We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.

참고 :이 6206는 avirulent 변형 17, 18 결핵균의 MC 바와 같이,이 프로토콜의 모든 작품은 바이오 안전성 레벨 2 시설 (BSL-2)에서 수행 할 수 있습니다. M. 결핵 엠씨 2 표현 녹색 형광 단백질 1. 문화 조건 6206 (MTB -GFP) 참고 : M. 결핵 H37Rv는 영양 요구 균주에게 6206 엠씨 (2) 유도 (Δ panCD, Δ leuCD) 플라스미드 pMN437을 발현하는 GFP가이 프로토콜 16에서 사용되는 형질 전환. 비 GFP 연구자의 변형 쉽게 접근 가능하도록 야생형 균주를 발현과 MTB -GFP의 피로를 대체 할 수있다. 그러나, GFP 발현은 식균 작용과 MTB / 대식 세포의 응집체 형성을 육안으로 확인 가능하게하는 것이 바람직하다. 경도의 경우g의 장기 저장은, MTB -GFP 20 % 글리세롤로 보충 (단계 1.1에서 설명) 전체 7H9 미디어에서 -80 ° C에서 동결했다. <ol><li> 10 % 미들 OADC, 0.02 %의 틸록 사폴, 24 μg의 / mL의 D-판토텐산, 50 μg의 / ML의 L-류신 50 μg의 / mL의 하이 그로 마이신 B를 함께 7H9 매체를 보완하여 완전한 MTB -GFP 미디어 (7H9-C)를 준비 </li><li> MTB -GFP의 한 유리 병을 해동하고, 30 mL의 사각형 바닥이 PETG 플라스크에 7H9-C 10 mL를 추가 할 수 있습니다. </li><li> 회전 진탕 (50 회전)에 37 ° C에서 품어. 광학 밀도 측정 (OD 600) 며칠마다 OD 600 1.0 (약 5-8일)에 도달 할 때까지 가져 가라. </li><li> OD를 0.2 ~ 600 사이에 유지하기 위해 희석 통로 배양. </li><li> 감염의 경우, 대수 성장 단계 (OD 0.6-1 600) 내에서 MTB -GFP의 확립 된 문화를 사용합니다. 큰 덩어리의 문화를 방지하기 위해 수조에 MTB 문화 플라스크를 초음파 처리 (130 W 3 × 5의 펄스) ONC전자 또는 일주일에 두 번 </li></ol> 2. 문화 조건 THP-1 세포 <ol><li> 10 % 열 - 불 활성화 소 태아 혈청, 2 mM L- 글루타민 및 10 mM의 HEPES과 RPMI 1640 배지를 보충함으로써 완전한 THP-1 세포 배지 (RPMI-C)를 준비한다. </li><li> 5 % CO 2의 가습 된 분위기 하에서 37 ℃에서 T-75 플라스크에서 세포를 부화. </li><li> 혈구를 사용하여 세포를 계산하거나 매일 유동 세포 계측법 0.1 mL를 당 만 0.6 사이의 밀도 THP-1 세포를 유지한다. </li></ol> 3. 감염 프로토콜 MTB / 대 식세포 집계 구조를 생성합니다 <ol><li> THP-1 세포의 준비 (96 웰 플레이트 당) <ol><li> 5 분 250 XG에 원심 분리기 7 × 10 6 THP-1 세포. 7 mL의 RPMI-C에 재현 탁. </li></ol></li><li> MTB -GFP 준비 <ol><li> 원심 분리기 2.8 × 10 8 MTB -GFP 스윙 - 양동이 원심 분리기에서 3,200 XG에 5 분에서 유OD 600 1.0 = 3 × 10 8 박테리아 / mL로의 변환을 노래. </li><li> 단계 3.2.1에서와 같이 RPMI-C와 원심 분리기로 한 번 씻으십시오. </li><li> 10 초 7 mL의 RPMI-C에 재현 탁하고 소용돌이. </li></ol></li><li> 감염 참고 : 템플릿 그림 1을 참조하십시오. <ol><li> 배지의 증발을 방지하기 위해 물 림 로우 A 및 H 및 칼럼 1, 12 멸균 물 200 μL를 첨가하여 96- 웰 플레이트를 준비한다. </li><li> 배경 제어 (빈)에 대한 2 열 (G2에 B2) 200 μL RPMI-C를 추가합니다. </li><li> 감염, THP-1 세포 현탁액 (3.1 단계)에 MTB -GFP 서스펜션 (단계 3.2)을 추가하고 피펫으로 잘 섞는다. 최종 THP-1 세포 밀도는 용액 당 5 × 105이고, 감염에 대응하는 다수의 40이다. </li><li> 25 mL의 저수지에 THP-1 / MTB -GFP 서스펜션을 따르십시오. </li><li> (G11를 통해 B3) THP-1 나머지 96 우물에 / MTB -GFP 서스펜션의 200 μL 날기 추가GA 멀티 채널 피펫. 참고 : 여러 96 웰 플레이트로 작업하는 경우, 정기적으로 더 혼합물을 각 웰에 첨가되어 있는지 확인하기 위해 탱크에 남아있는 THP-1 / MTB 서스펜션을 재현 탁. </li><li> 7-10일 5 % CO 2, 37 ℃에서 인큐베이션. </li><li> 천천히 (100)을 제거하여 2 일마다 미디어를 변경 μL 100 μL 멀티 채널 피펫을 사용하여 RPMI-C를 미리 예열 추가 잘 부드럽게 각각의 상단에서 미디어를 보냈다. 우물을 재현 탁하고 우물의 바닥에있는 MTB / 대 식세포 집계를 방해하지 마십시오. </li><li> 시각 MTB / 대식 세포 응집체의 크기의 노트를 가지고, 매일 형광 현미경 (4-10X 목적)에 의해 우물을 검사합니다. 하루에 7-10으로 MTB / 대 식세포 집계가 충분히 커야한다 약효 시험 (제 4 절) 동안 진행 (참조 그림 2 참조). </li><li> 원하는 경우, 자동 세포 이미징 시스템을 이용하여 96 웰 캡처 이미지 끼워밝은 필드와 GFP 필터 MTB / 대 식세포 골재 형성을 문서로 설정합니다. 주 : 사용자가 자동화 된 이미징 시스템에 대한 액세스 권한이없는 경우, 대표적인 웰 GFP 이미지 및 시야 기능을 가진 임의의 적당한 현미경을 사용하여 측정 할 수있다. </li></ol></li></ol> 4. 성장 억제 분석은 MTB / 대 식세포 집계에서 파생 의약품 효능에 대해 MTB 평가하기 <ol><li> 약물 준비 (두 약물에 대한 세중의 조건) 참고 : 템플릿도 1a를 참조하십시오. <ol><li> 별도의 96 웰 플레이트에서 G10까지 B2에 7H9-C 미디어의 125 μL를 추가합니다. </li><li> 단계 4.2.4의 희석을 고려하여 7H9-C의 1 mL에 이중 가장 원하는 최종 농도에서 두 약물을 준비합니다. </li><li> 세중 트리트먼트 각각 우물에 B11-C11-D11와 E11-F11-G11 각 약물의 250 μL를 추가합니다. </li><li> 멀티 채널 피펫을 사용하여 직렬 DILUTE 시험 약물 B10-G10에 B11-G11에서 125 μL를 이동하여 두 가지. 각 단계에서 5 회 피펫 팅하여 섞는다. </li><li> 플레이트에서 (오른쪽에서 왼쪽) 열에 열에서 125 μL를 이동하고, 열 4 후 중지를 계속합니다. </li><li> 4 열을 혼합 한 후, 폐기물 용기에 125 μL를 폐기합니다. 열 2와 3은 배경 (빈)과 긍정적 인 성장 컨트롤로 사용할 수 있도록 어떤 약물을 포함 할 수 없습니다. , 또는 테스트 약물 라이브러리도 1b에 템플릿을 따르 참고. 각각의 96 웰 플레이트는 단일 농도에서 58 약물을 수용 할 수있다. 이 단계 4.2.4 절반에 희석되기 때문에 이중 원하는 최종 농도를 사용하여이 약물 플레이트를 준비합니다. </li></ol></li><li> 약물 치료 : <ol><li> MTB를 포함하는 96 웰 플레이트는 대 식세포 (MTB / 대 식세포 집계) -infected 검색합니다. </li><li> 조심 멀티 채널 피펫과 관련된 모든 웰 (G11 내지 B2)을 디 캔트.두 단계의 방법이 수행 첫번째 플레이트 기울임없이 150 μL를 제거하고 플레이트를 기울 웰의 저부 에지에 피펫 팁을 삽입하여 나머지 미디어 (~ 50 μL)을 제거한다. 대 식세포 MTB / 집계가 잘 바닥에 부착 한, 어떤 자료가 손실 될 수 없습니다. </li><li> 조심스럽게 MTB / 대식 세포 집합체를 포함하는 판의 모든 관련 우물 (G11를 통해 B2)에 7H9-C 미디어의 100 μL를 추가합니다. </li><li> 멀티 채널 피펫을 사용하여 감염 판의 대응 웰에 96 웰 플레이트 (단계 4.1)을 함유하는 약물의 100 μL 옮긴다. </li><li> 밀폐 된 가방에 넣어 37 ℃에서 3 일간 배양한다. </li></ol></li></ol> 레사 주린 미세 적정 분석을 사용하여 정량 약물 효능의 (5) <ol><li> 0.8 mg의 최종 농도 원액 레사 주린 준비 / ㎖가 H 2 O.은 멸균 0.22 ㎛의 공극 크기의 PVDF 막 필터를 통해.</li><li> 1 : 1 비율의 2 레사 주린 원액, H 2 O 및 트윈 80 (H 2 O 20 % 용액)을 혼합하여 용액을 준비 작업 레사 주린. 최종 농도가 0.4 ㎎ / ㎖ 레사 주린의 5 % 트윈 80이다. </li><li> 플레이트 판독기 설치 530 nm의 여기 및 590 nm의 발광 37 ° C에서 24 시간 동안 매 30 분에서 형광을 판독 할 수있는 프로그램을 사용. 37 ° C까지 사전 따뜻한 플레이트 리더. </li><li> 멀티 채널 피펫을 사용하여 약물 치료 플레이트 (단계 4.11)의 모든 관련 우물 (G11를 통해 B2)에 레사 주린 작업 용액 20 μL를 추가합니다. </li><li> 플레이트 리더에 접시를 놓고 단계 5.3에서 설정 프로그램을 시작합니다. 주 : 분석 플레이트의 대량의 처리를 용이하게하기 위해, 37 ° C의 배양기에서 분석을 개발하고 단일 형광 24 시간마다 판독을 수행하기에 충분하다. 이것은 또한 운동 기능 배양 플레이트 리더에 대한 액세스 권한이없는 사용자들에 적용 가능하다. </li></ol>

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M., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+granuloma+in+expansion+and+dissemination+of+early+tuberculous+infection.">The role of the granuloma in expansion and dissemination of early tuberculous infection.</a> Cell. 136 (1), 37-49 (2009).</li><li>Collins, L., Franzblau, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplate+alamar+blue+assay+versus+BACTEC+460+system+for+high-throughput+screening+of+compounds+against+Mycobacterium+tuberculosis+and+Mycobacterium+avium.">Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.</a> Antimicrob Agents Chemother. 41 (5), 1004-1009 (1997).</li><li>Snewin, V. 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=Assessment+of+immunity+to+mycobacterial+infection+with+luciferase+reporter+constructs.">Assessment of immunity to mycobacterial infection with luciferase reporter constructs.</a> Infect Immun. 67 (9), 4586-4593 (1999).</li></ol>

The authors have nothing to disclose.

여기서는 상세히 약효 시험에 적합한 다른 MTB 감염 모델을 설명 하였다. 이 모델은 계정 초기 결핵 약물 개발 과정에서 더 고려를 부여해야 두 가지 주요 요인을 취 약물 침투와 감염시 MTB의 대사 변화에 생리 학적으로 관련 장벽의 존재. 우리는 이전에 우리 감염 모델의 이점을 도시 처리량 호환성 16 감염을 스케일링 가능성을 제시하고 있지만, 여기에서 우리는 96 ...

<html> 결핵 (TB)는 40 년 1 항 결핵 화학 요법의 유용성에도 불구하고 심각한 세계 보건 위협 남아있다. 이 비 준수 (2) 환자에 이르게 여러 약물 조합을 사용하여 6 개월의 긴 치료 기간에 대한 요구에 부분적으로 기인한다. 임상 적으로 승인 된 약물의 개발에 성공 3 사실상 존재이다 최근 약제 내성 결핵의 출현은 상기 전계의 문제를 배합했다. 사실, 철저한 항 결핵 약물 개발에도 불구하고, 단 하나의 약물은 FDA가 지난 40 년 4 임상 사용이 승인되었습니다. 따라서, 항 결핵 약물의 새로운 세대는 시급히이 문제를 해결하기 위해 필요하다. 결핵 신약 개발의 핵심 문제는 임상에서 효능을 시험 관내 활성을 가진 화합물에서 성공적으로 전송의 부족이다= &quot;외부 참조&quot;&gt; 5, 6, 7. 처음에, 목표 기반의 접근 방식은 전체 박테리아 세포로 번역하는 데 실패 방지 MTB 약 5,을 선별하기 위해 사용되었다. MTB 세포가 사용되는 경우에도, 그것은 종종 정확하게 생체 8,9 약물 효능을 예측할 수없는 배양액 성장 배양 물을 사용하여 수행된다. 이러한 문제가 인식되었고 MTB 잠복 또는 MTB 대 식세포를 함유하는 약물 스크리닝 분석은 성공적으로 8, 10, 11, 12을 확립하고있다. 그러나, 이들 고급 분석법 약물 비 혈관 폐 병변 발생 관통 장벽 충분한 고려를하고,하지 감염 부위의 괴사 병소이다. 과연심지어 첫번째 줄 TB 약물 리팜피신위한 차선 투여 인해 생체 조직 및 뇌척수액 (CSF)의 불충분으로 문제시 된 세포 MTB (8, 9)에 대해 효능을 감소뿐만 아니라 13 14 15 침투. 의심 할 여지없이 TB 신약 개발 노력을 향상시킬 것이다 초기 리드 개발 과정에서 계정에 이러한 매개 변수를 걸릴 것 같은, 새로운 모델과 분석으로. 이러한 요구를 해결하기 위해, 우리는 최근 MTB 약물 효능 검사 (16)에 대한 저렴하고 신속하고 BSL-2 호환 대안 감염 모델을 설립했다. 생리 학적으로 관련 세포 침투 장벽을 효과적으로 요약 및 대 식세포 계대를 생성 밀도가 생산 큰 식세포 집계 구조 내에서 MTB 포장이 감염 모델, <em&gt; 잠재 MTB를 닮은 변경된 생리 학적 상태와 MTB. 이 감염 모델로부터 유도 MTB 다른 세포 내 감염 모델과 일치하는 결과를 생성 약효를 평가하는 레사 주린 마이크로 타이 세이 (REMA)와 함께 혈중 농도에 비해 높은 CSF 농도를 달성하기 위해 공통 TB 약물의보고 능력을 잘 상관 관계 16. 여기서는 상세히 REMA를 사용 약제 감수성 테스트 식세포 계대 MTB 적합한 생산하는 MTB / 대식 세포 집합체 구조의 생성을 설명한다. 특히,이 감염 시스템을 후보 항 결핵 약물의 스크리닝과의 호환성을 위해 96- 웰 포맷으로 구성 될 수있는 방법을 보여준다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7H9</td>
			<td>BD Difco</td>
			<td>271310</td>
			<td>Follow manufacturer&#39;s recommendations</td>
		</tr>
		<tr>
			<td>Middlebrook OADC</td>
			<td>BD Biosciences</td>
			<td>212351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyloxapol</td>
			<td>Sigma</td>
			<td>T8761</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma</td>
			<td>P5710</td>
			<td>Prepare 24 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>MP Biomedicals</td>
			<td>194694</td>
			<td>Prepare 50 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>EMD Millipore</td>
			<td>400051</td>
			<td>Prepare 200 mg/ml&#160; stock solution in H2O</td>
		</tr>
		<tr>
			<td>Nalgene Square PETG media bottle</td>
			<td>Thermo Fisher</td>
			<td>2019-0030</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Hyclone</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning</td>
			<td>MT25005CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Hyclone</td>
			<td>SH30237.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 3 plate reader</td>
			<td>Biotek</td>
			<td></td>
			<td>Interchangable with any fluorescent plate reader and microscope</td>
		</tr>
		<tr>
			<td>Gen5 Software</td>
			<td>Biotek</td>
			<td></td>
			<td>Recording and analysis of rezasurin coversion</td>
		</tr>
		<tr>
			<td>Rifampicin&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP2679250</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Moxifloxacin Hydrochloride</td>
			<td>Acros Organics</td>
			<td>457960010</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Resazurin Sodium Salt</td>
			<td>Sigma</td>
			<td>R7017</td>
			<td>Prepare 800 &#956;g/mL stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher Scientific</td>
			<td>T164500</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
	</tbody>
</table>

a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

96 웰 플레이트 형식으로이 감염 모델을 적응의 견고성을 확인하기 위해, 우리는 여기에 그림에 주어진 템플릿에 따라 약물 우리의 96 웰에서 파생 된 MTB의 감수성 감염 리팜피신에 모델 (RIF) 및 목시 플록 사신 (MOXI를) 적응을 조사 (1A). 우리는이 분석의 핵심 MTB / 대식 세포 집합체 구조의 생성을 확실하게함으로써, 스루풋의 호환성 <s...

Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기<em&gt; 결핵균</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

a-high-throughput-compatible-assay-to-evaluate-drug-efficacy-against

Research

JoVE Journal

Immunology and Infection

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

7.7K 조회수.  University of Alabama at Birmingham. New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening. 

비디오: A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

의 성장 결핵은 Mycobacterium Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

연구

면역학 및 감염병학

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

자연의 전송에 결핵 말라리아 동시 감염을 연구하는 실험 모델 결핵균와 변형체 berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

다중 약물 내성을 보여<em&gt; 결핵균</em&gt; 증폭 마이크로 어레이

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

설립과 연구에 높은 처리량 설정 최적화<em&gt; 표피 포도 구균</em&gt;와<em&gt; Mycobacterium의 박테</em&gt; 감염 신약 개발의 모델로

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

면역 적격 설치류에서 강력한 폐렴 모델에 대해 항균 효능을 평가하기<i&gt; S. 폐렴</i&gt;<i&gt; H. 인플루엔자</i&gt;<i&gt; K. 폐렴</i&gt;<em&gt; P. 녹농균</em&gt; 또는<em&gt;를했다. baumannii가</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템<em&gt; 결핵균</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

기자 효소 형광와 쥐에서 이미징 결핵균

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

진 균 marinum 감염 성인 Zebrafish에 모델링 결핵

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기

요약

더 많은 비디오 탐색

이 비디오의 챕터

의 성장 결핵은 Mycobacterium Biofilms

자연의 전송에 결핵 말라리아 동시 감염을 연구하는 실험 모델 결핵균와 변형체 berghei

다중 약물 내성을 보여

설립과 연구에 높은 처리량 설정 최적화

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

면역 적격 설치류에서 강력한 폐렴 모델에 대해 항균 효능을 평가하기

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

기자 효소 형광와 쥐에서 이미징 결핵균

진 균 marinum 감염 성인 Zebrafish에 모델링 결핵