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. Préparation des milieux de culture, des antibiotiques, et 2- [2-nitro-4- (trifluorométhyl) benzoyl] -1,3-cyclohexanedione (NTBC) <ol><li> Ajouter du bouillon LB (1% de tryptone, 0,5% d&#39;extrait de levure, 0,25% de NaCl dans H 2 O) et aliquote dans des volumes appropriés. Stériliser par autoclave. Magasin à la température ambiante. </li><li> Ajouter 100 ml de gélose LB (1% de tryptone, 0,5% d&#39;extrait de levure, 0,25% de NaCl, 1,5% de gélose dans H 2 O) dans des flacons de 250 ml. Stériliser par autoclave et conserver à la température ambiante. Assurez-vous que l&#39;agar-agar est fondu avant de verser dans des assiettes. REMARQUE: Les flacons contenant 100 ml de gélose LB donnera 4 assiettes. La quantité de gélose LB peut être modifiée pour correspondre au nombre de plaques nécessaires pour le dosage. </li><li> Assurez-PBS (137 mM NaCl, KCl 2,7 mM, 10 mM de Na 2 HPO 4, 2 mM KH 2 PO 4) 16. Stériliser par autoclave ou par filtration et stocker à température ambiante. </li><li> Préparer les solutions d&#39;achat d&#39;actions aux antibiotiques pour la gentamicine, la kanamycine, und tobramycine. <ol><li> Préparer les concentrations de stocks d&#39;antibiotiques appropriés pour P. aeruginosa souches contenant 100 mg / ml de gentamicine, 30 mg / ml de kanamycine et 10 mg / ml de tobramycine. Dissoudre les antibiotiques dans l&#39;eau, stériliser par filtration (0,2 um), et conserver à 4 ° C. Modifier les antibiotiques et les concentrations en fonction de la bactérie étudiée. </li></ol></li><li> Préparer les solutions mères NTBC. Dissolvez 10 mg de NTBC dans 400 pi de DMSO. Cela donne une concentration de 75,9 mM NTBC. Magasin NTBC solutions stock à -20 ° C. Solutions décongeler à la température ambiante, au besoin. Remarque: Les différentes sources de NTBC ont des différences de solubilité. Déterminer le véhicule approprié pour dissoudre le NTBC basée sur les recommandations du fabricant et ajuster l&#39;étape 1.5 en conséquence. </li></ol> 2. NTBC titrages de souches bactériennes <ol><li> Mettre en place des cultures de la nuit des souches à tester. Ajouter 2 ml de LB tube à essai bouillon à 16 x 150 mms (un par souche) et ensemencer avec 1 colonie isolée de chaque souche. Incuber pendant une nuit à 37 ° C avec aération sur un rotateur de culture de tissu dans un incubateur à air. </li><li> Le lendemain, préparer titrages de NTBC dans un bouillon LB. Utilisez une gamme initiale de 0 à 900 uM NTBC depuis différentes souches ont des différences de sensibilité à NTBC. <ol><li> Ajouter 1 ml de bouillon LB à 4 à 5 tubes à essai (16 x 150 mm) par souche. </li><li> Ajouter le bouillon solution NTBC (75,9 mM) pour les tubes à essai (16 x 150 mm) dans une gamme de concentrations. Voir le tableau 1 pour les concentrations NTBC et les volumes d&#39;achat d&#39;actions correspondantes à ajouter à 1 ml de bouillon LB. </li></ol></li><li> Mesurer la DO 600 des cultures de la nuit. Laver les cultures avant de prendre OD 600 lectures pour éliminer pyomelanin présente dans les médias. <ol><li> Laver les cultures par centrifugation 1 ml de la culture dans une microcentrifugeuse à 16 000 g pendant 2 min. Retirer le surnageant et des cellules faiblement granulés avecune micropipette et remettre en suspension le culot cellulaire solide dans 1 ml LB. </li></ol></li><li> Ensemencer les tubes de titrage à DO 600 0,05. Calculez le montant de la culture lavé nécessaire pour inoculer les tubes. REMARQUE: Utilisez les cultures lavées pour inoculations depuis pyomelanin ne devraient pas être présents. </li><li> Incuber les tubes de titrage pour environ 24 heures à 37 ° C avec aération à l&#39;aide d&#39;un rotateur de culture tissulaire dans un incubateur à air. </li><li> Photographier les tubes de titrage et de comparer la production de pigments à l&#39;intérieur et entre les souches afin de déterminer la quantité de NTBC à utiliser pour MIC et le stress oxydatif dosages. Utilisez OD 600 lectures pour déterminer le montant de pyomelanin dans la cellule libre surnageant de culture et pour déterminer la densité cellulaire. Remarque: Le ratio DO 600 de pyomelanin dans le surnageant de culture de cellules peut être calculée à quantifier les différences de production pyomelanin après le traitement avec NTBC. </li></ol> 3. antibiotiques INHIBI minimumConcentration Tory (MIC) Dosage des plaques à 96 puits <ol><li> Mettre en place des cultures de la nuit des souches à tester dans du LB avec et sans NTBC. NOTE: Ce protocole est décrit en utilisant le niveau représentatif de 300 um NTBC. Le niveau de NTBC approprié pour être utilisé dans l&#39;étape est déterminé 2.6. <ol><li> Ajouter 300 um NTBC à 2 ml LB. Ajouter un volume équivalent de véhicule (DMSO) à 2 ml de LB pour la condition de ne NTBC. </li><li> Utiliser des cure-dents stériles, ensemencer des tubes avec une colonie isolée de bactéries. Il y aura une culture avec NTBC et une culture sans NTBC pour chaque souche. Incuber pendant une nuit à 37 ° C avec aération à l&#39;aide d&#39;un rotateur de culture tissulaire dans un incubateur à air. </li></ol></li><li> Le lendemain, faire LB + NTBC et des solutions de maître LB + DMSO pour la mise en place du test MIC. Ajouter NTBC à une concentration de 600 uM que ce sera diluée deux fois lorsque l&#39;inoculum est ajouté, donnant une concentration finale de 300 um. <ol><li> Pour tester un Antibiotique pour une souche, ajouter 600 um NTBC à 2 ml de LB et mélanger pour rendre la solution maître NTBC. Ajouter un volume équivalent de véhicule (DMSO) à 2 ml de LB et mélanger pour faire le pas de solution de maître NTBC. Utiliser ces solutions pour créer des solutions mères d&#39;antibiotiques, ainsi que pour mettre en place la série de dilutions dans des plaques à 96 puits. NOTE: Les formulations de solutions de maître seront obtenir une solution supplémentaire pour tenir compte des erreurs de pipetage. Les solutions de base peuvent être mises à l&#39;échelle supérieure ou inférieure selon les besoins en fonction du nombre des antibiotiques et des souches testées. </li></ol></li><li> Préparer les solutions d&#39;antibiotiques dans les solutions de maître LB + NTBC ou LB + DMSO. NOTE: La concentration de l&#39;antibiotique dans ces solutions devrait être le double de la concentration souhaitée finale. Assez solution doit être fait pour transférer 100 pi de quatre puits dans une plaque de 96 puits. <ol><li> Préparer la gentamicine +/- solution stock NTBC à 64 pg / ml. Pour rendre cette solution, ajouter 0,288 ul d&#39;actions gentamicine (100 mg / ml) à 450ul LB + NTBC ou une solution de maître LB + DMSO. NOTE: La concentration maximale de gentamicine pendant P. aeruginosa est PAO1 32 ug / ml. </li><li> Faire de la kanamycine +/- solution stock NTBC à 256 pg / ml. Pour rendre cette solution, ajouter 3,84 pi de kanamycine de stock (30 mg / ml) à des solutions de maître LB + NTBC ou LB + DMSO 450 pi. NOTE: La concentration maximale de kanamycine pour P. aeruginosa est PAO1 128 ug / ml. </li><li> Préparer tobramycine +/- solution stock NTBC à 8 pg / ml. Pour rendre cette solution, ajouter 0,36 pi de tobramycine de stock (10 mg / ml) à des solutions de maître LB + NTBC ou LB + DMSO 450 pi. NOTE: Pour P. PAO1 aeruginosa, la concentration maximale de tobramycine est de 4 ug / ml. REMARQUE: Les antibiotiques et les concentrations peuvent être ajustés pour les bactéries à tester. </li></ol></li><li> Ajouter 100 ul de chaque solution antibiotique 2x à quatre puits dans une plaque à 96 puits. Placez ces solutions dans la ligne A. Pour example, la gentamicine doit être placé dans A1 à A4, kanamycine doit être placé dans A5 à A8, et la tobramycine doit être placé dans A9 travers A12. Voir Figure 1A pour un schéma d&#39;une plaque de 96 puits mis en place. NOTE: Plusieurs antibiotiques peuvent être testés dans une plaque, mais une seule souche doivent être testés par plaque pour éliminer le risque de contamination croisée à partir d&#39;autres souches. </li><li> Ajouter 50 pi de la solution de maître LB + NTBC ou LB + DMSO aux rangées B à H de la plaque de 96 puits. Veiller à ce que une plaque est LB + NTBC et une plaque est LB + DMSO. Voir Figure 1A. <ol><li> Utilisez LB + NTBC pour les antibiotiques dans LB + NTBC. Utilisation LB + DMSO pour les antibiotiques dans LB + DMSO. </li></ol></li><li> Aide d&#39;un micro effectuer deux dilutions en série pliage des antibiotiques en transférant 50 pi de la solution de la ligne A à la ligne B. Mélangez la solution, changer les embouts de pipette et transférer 50 pi de la solution de la ligne B à ramer C. Répétez l&#39;opération pour l&#39;remainirangées Ng. Après dilution rangée G, retirez 50 pi de la solution de cette ligne et de défausse. Utilisez la rangée H comme aucun contrôle antibiotique pour la croissance bactérienne. Voir Figure 1B. REMARQUE: Chaque puits dans les rangées A à G contient maintenant 50 pi d&#39;antibiotiques dans LB + NTBC ou LB + DMSO à 2X la concentration souhaitée finale. Rangée H contient NTBC LB + LB + DMSO ou sans antibiotiques. </li><li> Mesurer la DO 600 des cultures de la nuit. Lavez toutes les cultures avant de prendre OD 600 lectures pour éliminer pyomelanin présente dans les médias. <ol><li> Laver les cultures par centrifugation 1 ml de la culture dans une microcentrifugeuse à 16 000 g pendant 2 min. Retirer le surnageant avec une micropipette et remettre en suspension le culot cellulaire dans 1 ml de LB. </li></ol></li><li> Diluer les cultures d&#39;une nuit à 2.75x10 5 UFC / ml en LB. REMARQUE: On suppose qu&#39;une unité OD 600 est l&#39;équivalent de 1x10 9 UFC / ml de P. aeruginosa. OD UFC / ml conversions peut être DDIFFÉRENTES dans d&#39;autres bactéries. </li><li> Ajouter 50 ul de la culture bactérienne diluée au puits approprié. REMARQUE: Les cultures cultivées dans NTBC doivent être ajoutés dans les puits contenant NTBC et cultures cultivées dans du DMSO doit être ajouté dans les puits contenant le DMSO. Voir Figure 1B. <ol><li> Ajouter bactéries trois puits pour chaque souche et la concentration antibiotique. Ajouter 50 pi de LB pour la quatrième et à agir en tant que témoin de la contamination bactérienne. Voir Figure 1B. </li><li> Utilisez une micropipette multi-canal pour inoculer les puits. Veiller à ce que la pipette conseils sont près du fond des puits lors de l&#39;ajout inoculum pour prévenir la contamination des puits voisins. REMARQUE: Ajout de culture bactérienne dans les puits va diluer les concentrations d&#39;antibiotiques et NTBC double. </li></ol></li><li> Couvrir les plaques à 96 puits avec du parafilm et incuber environ 24 heures à 37 ° C. Incuber les plaques à 96 puits de façon statique, dans un incubateur à air. </li><li> Examinerles plaques pour la croissance bactérienne dans les puits. La CIM est la concentration la plus faible de l&#39;antibiotique à laquelle aucune croissance bactérienne est observée pour les trois répétitions de chaque souche. <ol><li> Examiner la plaque de croissance ou de lire en utilisant un lecteur de plaque d&#39;ensemble DO 600. </li></ol></li></ol> 4. Coin plaque de dosage pour réponse au stress oxydatif <ol><li> Mettre en place des cultures de la nuit des souches à tester dans du LB avec et sans NTBC comme décrit dans l&#39;étape 3.1. </li><li> Le jour suivant, la préparation des plaques de gélose LB contenant H 2 O 2 comme un facteur de stress oxydatif. Une gamme de H 2 O 2 concentrations de 0 à 1 mm est un bon point de départ. <ol><li> Faire fondre les flacons de gélose LB. Refroidir les médias à environ 50 ° C à la température ambiante. </li><li> Ajouter H 2 O 2 directement aux médias refroidis aux concentrations désirées. Flacons Agiter pour mélanger. Voir le tableau 2 pour les concentrations de H 2 O 2et les volumes de H 2 O 2 concentré à ajouter. Ces valeurs sont basées sur 100 ml de LB agar. </li><li> Verser plaques immédiatement après l&#39;addition de H 2 O 2 et de la flamme de la surface pour éliminer les bulles. Le rendement est de 4 plaques par 100 ml de gélose LB. Marquer les plaques avec la concentration de H 2 O 2. </li><li> Placez les plaques découverts dans une hotte à flux biologique avec le ventilateur en marche pendant 30 min pour éliminer l&#39;excès d&#39;humidité des plaques. REMARQUE: Utilisez les plaques de la même journée, ils sont préparés. Ne pas le faire peut conduire à des données incohérentes. NOTE: les facteurs de stress oxydatif tels que le paraquat peut être remplacé par H 2 O 2 dans cet essai. Les concentrations utilisées pour les facteurs de stress oxydatif d&#39;autres peuvent être différents de ceux utilisés pour H 2 O 2. </li></ol></li><li> Lavez et mesurer l&#39;OD 600 des cultures de la nuit tel que décrit dans l&#39;étape 3.7. </li><li> Normaliser l&#39;OD 600 de toute la nuit cultures à la plus faible valeur pour l&#39;ensemble des souches testées. P. aeruginosa présente généralement une DO600 d&#39;environ 2,5 lorsqu&#39;il est cultivé pendant la nuit dans LB + NTBC ou LB + DMSO. <ol><li> Déterminer le volume de culture nécessaire pour diluer la culture à la densité optique la plus basse 600 dans un volume total de 1 ml. Par exemple, si une culture a une DO 600 de 3 et le plus bas OD 600 pour l&#39;ensemble des souches est de 2,5, effectuer le calcul suivant: (2.5) (1 ml) = (3) (x). x = 0.833 ml. 0.833 ml de culture seront placés dans un tube de micro. </li><li> Calculer la quantité de LB + NTBC (300 uM) ou de LB + DMSO nécessaire pour porter le volume de culture de 1 ml. Pour l&#39;exemple à l&#39;étape 4.4.1, la quantité de LB + NTBC ou LB + DMSO ajouté à la culture serait 0,167 ml (1 ml de volume total - 0,833 ml de culture). Faire des solutions mères de LB + NTBC et LB + DMSO à utiliser pour ces dilutions basé sur le volume nécessaire pour diluer toutes les souches. </li><li> Mélanger la culture et LB + NTBC ou LB + DMSO au vortex. </li></ol></li><li> Afin de maintenir les cultures à une concentration constante de NTBC ou le DMSO, des dilutions en série de dix effectuer pliage des cultures d&#39;une nuit dans du PBS + normalisées NTBC ou PBS + DMSO. <ol><li> Faire des solutions d&#39;achat d&#39;actions de PBS + NTBC et PBS + DMSO. Pour un ensemble de dilutions pour une souche, mélanger 300 um NTBC ou un volume équivalent de DMSO avec du PBS pour obtenir un volume total de 720 ul. Échelle ces stocks haut ou le bas selon le nombre de souches sont testées. </li><li> Étiquette tubes de microcentrifugation à 10 -1 à -7 10 dilutions en série. Ajouter 90 pl de PBS ou du PBS + NTBC + DMSO dans les tubes appropriés. Utilisation du PBS + NTBC pour les souches cultivées dans du milieu LB + NTBC et en utilisant du PBS + DMSO pour les souches cultivées dans du milieu LB + DMSO. </li><li> Ajouter 10 ul de la culture à la 10 -1 tube de dilution appropriée. Mélanger par tourbillonnement et transférer 10 pi de la dilution 10 -1 à 10 -2 tube de dilution. Répétez jusqu&#39;à ce que toutes les dilutions ont été performée. Changer conseils pipette entre dilutions. </li></ol></li><li> Spot 5 pi de la 10 -3 à travers 10 -7 dilutions sur LB + H 2 O 2 plaques en double pour chaque souche. Utilisez une pipette si des points sont étalées de plus dilué à moins diluée (10 -7 à 10 -3). Ne pas basculer ou incliner la plaque jusqu&#39;à ce que le liquide a séché dans la plaque. </li><li> Incuber les plaques pendant 24-48 h à 37 ° C (incubateur à air), en fonction de la souche. REMARQUE: P. aeruginosa PAO1 aura de bonnes colonies de taille sur LB après 24 heures d&#39;incubation. Incuber les souches jusqu&#39;à ce qu&#39;ils aient colonies environ la même taille que PAO1. </li><li> Photographier les plaques à l&#39;aide d&#39;une caméra CCD-dessus d&#39;une transluminator. Eventuellement, modifier les photos pour le contraste et la culture à la même taille. Comptez le nombre de colonies dans chaque endroit pour déterminer les changements dans la sensibilité au stress oxydatif. </li></ol>

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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.

Titrages NTBC Les titrages NTBC ont été utilisés pour déterminer si NTBC était en mesure de réduire la production de pyomelanin dans P. aeruginosa, et également d&#39;identifier la concentration de NTBC qui élimine ou réduit pyomelanin production pour utilisation dans des essais supplémentaires. Il peut y avoir des variations dans les niveaux de pyomelanin produite dans différentes répétitions, mais les tendances générales restent constants. L&#39;essai de ti...

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).

Méthodes pour inhiber bactérienne Pyomelanin production et de déterminer l&#39;augmentation correspondante de la sensibilité au stress oxydatif

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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JoVE Journal

Immunology and Infection

8.1K vues.  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).

Vidéo: 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.

Présentation de contrainte de cisaillement dans l&#39;étude de l&#39;adhésion bactérienne

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

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Immunologie et infection

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.

Utilisation de l&#39;Essai de superposition pour mesurer qualitativement la production bactérienne et la sensibilité à pneumocoque bactériocines

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.

Méthodes pour augmenter la sensibilité de la haute résolution de fusion Single Nucleotide Polymorphism génotypage dans le paludisme

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 et in vivo modèle pour étudier l&#39;adhésion bactérienne au navire muraux dans des conditions de débit

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.

Neutrophiles Isolement et analyse pour déterminer leur rôle dans le lymphome cellulaire Sensibilité aux agents thérapeutiques

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.

Une méthode rapide pour le traitement du sang pour augmenter le rendement des niveaux peptidiques plasma dans le sang humain

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.

La surexpression et la purification de l&#39;être humain<em&gt; Cis</em&gt; -prenyltransférase dans<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.

Isolement de Salmonella typhimurium-contenant des Phagosomes des Macrophages

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

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

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

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

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

Un nouveau système exempt d’alimentation pour la Production de masse des cellules tueuses naturelles murins 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.

La cartographie métabolique : Cytochimie enzymatique Quantitative et histochimie pour déterminer l’activité des déshydrogénases dans les cellules et tissus

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