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.

배양기 항생제 1. 제조, 및 2- [2- 니트로 -4- (트리 플루오로 메틸) 벤조일] -1,3- 시클로 헥산 디온 (NTBC) <ol><li> 적절한 볼륨으로 LB 액체 배지 (1 % 트립 톤, 0.5 % 효모 추출물, 0.25 %의 NaCl의 H 2 O)과 분취 액을 만든다. 오토 클레이브 소독. 실온에서 보관하십시오. </li><li> 100 ㎖ LB 한천 (1 % 트립 톤, 0.5 % 효모 추출물, 0.25 % 염화나트륨, H 2 O 1.5 % 한천) 250 mL의 플라스크를 만든다. 실온에서 오토 클레이브 및 저장에 의해 소독. 한천 플레이트에 쏟아져 전에 용융되어 있는지 확인합니다. 주 : 4 판을 수득한다 LB 한천 100 ㎖을 함유하는 플라스크. LB 한천의 양이 분석에 필요한 플레이트의 수에 대응하도록 변경 될 수있다. </li><li> (137 mM의 염화나트륨, 2.7 밀리미터의 KCl, 10 mM의 나 2 HPO 4, 2 mM의 KH 2 PO 4) (16) PBS를 확인합니다. 실온에서 오토 클레이브 또는 여과 및 저장에 의해 소독. </li><li> 겐타 마이신에 대한 항생제 재고 솔루션을 준비, 카나마이신,D 토 브라 마이신. <ol><li> 피에 대한 적절한 항생제 재고 농도를 준비 100 ㎎ / ㎖ 젠타 마이신 30 ㎎ / ㎖의 카나마이신 및 10 ㎎ / ㎖의 토 브라 마이신을 함유 루기 노자 균주. 4 ° C에서 물 필터 소독 (0.2 μm의)와 저장소에 항생제를 녹인다. 연구 박테리아에 따라 항생제 농도를 변경합니다. </li></ol></li><li> NTBC 재고 솔루션을 준비합니다. DMSO 400 μL에 NTBC 10 mg을 녹이고. 이것은 75.9 mM의 NTBC의 농도를 산출한다. -20 ° C에서 보관 NTBC 스톡 솔루션을 제공합니다. 실온에서 해동 용액은 필요에 따라. 참고 : NTBC의 다른 소스는 용해도의 차이가 있습니다. 제조업체의 권장 사항에 따라 NTBC을 용해 단계 1.5 따라서를 조정하는 적절한 차량을 결정합니다. </li></ol> 2. 세균 균주의 NTBC 적정의를 <ol><li> 균주의 설정 하룻밤 문화를 테스트합니다. 2 ㎖의 LB 배지에 16 × 150mm 시험관 추가S (주 당 하나) 및 각 변형에서 1 절연 식민지 접종. 공기 인큐베이터에서 조직 배양 회전기에 통기로 37 ℃에서 밤새 배양한다. </li><li> 다음 날, LB 배지에 NTBC의 적정을 준비합니다. 다른 균주 NTBC에 대한 민감도에 차이가 있기 때문에 0 ~ 900 μm의 NTBC에 초기 범위를 사용합니다. <ol><li> 변형 당 4 ~ 5 시험관 (16 × 150mm)에 1 ㎖의 LB 배지를 추가합니다. </li><li> 농도 범위에서 시험관 (16 × 150mm)에 NTBC 스톡 용액 (75.9 mm)를 추가한다. LB 액체 배지 1 ㎖에 추가 NTBC 농도 및 해당 재고 수량은 표 1을 참조하십시오. </li></ol></li><li> 하룻밤 문화의 OD 600을 측정합니다. 미디어에 존재 pyomelanin 제거하기 위해 OD 600 판독을 복용하기 전에 문화를 씻으십시오. <ol><li> 2 분 동안 16,000 XG에서의 microcentrifuge에 문화 1 ㎖을 원심 분리하여 문화를 씻으십시오. 뜨는 및 느슨하게 펠렛 세포를 제거micropipettor 및 1 ml의 (LB)에 고체 세포 펠렛을 재현 탁 </li></ol></li><li> OD 600 0.05 적정 튜브를 접종. 튜브에 접종하는 데 필요한 세척하고 배양의 양을 계산한다. 참고 : 존재하지 않아야 pyomelanin 때문에 예방 접종에 대한 세척 문화를 사용합니다. </li><li> 폭기는 공기 인큐베이터에서 조직 배양 회전기를 사용하여 37 ℃에서 약 24 시간 동안 적정 튜브 부화. </li><li> 적정 튜브 사진 및 MIC 및 산화 스트레스 분석에 사용할 NTBC의 양을 결정하기 위해 내부 및 균주간에 색소 생산을 비교한다. 무 세포 배양 상층 액에서 pyomelanin의 양을 결정하고 세포 밀도를 결정하기 위해 600 OD 판독을 사용한다. 주 : 세포 배양 상청액에서 pyomelanin OD 600의 비율로 처리 후 NTBC pyomelanin 생산에서의 차이를 정량화하기 위해 계산 될 수있다. </li></ol> 3. 항생제 최소 Inhibi96 웰 플레이트에 토리 농도 (MIC) 분석 <ol><li> 균주의 설정 하룻밤 문화와 NTBC없이 LB에서 시험한다. 주 :이 프로토콜은 300 μM NTBC의 대표적인 레벨을 사용하여 설명한다. 사용될 NTBC의 적절한 수준은 단계 260에서 결정된다. <ol><li> 2 ml의 (LB) 300 μm의 NTBC 추가 더 NTBC 조건에 대해 2 ml의 LB에 차량 (DMSO)의 동등한 볼륨을 추가합니다. </li><li> 멸균 이쑤시개를 사용하여 박테리아의 하나 고립 된 식민지와 튜브를 접종. NTBC 하나의 문화와 각 변형에 대한 NTBC없이 하나의 문화가 될 것입니다. 폭기는 공기 인큐베이터에서 조직 배양 회전기를 사용하여 37 ℃에서 밤새 배양한다. </li></ol></li><li> 다음 날, MIC 분석을 설정하는 LB + NTBC 및 LB + DMSO 마스터 솔루션을합니다. 접종 물이 추가 될 때,이 600 μM의 농도로 추가하기 NTBC 300 μM의 최종 농도를 산출 두 배 희석한다. <ol><li> 하나 antibi을 테스트하려면하나의 변형, 2 ㎖의 LB 600 μm의 NTBC을 추가하고 NTBC 마스터 솔루션을 만들기 위해 혼합에 대한 귀의. 2 ㎖의 LB에 차량 (DMSO)의 동등한 볼륨을 추가하고 더 NTBC 마스터 솔루션을하지 않습니다 섞는다. 항생제 원액을 만들뿐만 아니라 96 웰 플레이트에서 희석 시리즈를 설정하는 이러한 솔루션을 사용한다. 참고 : 마스터 용액 제형은 피펫 오류를 설명하는 별도의 솔루션을 얻을 것입니다. 시험 균주의 항생제와 수에 따라서 필요에 따라 마스터 솔루션은 상하 또는 축소 될 수있다. </li></ol></li><li> LB + NTBC 또는 LB + DMSO 마스터 솔루션의 항생제 솔루션을 준비합니다. 참고 :이 솔루션의 항생제 농도가 두 배 최종 원하는 농도이어야한다. 충분한 용액을 96 웰 플레이트에 웰에 네 100 μl를 전송해야한다. <ol><li> 겐타 마이신 + 64 ㎍ / ml의에서 NTBC 주식 솔루션을 준비합니다. 겐타 마이신 (450)의 콘텐츠 0.288 μL (100 ㎎ / ㎖)을 추가하고,이 용액을 만드는μL LB + NTBC 또는 LB + DMSO 마스터 솔루션입니다. 참고 : P.에 대한 겐타 마이신의 최대 농도 녹농균 PAO1는 32 μg의 / ㎖이다. </li><li> 카나마이신 + 256 μg의 / ㎖에서 NTBC 주식 솔루션을 확인하십시오. 450 μL LB + NTBC 또는 LB + DMSO 마스터 솔루션 카나마이신 주식의 3.84 μL (30 ㎎ / ㎖)를 추가,이 솔루션을 확인합니다. 참고 : P.에 대한 카나마이신의 최대 농도 녹농균 PAO1 128 μg의 / ㎖이다. </li><li> 토 브라 마이신 + 8 μg의 / ㎖에서 NTBC 주식 솔루션을 준비합니다. 이 솔루션을 450 μL LB + NTBC 또는 LB + DMSO 마스터 솔루션에 토 브라 마이신 주식의 0.36 μL (10 ㎎ / ㎖)를 추가합니다. 참고 : P. 들어 aeruginosa의 PAO1, 토 브라 마이신의 최대 농도는 4 μg의 / ㎖이다. 주 : 박테리아 시험되어야하는 항생제 농도가 조절 될 수있다. </li></ol></li><li> 96 웰 플레이트에서 네 개의 우물에 각각 2 배 항생제 용액 100 μl를 추가합니다. exampl에 대한 행 A의 이러한 솔루션을 배치즉, 젠타 A4 통해 A1에 배치해야 카나마이신을 통해 A8 A5에 배치되어야하고, 토 브라 마이신을 통해 A12 A9에 배치되어야한다. 설정 96 웰 플레이트의 그림에 대한 그림 1A를 참조하십시오. 주 : 다중 항생제 한 플레이트에서 시험 할 수 있지만, 단지 하나의 변형은 다른 균주에서 교차 오염의 가능성을 제거하기 위해 접시 당 테스트되어야한다. </li><li> 96 웰 플레이트의 H​​를 통해 행 B에 LB + NTBC 또는 LB + DMSO 마스터 용액 50 μl를 추가합니다. 그 하나의 판을 확인 LB + NTBC이고 하나 플레이트 LB + DMSO입니다. 그림 1A를 참조하십시오. <ol><li> LB + NTBC에서 항생제에 대한 LB + NTBC을 사용합니다. LB + DMSO의 항생제에 대한 LB + DMSO를 사용합니다. </li></ol></li><li> micropipettor가 피펫 팁을 변경, B를 믹스에게 솔루션을 행하기 위해 행에서 용액 50 μl를 전송하여 항생제의 두 배 연속 희석을 수행하여, 및 행 (B)에서 용액 50 μl를 전송하기위한 C. 반복을 행합니다 remainiNG 행. 행 G 희석 한 후, 그 행과 폐기의 용액 50 μl를 제거합니다. 박테리아 성장을위한 항생제 컨트롤로 행 (H)를 사용합니다. 그림 1B를 참조하십시오. 참고 : G를 통해 각각의 잘 행은 지금 배에서 LB + NTBC 또는 LB + DMSO 항생제의 50 μL 최종 원하는 농도가 포함되어 있습니다. 행 H없이 항생제로 LB + NTBC 또는 LB + DMSO가 포함되어 있습니다. </li><li> 하룻밤 문화의 OD 600을 측정합니다. 미디어에 존재 pyomelanin을 제거하기 위해 OD 600 판독을 복용하기 전에 모든 문화를 씻으십시오. <ol><li> 2 분 동안 16,000 XG에서의 microcentrifuge에 문화 1 ㎖을 원심 분리하여 문화를 씻으십시오. micropipettor으로 뜨는을 제거하고 1 ㎖의 (LB)에있는 세포 펠렛을 resuspend </li></ol></li><li> (LB)에서 2.75x10 5 CFU / ㎖로 하룻밤 문화를 희석 참고 : 하나의 OD 600 단위 (P)에 대한 1 × 9 CFU / ㎖의 상응이라고 가정 녹농균. CFU / ㎖ 변환에 OD는 거라고 할 수있다다른 박테리아에 ifferent. </li><li> 적절한 웰에 희석 세균 배양의 50 μl를 추가합니다. 주 : NTBC 성장 DMSO 배양에서 성장 및 배양 NTBC DMSO를 함유하는 웰에 첨가한다 함유 웰에 첨가한다. 그림 1B를 참조하십시오. <ol><li> 각 균주와 항생제 농도 세 우물에 세균을 추가합니다. 세균 오염에 대한 제어 역할을하는 네 번째 우물에 LB의 50 μl를 추가합니다. 그림 1B를 참조하십시오. </li><li> 웰 접종 멀티 채널 micropipettor를 사용한다. 이웃하는 웰의 오염을 방지하기 위해 추가 접종 할 때 팁을 웰의 바닥 근처에 있음을 보장 피펫. 참고 : 우물에 세균 배양을 추가하면 항생제와 NTBC 농도의 두 배를 희석합니다. </li></ol></li><li> 파라 필름으로 96 웰 플레이트를 덮고, 37 ℃에서 약 24 시간 배양한다. 공기 인큐베이터에서 정적으로 96 웰 플레이트를 품어. </li><li> 검사우물에 세균 성장​​을위한 판. MIC는 세균의 성장에는 각 균주의 복제에 대해 세 보지되는 항생제의 최소 농도이다. <ol><li> 시각적으로 성장 판을 검사하거나 (600) 중독에 플레이트 리더 세트를 사용하여 읽기. </li></ol></li></ol> 산화 스트레스 응답 4. 스팟 플레이트 분석 <ol><li> 3.1 단계에 설명 된대로 균주의 설정 하룻밤 문화 NTBC와 LB와없이 테스트 할 수 있습니다. </li><li> 다음날, 산화성 스트레스로 H 2 O 2를 함유하는 LB 아가 플레이트를 준비한다. 0 내지 1 mM의 H의 범위는 2 O 2 농도는 좋은 출발점이다. <ol><li> LB 한천 플라스크 용융. 실온에서 약 50 ° C로 냉각 매체. </li><li> 원하는 농도로 냉각 된 미디어에 직접 O 2 H 2를 추가합니다. 소용돌이 플라스크 혼합합니다. H의 농도는 표 2를 참조하십시오 2 O 2농축 H 2 O 2의 볼륨을 추가 할 수 있습니다. 이러한 값은 LB 한천 100 ㎖에 기초한다. </li><li> 바로 거품을 제거하기 위해 H 2 O 2 불꽃을 표면을 추가 한 후 번호판을 붓고. 수율은 LB 한천의 100 ㎖ 당 4 판입니다. H 2 O 2 농도가 판을 표시합니다. </li><li> 플레이트에서 과도한 수분을 제거하는 30 분 동안 실행 팬 생물학적 흐름 후드에서 발견 판을 놓습니다. 참고 : 판들이 준비되어 같은 날을 사용합니다. 그렇게하지 ​​않으면 일치하지 않는 데이터가 발생할 수 있습니다. 참고 사항 : 파라쿼트 같은 산화 스트레스는이 분석에서 H 2 O 2를 대체 할 수 있습니다. 다른 산화성 스트레스에 사용 농도는 H 2 O 2에 사용되는 것과 다를 수있다. </li></ol></li><li> 세척 단계 3.7에 설명 된대로 외경에게 하룻밤 문화 (600)을 측정한다. </li><li> 모든 하룻밤 CU의 OD 600 정상화균주의 집합의 가장 낮은 값 ltures 테스트중인. P. 녹농균은 일반적으로 LB + NTBC 또는 LB + DMSO에 하룻밤 성장 약 2.5의 OD 600이 있습니다. <ol><li> 1 ml의 총 부피에서 낮은 OD 600 문화를 희석하는 데 필요한 배양의 볼륨을 결정한다. 배양 OD (3)의 600 균주의 세트는 낮은 OD (600)가있는 경우, 예를 들면 2.5 인, 다음의 계산을 수행 (2.5) (1 ㎖)을 = (3) (X). X는 = 0.833 ml의. 문화의 0.833 mL의 미세 원심 분리 튜브에 배치됩니다. </li><li> 1 ml의 배양 부피를 가지고 필요 LB + NTBC (300 μM) 또는 DMSO LB +의 양을 계산한다. 단계 4.4.1에서 예를 들어, LB + NTBC 또는 LB + DMSO의 양이 0.167 ㎖ (- 0.833 ml의 문화 1 ml의 총 부피) 될 문화에 추가됩니다. LB + NTBC 및 LB + DMSO의 재고 솔루션은 모든 균주를 희석에 필요한 볼륨 근거로이 희석에 사용합니다. </li><li> 문화와 LB + N 믹스텍싱에 의해 TBC 또는 LB + DMSO. </li></ol></li><li> NTBC 또는 DMSO의 일정한 농도의 문화를 유지하기 위해, PBS + NTBC 또는 PBS + DMSO의 정규화 하룻밤 문화의 열 배 연속 희석을 수행합니다. <ol><li> PBS + NTBC과 PBS + DMSO의 재고 솔루션을합니다. 하나의 균주에 대한 희석 한 세트의 경우, 720 ㎕의 총 부피를 수득 300 μM의 NTBC 또는 PBS와 DMSO의 동등한 양을 혼합한다. 테스트 얼마나 많은 균주에 따라 위 또는 아래로이 주식을 확장 할 수 있습니다. </li><li> 10-7 연속 희석을 통해 10 -1에 대한 라벨의 microfuge 튜브. 적절한 튜브에 PBS + NTBC 또는 PBS + DMSO의 90 μl를 추가합니다. LB + NTBC에서 재배 변종에 대한 PBS + NTBC를 사용하여 LB + DMSO에 성장 균주에 대한 PBS + DMSO를 사용합니다. </li><li> 해당 10-1 희석 관에 문화의 10 μl를 추가합니다. 텍싱하여 혼합하고 10 -2 희석 튜브에 10 -1 희석의 10 μl를 전송합니다. 모든 희석이 PERFO 때까지 반복rmed. 희석 사이에 피펫 팁을 변경합니다. </li></ol></li><li> 각 변형에 대한 중복에 LB + H 2 O 2 판에 10-7 희석을 통해 10 -3의 자리는 5 μL. 관광 명소 도금하는 경우 대부분은 적어도 희석 (10-7 -3 10) 희석에서 한 피펫 팁을 사용합니다. 팁 또는 액체가 플레이트에 건조 될 때까지 판을 기울이지 마십시오. </li><li> 변형에 따라 37 ° C (공기 인큐베이터)에서 24 ~ 48 시간 동안 접시를 품어. 참고 : P. 녹농균 PAO1는 배양 24 시간 후 LB에 좋은 크기의 식민지를해야합니다. 그들은 콜로니를 PAO1 대략 동일한 크기를 가질 때까지 균주를 배양한다. </li><li> transluminator 위의 CCD 카메라를 사용하여 번호판을 사진. 선택적으로, 같은 크기 대비 및 작물에 대한 사진을 편집 할 수 있습니다. 산화 스트레스에 대한 민감도에서의 변화를 결정하기 위해 각각의 스폿에서의 콜로니 수를 카운트. </li></ol>

<ol>
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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 적정 NTBC가 경찰에 pyomelanin 생산을 줄일 수 있다면 NTBC 적정을 결정하는 데 사용되었다 애 루기 노사, 또한 제거 또는 추가 분석에 사용하기 위해 생산을 감소 pyomelanin NTBC의 농도를 확인한다. 이 다른 복제에서 생성 pyomelanin의 수준에서 변화 될 수 있지만 일반적인 경향은 일정하게 유지 될 수 있습니다. NTBC 적정 분석은 다른 세균 안료 생산에 영향을 미칠 수...

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).

방법은 세균 Pyomelanin 생산을 억제하고 산화 스트레스에 감도에 대응 증가를 확인하는 방법

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 조회수.  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).

비디오: 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.

세균성 접착 연구에 전단 응력을 소개합니다

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

연구

면역학 및 감염병학

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.

오버레이 분석을 사용하여 성적으로 폐렴 구균 박테리오신에 대한 세균의 생산 및 감도를 측정하는

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.

방법은 말라리아에 단일 염기 다형성 유전자형 녹는 고해상도의 감도를 증가

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.

생체 외 및 생체 내 모델에서 혈관 벽에서 흐름 조건에 세균 부착을 연구하기 위해

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.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

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.

혈액 처리를위한 빠른 방법은 인간의 혈액에서 혈장 펩타이드 수준의 수율을 증가하는

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.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</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.

소설 급지대 무료 시스템 대량 생산을 위한 생체 외에서 Murine 자연 킬러 세포의

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.

신진 대사 매핑: 양적 효소 Cytochemistry 및 Histochemistry Dehydrogenases 세포와 조직에서의 활동을 결정 하

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.

조직학 정량화 폐 실험 Aspergillosis에 곰 팡이 부담을 결정 하

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