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. Herstellung von Kulturmedien, Antibiotika, und 2- [2-Nitro-4- (trifluormethyl) benzoyl] -1,3-cyclohexandion (NTBC) <ol><li> Machen LB-Brühe (1% Trypton, 0,5% Hefeextrakt, 0,25% NaCl in H 2 O) und Aliquot in angemessenen Mengen. Sterilisieren Sie durch Autoklavieren. Lagerung bei Raumtemperatur. </li><li> Auf 100 ml LB-Agar (1% Trypton, 0,5% Hefeextrakt, 0,25% NaCl, 1,5% Agar in H 2 O) in 250 ml-Kolben. Sterilisieren Sie durch Autoklavieren und bei Raumtemperatur lagern. Stellen Sie sicher, dass der Agar wird vor dem Gießen in Platten geschmolzen. HINWEIS: Kolben, die 100 ml LB-Agar-Platten werden 4 ergeben. Die Menge des LB-Agar kann verändert werden, um der Anzahl von Platten für den Assay benötigt entsprechen. </li><li> Machen PBS (137 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4, 2 mM KH 2 PO 4) 16. Sterilisieren Sie durch Autoklavieren oder Filtration und bei Raumtemperatur lagern. </li><li> Bereiten Sie die Antibiotika-Stammlösungen für Gentamicin, Kanamycin, eind Tobramycin. <ol><li> Bereiten entsprechenden Antibiotikum stock Konzentrationen für P. aeruginosa-Stämme, die 100 mg / ml Gentamicin, 30 mg / ml Kanamycin und 10 mg / ml Tobramycin. Lösen Sie den Antibiotika in Wasser, Filter zu sterilisieren (0,2 um), und bei 4 ° C. Ändern Sie die Antibiotika und Konzentrationen je nach Bakterium untersucht. </li></ol></li><li> Bereiten Sie die NTBC Stammlösungen. Auflösen 10 mg NTBC in 400 ul DMSO. Dies ergibt eine Konzentration von 75,9 mM NTBC. Shop NTBC Stammlösungen bei -20 ° C. Tau-Lösungen bei Raumtemperatur nach Bedarf. HINWEIS: Verschiedene Quellen von NTBC haben Unterschiede in der Löslichkeit. Bestimmen Sie das passende Fahrzeug, in dem die NTBC basierend auf den Empfehlungen des Herstellers zu lösen und anpassen Schritt 1.5 entsprechend. </li></ol> 2. NTBC Titrationen von Bakterienstämmen <ol><li> Eingerichtet Übernachtkulturen der Stämme zu testen. 2 ml LB-Brühe zu 16 x 150 mm Teströhrchens (einer pro-Stamm) und zu impfen mit 1 isoliert Kolonie aus jedem Stamm. Inkubieren über Nacht bei 37 ° C unter Belüftung auf einem Gewebekultur-Rotator in einem Luft-Inkubator. </li><li> Am folgenden Tag vorzubereiten Titrationen von NTBC in LB-Brühe. Starten Sie mit einem Bereich von 0 bis 900 um NTBC da verschiedene Stämme haben Unterschiede in der Empfindlichkeit gegenüber NTBC. <ol><li> 1 ml LB-Medium auf 4 bis 5 Teströhrchen (16 x 150 mm) pro Stamm. </li><li> Fügen Sie den NTBC Stammlösung (75,9 mm), um die Teströhrchen (16 x 150 mm) in einem Bereich von Konzentrationen. Siehe Tabelle 1 für NTBC Konzentrationen und entsprechende Aktienvolumen auf 1 ml LB-Brühe hinzufügen. </li></ol></li><li> Messen Sie die OD600 der Übernachtkulturen. Kulturen vor der Einnahme von OD 600 Lesungen zu beseitigen in den Medien präsent pyomelanin waschen. <ol><li> Die Kulturen durch Zentrifugieren von 1 ml der Kultur in einer Mikrozentrifuge bei 16.000 × g für 2 min waschen. Entfernen Sie den Überstand und keine lose pelletierten Zellen miteine Mikropipette und Resuspendieren der festen Zellpellet in 1 ml LB </li></ol></li><li> Impfen Titration Röhrchen bei OD 600 0.05. Berechnung der Menge der Kultur gewaschen erforderlich, um die Rohre anzuimpfen. HINWEIS: Verwenden Sie die gewaschenen Kulturen für Impfungen seit pyomelanin sollte nicht vorhanden sein. </li><li> Die Titration Rohre etwa 24 h bei 37 ° C inkubieren mit Belüftung unter Verwendung einer Gewebekultur-Rotator in einem Luft-Inkubator. </li><li> Fotografieren Sie die Titration Rohre und vergleichen Pigmentproduktion innerhalb und zwischen den Stämmen, die Menge an NTBC bestimmen, die für MIC und oxidativer Stress-Tests zu verwenden. Verwenden OD 600 Lesungen, die Menge an pyomelanin in zellfreien Kulturüberstand zu bestimmen, und um die Zelldichte zu bestimmen. HINWEIS: Der OD 600-Verhältnis von pyomelanin in Kulturüberstand von Zellen berechnet werden kann, um Unterschiede in pyomelanin Produktion nach Behandlung mit NTBC quantifizieren. </li></ol> 3. Antibiotika Mindest InhibiTory-Konzentration (MIC) Assay in 96-Well-Platten <ol><li> Eingerichtet Übernachtkulturen der Stämme in LB mit und ohne NTBC getestet werden. HINWEIS: Dieses Protokoll wird mit dem repräsentativen Niveau von 300 um NTBC beschrieben. Die angemessene Höhe der NTBC verwendet werden in Schritt 2.6 festgelegt. <ol><li> In 300 um NTBC bis 2 ml LB Fügen Sie eine gleichwertige Menge von Vehikel (DMSO) auf 2 ml LB für die kein NTBC Zustand. </li><li> Mit einer sterilen Zahnstocher, zu impfen Rohre mit einem isolierten Bakterienkolonie. Es wird eine Kultur mit NTBC und einer Kultur ohne NTBC für jeden Stamm sein. Inkubieren über Nacht bei 37ºC unter Belüftung mit einem Gewebekultur-Rotator in einem Luft-Inkubator. </li></ol></li><li> Am nächsten Tag machen LB + NTBC und LB + DMSO Stammlösungen für die Einrichtung des MIC-Assay. Hinzufügen NTBC bei einer Konzentration von 600 &amp; mgr; M, wie dies zweifach verdünnt, als Impfmaterial zugesetzt werden, wodurch man eine Endkonzentration von 300 uM. <ol><li> Einem antibi testenOhr für einen Stamm, fügen 600 um NTBC bis 2 ml LB und mischen, um den NTBC Stammlösung zu machen. Fügen Sie eine gleichwertige Menge von Vehikel (DMSO) auf 2 ml LB und mischen, die keine NTBC Stammlösung zu machen. Mit diesen Lösungen für die Erstellung Antibiotikum Stammlösungen als auch für den Aufbau der Verdünnungsreihe in 96-Well-Platten. HINWEIS: Die Stammlösung Formulierungen zusätzliche Lösung für Pipettierfehler-Konto zu erhalten. Die Stammlösungen kann nach oben oder unten skaliert, wie abhängig von der Anzahl von Antibiotika und getesteten Stämmen erforderlich. </li></ol></li><li> Bereiten Sie die Antibiotika-Lösungen in den LB + NTBC oder LB + DMSO Stammlösungen. HINWEIS: Die Konzentration des Antibiotikums in diesen Lösungen sollte das Doppelte der gewünschten Endkonzentration sein. Genug Lösung sollte, um 100 &amp; mgr; l bis vier Vertiefungen einer Platte mit 96 Vertiefungen übertragen. <ol><li> Bereiten Gentamicin +/- NTBC Stammlösung bei 64 ug / ml. Um diese Lösung zu machen, fügen Sie 0,288 &amp; mgr; l von Gentamicin Lager (100 mg / ml) auf 450ul LB + NTBC oder LB + DMSO Stammlösung. HINWEIS: Die maximale Konzentration von Gentamicin für P. aeruginosa PAO1 beträgt 32 &amp; mgr; g / ml. </li><li> Machen Sie das Kanamycin +/- NTBC Stammlösung bei 256 ug / ml. Um diese Lösung zu machen, fügen 3,84 &amp; mgr; l Kanamycin Lager (30 mg / ml) und 450 &amp; mgr; l LB + NTBC oder LB + DMSO Stammlösungen. HINWEIS: Die maximale Konzentration von Kanamycin für P. aeruginosa PAO1 beträgt 128 ug / ml. </li><li> Bereiten Tobramycin +/- NTBC Stammlösung bei 8 &amp; mgr; g / ml. Um diese Lösung zu machen, fügen 0,36 &amp; mgr; l von Tobramycin Lager (10 mg / ml) und 450 &amp; mgr; l LB + NTBC oder LB + DMSO Stammlösungen. HINWEIS: Für P. aeruginosa PAO1, die maximale Konzentration von Tobramycin beträgt 4 &amp; mgr; g / ml. HINWEIS: Die Antibiotika und Konzentrationen eingestellt, damit das Bakterium getestet werden kann. </li></ol></li><li> Füge 100 ul je 2x Antibiotikalösung zu vier Vertiefungen einer Platte mit 96 Vertiefungen. Legen Sie diese Lösungen in der Reihe A. Für example sollte Gentamycin in den A1 bis A4 platziert werden sollte Kanamycin in A5 bis A8 angeordnet werden und Tobramycin sollte A9 bis A12 gelegt werden. Siehe 1A für ein Schaltbild einer 96-Well-Platte einzurichten. HINWEIS: Mehrere Antibiotika kann in einer Platte getestet werden, aber nur ein Stamm sollte pro Platte getestet, um das Risiko einer Kreuzkontamination von anderen Stämmen zu beseitigen. </li><li> In 50 ul der LB + NTBC oder LB + DMSO Stammlösung, um Zeilen B bis H der 96-Well-Platte. Stellen Sie sicher, dass eine Platte LB + NTBC und eine Platte LB + DMSO. Siehe 1A. <ol><li> Verwenden Sie LB + NTBC für die Antibiotika in LB + NTBC. Verwenden Sie LB + DMSO für die Antibiotika in LB + DMSO. </li></ol></li><li> Mit einer Mikroführen zweifachen Reihenverdünnungen der Antibiotika durch die Übertragung von 50 ul der Lösung von Reihe zu Reihe A B. Mischen Sie die Lösung, ändern Sie die Pipettenspitzen, und übertragen Sie 50 ul der Lösung von Reihe B C. Wiederholen Sie zum zu rudern die remaining Zeilen. Nach Verdünnen Reihe G, entfernen Sie 50 ul der Lösung aus dieser Reihe und entsorgen. Verwenden Reihe H als ohne Antibiotika-Steuerung für das Bakterienwachstum. Siehe Abbildung 1B. HINWEIS: Jedes der gut in den Reihen A bis G enthält nun 50 ul von Antibiotika in LB + NTBC oder LB + DMSO bei 2X die endgültige gewünschte Konzentration. Row H enthält LB + NTBC oder LB + DMSO ohne Antibiotika. </li><li> Messen Sie die OD600 der Übernachtkulturen. Alle Kulturen vor der Einnahme von OD 600 Lesungen in den Medien präsent pyomelanin beseitigen waschen. <ol><li> Die Kulturen durch Zentrifugieren von 1 ml der Kultur in einer Mikrozentrifuge bei 16.000 × g für 2 min waschen. Entfernen Sie den Überstand mit einer Mikropipette und Zellpellet in 1 ml LB </li></ol></li><li> Verdünnen Sie die Übernachtkulturen zu 2.75x10 5 CFU / ml in LB. HINWEIS: Es sei angenommen, dass eine OD 600-Einheit ist das Äquivalent von 1x10 &amp; sup9; CFU / ml von P. aeruginosa. OD, um CFU / ml Umwandlungen können d werdenifferent in anderen Bakterien. </li><li> Dann werden 50 ul der verdünnten Bakterienkultur zu dem geeigneten gut. HINWEIS: Kulturen in NTBC wachsen sollten an die Vertiefungen, die NTBC und Kulturen in DMSO wachsen sollten an die Vertiefungen, die DMSO hinzugefügt werden, hinzugefügt werden. Siehe Abbildung 1B. <ol><li> In Bakterien drei Brunnen für jeden Stamm und Antibiotikakonzentration. Dann werden 50 ul LB dem vierten gut, um als Kontrolle für die bakterielle Kontaminierung zu handeln. Siehe Abbildung 1B. </li><li> Verwenden Sie eine Mehrkanal-Mikropipette, um die Brunnen zu impfen. Sicher, dass die Pipettenspitzen sich in der Nähe der Boden der Vertiefungen beim Hinzufügen Inokulum um eine Kontamination benachbarter Vertiefungen zu verhindern. HINWEIS: Durch Hinzufügen Bakterienkultur in die Vertiefungen wird der Antibiotikum und NTBC Konzentrationen zu verdünnen zweifach. </li></ol></li><li> Decken Sie die 96-Well-Platten mit Parafilm und Inkubation etwa 24 Stunden bei 37 ° C. Inkubieren Platten mit 96 Vertiefungen statisch in einem Luft Inkubator. </li><li> Untersuchendie Platten für das Bakterienwachstum in den Vertiefungen. Die MHK ist die niedrigste Konzentration des Antibiotikums in der kein Bakterienwachstum wird für alle drei Wiederholungen jedes Stammes gesehen. <ol><li> Optisch prüfen Sie die Platte für das Wachstum oder lesen unter Verwendung eines Plattenlesegerät-Set bis zu einer OD 600. </li></ol></li></ol> 4. Spot-Platten-Assay für die oxidative Stressreaktion <ol><li> Eingerichtet Übernachtkulturen der Stämme in LB mit und ohne NTBC wie in Schritt 3.1 beschrieben, getestet werden. </li><li> Am nächsten Tag vorzubereiten LB-Agarplatten, die H 2 O 2 als oxidative Stressor. Eine Reihe von H 2 O 2 -Konzentrationen von 0 auf 1 mM ist ein guter Ausgangspunkt. <ol><li> Schmelzen Sie die LB-Agar-Kolben. Kühles Medium auf etwa 50 ° C bei Raumtemperatur. </li><li> Hinzuzufügen H 2 O 2 direkt an den gekühlten Medien in den gewünschten Konzentrationen. Swirl Flaschen zu mischen. Siehe Tabelle 2 für die Konzentration von H 2 O 2und Volumina konzentrierter H 2 O 2 zu. Diese Werte beziehen sich auf 100 ml LB-Agar basiert. </li><li> Platten gießen unmittelbar nach Zugabe von H 2 O 2 und Flamm der Oberfläche, um Blasen zu entfernen. Die Ausbeute beträgt 4 Platten pro 100 ml LB-Agar. Markieren die Platten mit dem H 2 O 2 -Konzentration. </li><li> Legen Sie die Platten in einer biologischen Flow-Haube mit dem Lüfter läuft für 30 Minuten unbedeckt, um überschüssige Feuchtigkeit aus den Platten zu entfernen. HINWEIS: Verwenden Sie die Platten am selben Tag sie hergestellt werden. Bei Nichtbeachtung kann zu inkonsistenten Daten führen. HINWEIS: Oxidativer Stressfaktoren wie Paraquat für H 2 O 2 in diesem Test ausgewechselt werden. Die für andere oxidative Stressoren verwendeten Konzentrationen kann anders als die für H 2 O 2 verwendet wird. </li></ol></li><li> Waschen Sie und Messen der OD 600 der Übernacht-Kulturen wie in Schritt 3.7 beschrieben. </li><li> Normalisieren die OD 600 von der ganzen Nacht cultures auf den niedrigsten Wert für die Menge der Stämme getestet. P. aeruginosa hat im allgemeinen eine OD 600 von etwa 2,5, wenn über Nacht in LB + NTBC oder LB + DMSO gewachsen. <ol><li> Bestimmung der Kulturvolumen benötigt wird, um die Kultur auf die niedrigste OD 600 in einem Gesamtvolumen von 1 ml zu verdünnen. Zum Beispiel, wenn eine Kultur eine OD 600 von 3 und der niedrigste OD 600 für die Menge der Stämme ist 2.5, die folgende Rechnung aus: (2,5) (1 ml) = (3) (x). x = 0,833 ml. 0,833 ml der Kultur in ein Mikrozentrifugenröhrchen platziert werden. </li><li> Berechnung der Menge an LB + NTBC (300 uM) oder LB + DMSO benötigt, um das Kulturvolumen auf 1 ml zu bringen. Zum Beispiel in Schritt 4.4.1 wird die Menge an LB + NTBC oder LB + DMSO zu der Kultur gegeben wäre 0,167 ml (1 ml Gesamtvolumen - 0.833 ml Kultur) sein. Machen Stammlösungen LB + NTBC und LB + DMSO für diese Verdünnungen auf der Basis des zum Verdünnen aller Stämme benötigte Volumen zu verwenden. </li><li> Mischen Sie die Kultur und LB + NTBC oder LB + DMSO durch Vortexen. </li></ol></li><li> Kulturen in einer konstanten Konzentration von NTBC oder DMSO zu erhalten, auszuführen zehnfache serielle Verdünnungen der normalisierten Übernachtkulturen in PBS + NTBC oder PBS + DMSO. <ol><li> Machen Stammlösungen von PBS + NTBC und PBS + DMSO. Für einen Satz von Verdünnungen für einen Stamm, mix 300 um NTBC oder eine gleichwertige Menge von DMSO mit PBS auf ein Gesamtvolumen von 720 &amp; mgr; l zu ergeben. Skalieren Sie diese Aktien nach oben oder unten, je nachdem, wie viele Stämme getestet. </li><li> Label-Mikrozentrifugenröhrchen für 10 -1 bis 10 -7 Verdünnungsreihen. Werden 90 &amp; mgr; l PBS + NTBC oder PBS + DMSO in die entsprechenden Tuben. Verwenden PBS + NTBC für die Stämme in LB + NTBC gewachsen und benutzen PBS + DMSO für die Stämme in LB + DMSO gewachsen. </li><li> Zugabe von 10 &amp; mgr; l der Kultur in die entsprechende 10 -1 Verdünnungsröhrchen. Vortexen und übertragen 10 ul der 10 -1 Verdünnung auf 10 -2 Verdünnungsröhrchen. Wiederholen, bis alle Verdünnungen wurden perfoRMED. Ändern Pipettenspitzen zwischen Verdünnungen. </li></ol></li><li> Spot 5 ul der 10 -3 bis 10 -7 Verdünnungen auf LB + H 2 O 2 Platten in doppelter Ausführung für jeden Stamm. Verwenden Sie eine Pipettenspitze, wenn Flecken überzogen von den meisten zu verdünnen, um mindestens verdünnter (10 -7 bis 10 -3). Nicht kippen oder neigen Sie die Platte, bis die Flüssigkeit in die Platte getrocknet. </li><li> Die Platten für 24-48 h bei 37 ° C (Luftinkubator) in Abhängigkeit von der Belastung. HINWEIS: P. aeruginosa PAO1 wird recht großen Kolonien auf LB nach 24 Stunden Inkubation haben. Stämmen inkubieren, bis sie Kolonien etwa die gleiche Größe wie PAO1. </li><li> Fotografieren Sie die Platten unter Verwendung einer CCD-Kamera über einem transluminator. Bearbeiten Sie optional die Fotos für Kontrast und Ernte auf die gleiche Größe. Zählen die Anzahl der Kolonien in jedem Spot zu Veränderungen in der Empfindlichkeit gegenüber oxidativem Stress zu bestimmen. </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> Molecular cloning: a laboratory manual. , (2001).</li><li>Roy-Burman, 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=Type+III+protein+secretion+is+associated+with+death+in+lower+respiratory+and+systemic+Pseudomonas+aeruginosa+infections.">Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections.</a> J Infect Dis. 183, 1767-1774 (2001).</li><li>Jacobs, M. 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=Comprehensive+transposon+mutant+library+of+Pseudomonas+aeruginosa.">Comprehensive transposon mutant library of Pseudomonas aeruginosa.</a> Proc Natl Acad Sci USA. 100, 14339-14344 (2003).</li><li>Youngchim, S., Pornsuwan, S., Nosanchuk, J. 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.

NTBC Titrationen Die NTBC Titrationen wurden verwendet, um festzustellen, ob NTBC konnte pyomelanin in P. reduzieren aeruginosa, und auch die Konzentration von NTBC, die eliminiert oder reduziert pyomelanin Produktion für die Verwendung in weiteren Assays identifizieren. Es können Variationen in der Höhe der pyomelanin in verschiedenen Replikationen hergestellt sein, aber allgemeine Trends konstant bleiben. Die NTBC Titrationsassay könnte auch modifiziert werden, um and...

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).

Verfahren zur Inhibierung Bakterielle Pyomelanin Produktion und Bestimmen der entsprechenden Erhöhung der Empfindlichkeit gegenüber oxidativem Stress

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

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

Research

JoVE Journal

Immunology and Infection

8.1K Aufrufe.  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).

Serie: 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.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

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

Forschung

Immunologie und Infektion

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.

Verwendung des Overlay-Assay zur qualitativen Messung bakterielle Produktion und die Sensibilität für Pneumokokken Bacteriocine

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.

Methoden, um die Sensitivität der High Resolution Melting Single Nucleotide Polymorphism Genotypisierung in Malaria erhöhen

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- und In-vivo-Modell, um die bakterielle Adhäsion an der Gefäßwand unter Strömungsbedingungen Studieren

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.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

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.

Eine schnelle Methode zur Blutverarbeitung die Ausbeute an Plasma-Peptidkonzentrationen im menschlichen Blut zu erhöhen

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.

Überexpression und Reinigung des Menschen<em&gt; Cis</em&gt; -prenyltransferase in<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 ...

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.

Ein neuartiges Feeder-freie System für die Massenproduktion von murinen 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.

Metabolische Mapping: Quantitative Enzym Cytochemistry und Histochemie die Aktivität des Dehydrogenases in Zellen und Geweben zu bestimmen

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

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

Histologischen Quantifizierung Lunge Pilz Belastung in experimentellen Aspergillose bestimmen

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...