve ser adicionada aos poços contendo NTBC e culturas cultivadas em DMSO deve ser adicionado às cavidades contendo DMSO. Ver Figura 1B. <ol><li> Adicionar bactérias a três poços para cada estirpe e concentração do antibiótico. Adicionar 50 ul de LB para o quarto poço para actuar como um controlo para a contaminação bacteriana. Ver Figura 1B. </li><li> Usar uma pipeta de multi-canal para inocular os poços. Assegurar que pipeta pontas estão perto do fundo dos poços, quando a adição de inoculo para evitar a contaminação de poços vizinhos. NOTA: A adição de cultura bacteriana aos poços irá diluir as concentrações de antibiótico e NTBC duas vezes. </li></ol></li><li> Cobrir as placas de 96 poços com parafilme e incubar a cerca de 24 horas a 37 ° C. Incubar as placas de 96 poços estaticamente, numa incubadora de ar. </li><li> Examinaras placas para o crescimento bacteriano nas cavidades. A MIC é a concentração mais baixa de antibiótico em que nenhum crescimento bacteriano é visto para todas as três repetições de cada estirpe. <ol><li> Examine visualmente a placa de crescimento ou ler usando um conjunto leitor de placas de DO600. </li></ol></li></ol> 4. Ponto Ensaio Placa de resposta ao estresse oxidativo <ol><li> Defina-se culturas nocturnas das estirpes a ser testado em meio LB com ou sem NTBC tal como descrito na etapa 3.1. </li><li> No dia seguinte, para preparar as placas de agar LB contendo H 2 O 2 como um estressor oxidativo. Uma gama de H 2 O 2 concentrações de 0 a 1 mM é um bom ponto de partida. <ol><li> Derreter os frascos de LB-agar. Super meios para aproximadamente 50 ° C à temperatura ambiente. </li><li> Adicionar H 2 O 2 directamente sobre o suporte arrefecidos nas concentrações desejadas. Frascos agite para misturar. Ver Tabela 2 para as concentrações de H 2 O 2e volumes de concentrado H 2 O 2 a acrescentar. Estes valores baseiam-se em 100 ml de LB com agar. </li><li> Verter em placas imediatamente após a adição de H 2 O 2 e a superfície da chama para remover as bolhas. O rendimento é de 4 placas por 100 ml de LB com agar. Marcar as placas com a concentração de H 2 O 2. </li><li> Colocar as placas descoberto numa câmara de fluxo biológico com o ventilador corrida durante 30 min para remover o excesso de humidade das placas. NOTA: Use as placas no mesmo dia em que são preparados. Não fazer isso pode resultar em dados inconsistentes. NOTA: factores de stress oxidativo, tais como o paraquat pode ser substituído por H 2 O 2 neste ensaio. As concentrações utilizadas para outros factores de stress oxidativo pode ser diferente do que os usados ​​para a H 2 O 2. </li></ol></li><li> Lavar e medir a DO 600 das culturas durante a noite, tal como descrito na etapa 3.7. </li><li> Normalizar o OD 600 de todo o cu durante a noiteltures para o menor valor para o conjunto de estirpes que está sendo testado. P. aeruginosa tem geralmente uma DO600 de aproximadamente 2,5, quando crescidas durante a noite em LB ou LB + NTBC + DMSO. <ol><li> Determinar o volume de cultura necessárias para diluir a cultura para o menor OD 600 num volume total de 1 ml. Por exemplo, se uma cultura tem um OD 600 de 3 e menor do OD 600 para o conjunto de estirpes é de 2,5, executar o seguinte cálculo: (2,5) (1 ml) = (3) (X). x = 0.833 ml. 0,833 ml de cultura vai ser colocado num tubo de microcentrífuga. </li><li> Calcular a quantidade de LB + NTBC (300 uM) ou DMSO de LB + necessária para trazer o volume de cultura para 1 ml. Para o exemplo no passo 4.4.1, a quantidade de LB + NTBC ou LB + DMSO adicionado à cultura seria 0.167 ml (1 ml de volume total de 0,833 ml - de cultura). Adicione as soluções concentradas de LB + NTBC e LB + DMSO para utilizar para estes diluições com base no volume necessário para diluir todas as estirpes. </li><li> Misture a cultura e LB + NTBC ou LB + DMSO em vórtice. </li></ol></li><li> Para manter as culturas numa concentração constante de NTBC ou DMSO, realizar diluições em série de dez vezes das culturas durante a noite em PBS + normalizados NTBC ou PBS + DMSO. <ol><li> Faça as soluções concentradas de PBS + NTBC e PBS + DMSO. Para um conjunto de diluições de uma estirpe, misturar 300 NTBC uM ou um volume equivalente de DMSO com PBS para se obter um volume total de 720 ul. Escalar esses stocks cima ou para baixo, dependendo de quantas cepas são testadas. </li><li> Etiqueta para tubos de microcentrífuga através de diluições 10 -1 10 -7 série. Adicionar 90 ul de PBS + NTBC ou PBS + DMSO para os tubos apropriados. Use PBS + NTBC para as estirpes cultivadas em LB + NTBC e usar PBS + DMSO para as estirpes cultivadas em LB + DMSO. </li><li> Adicionar 10 ul de cultura para o tubo 10 -1 de diluição apropriado. Misturar em vortex e transferir 10 ul da diluição 10 -1 para o tubo de diluição 10 -2. Repetir até que todas as diluições foram Performou. Alterar pontas de pipeta entre diluições. </li></ol></li><li> Spot 5 ul de 10 -3 a 10 -7 através diluições em placas de LB + H 2 O 2 placas em duplicado para cada estirpe. Use uma ponta de pipeta se manchas são banhados de mais diluída para menos diluído (10 -7 a 10 -3). Não ponta ou incline a placa até que o líquido tenha secado na placa. </li><li> Incubar as placas durante 24-48 horas a 37 ° C (incubadora de ar), dependendo da estirpe. NOTA: P. aeruginosa PAO1 terá bons colônias porte sobre LB após 24 horas de incubação. Incubar estirpes até que colónias têm aproximadamente o mesmo tamanho que PAO1. </li><li> Fotografar as placas usando uma câmera CCD acima de um transluminador. Opcionalmente, editar as fotos para o contraste e cultura para o mesmo tamanho. Contar o número de colónias em cada local para determinar alterações na sensibilidade ao estresse oxidativo. </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.

Titulações NTBC As titulações NTBC foram utilizados para determinar se NTBC foi capaz de reduzir a produção em P. pyomelanin aeruginosa, e também identificar a concentração de NTBC que elimina ou reduz a produção pyomelanin para utilização em ensaios adicionais. Pode haver variações nos níveis de pyomelanin produzida em diferentes repetições, mas as tendências gerais permanecer constante. O ensaio de titulação NTBC também poderia ser modificado para te...

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).

Métodos para inibir bacteriana Pyomelanin Produção e determinar o correspondente aumento de sensibilidade ao estresse oxidativo

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 visualizações.  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).

Vídeo: 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.

Apresentando tensão de cisalhamento em o Estudo da adesão bacteriana

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

Imunologia e Infecção

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.

Usando a sobreposição de ensaio para medir qualitativamente produção bacteriana e sensibilidade para pneumocócica Bacteriocinas

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.

Métodos para aumentar a sensibilidade de alta resolução de derretimento Single Nucleotide Polymorphism Genotipagem em Malária

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 modelo para estudar a adesão bacteriana para o Vaso de parede em condições de escoamento

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.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

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.

Um método rápido para processamento de sangue para aumentar o rendimento dos níveis plasmáticos de peptídeos no sangue humano

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.

Sobreexpressão e purificação do ser humano<em&gt; Cis</em&gt; -preniltransferase em<em&gt; Escherichia 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 de Salmonella typhimurium-contendo Phagosomes de macrófagos

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.

Um novo sistema alimentador-livre para produção em massa de murino 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 ...

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.

Mapeamento metabólico: Citoquímica quantitativa enzimática e histoquímica para determinar a atividade de desidrogenases em células e tecidos

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