저자들은 이 연구에 자금을 지원한 낭포성 섬유증 재단(Cystic Fibrosis Foundation)과 항-Psl0096 항체를 아낌없이 기증한 MedImmune에 감사의 뜻을 전합니다. 이 연구를 위해 이미징은 토론토 대학의 CAMiLoD 이미징 시설에서 수행되었습니다.

연구 윤리 위원회(REB)의 승인은 인간 피험자로부터 객담 샘플을 수집하고 보관하는 데 필요합니다. 본 연구에 제시된 연구는 Hospital for Sick Children REB#1000058579의 승인을 받았습니다.
1. 객담 채취
<ol><li>객담을 멸균 채취 컵에 보관하고 즉시 4°C에서 최대 24시간 동안 보관한 후 고정합니다. 알림: 가래를 고정하지 않고 4°C에서 너무 오래 방치하면 세포 분해, 특히 백혈구 분해가 발생할 수 있습니다. 가능한 한 빨리 고정하는 것이 좋습니다.</li>
	<li>객담 검체를 멸균된 15mL 튜브로 옮깁니다.</li>
	<li>객담 샘플에 동일한 부피의 4% 파라포름알데히드(PFA)를 추가합니다. 예를 들어, 객담 샘플이 0.5mL인 경우 0.5mL의 4% PFA를 추가합니다. 부드러운 반전으로 섞는다.</li>
	<li>4 °C에서 밤새 가래를 배양합니다.</li>
	<li>고정 객담 2mL 각각에 5mL의 인산염 완충 식염수(PBS)를 추가하여 샘플을 세척합니다. 알림: 이 세척 단계에는 원심분리가 필요하지 않습니다.</li>
	<li>피펫으로 상층액을 조심스럽게 제거합니다. 알림: 팁을 가래에서 멀어지게 하여 피펫으로 가래를 빨아들이지 말고 표면 액체에서 매우 천천히 흡인하십시오. 세척 단계에는 가래 플러그의 구조적 무결성을 방해하지 않도록 원심 분리가 포함되지 않습니다.</li>
	<li>1.6-1.7단계를 두 번 더 반복합니다.</li>
	<li>펠릿을 PBS 부피의 2배에 0.01%(w/v) 아지드화나트륨으로 재현탁시킵니다.</li>
	<li>객담은 4 °C에서 보관한다.</li>
</ol>2. 객담의 MiPACT(조직 청소 기술) 처리
<ol><li>하이드로겔 성분(30% 29:1 아크릴아미드:비스-아크릴아미드, 경화제 및 PBS)을 혐기성 팩이 들어 있는 밀봉된 용기에서 72시간 전에 가스를 제거합니다. 알림: 산소가 아크릴아마이드 중합을 억제하기 때문에 혐기성 팩과 밀봉된 용기가 필요합니다. 대안적으로, 산소는 혐기성 후드 내에서 이 단계를 수행하거나, 밀봉된 용기에 진공을 적용하거나, 아크릴아마이드 혼합물을 통해N2 가스를 버블링함으로써 제거될 수 있다.<ol><li>밀봉된 혐기성 용기 내부의 뚜껑을 닫은 상태에서 30% 29:1 아크릴아마이드:비스-아크릴아미드 용액 2mL를 15mL 튜브에 추가합니다.</li>
		<li>15mL 튜브에 PBS 5mL에 경화제 0.5g을 첨가하여 10%(w/v) 경화제의 농축액을 만듭니다. 튜브의 뚜껑을 닫고 밀봉된 혐기성 용기에 보관하십시오.</li>
		<li>멸균 PBS 몇 mL를 혐기성 용기 내부의 뚜껑을 닫은 상태로 튜브에 그대로 둡니다.</li>
	</ol></li>
	<li>15mL 튜브에 PBS의 0.2% 경화제와 4% 29:1 아크릴아마이드:비스-아크릴아미드의 최종 농도로 하이드로겔 용액 5mL를 만듭니다. 반전으로 혼합하고 필터 살균합니다.</li>
	<li>냉장고에서 가래 샘플을 꺼낸 다음 메스로 멸균 상태에서 작은 부분(직경 약 5mm)으로 자릅니다. 알림: 가래가 매우 유동적인 경우에도 이 단계를 수행할 수 있지만 메스를 사용하여 원하는 분획을 분리하기 전에 핀셋으로 보관 용액에서 가래를 제거하려면 더 많은 인내심과 연습이 필요합니다.</li>
	<li>절단된 객담 샘플을 8개의 챔버가 있는 커버글라스 슬라이드의 우물 안에 넣습니다.</li>
	<li>300단계에서 필터 멸균 하이드로겔 용액 2.2μL를 객담이 포함된 각 웰에 추가합니다.</li>
	<li>혐기성 팩이 들어 있는 밀폐된 용기 안에 8개의 챔버 커버글라스를 37°C에서 3시간 동안 보관합니다. 알림: 일단 중합되면 가래가 내장된 하이드로겔은 단단한 겔의 농도여야 합니다.</li>
	<li>응고된 하이드로겔 객담 샘플을 pH 8의 8% 도데실 황산나트륨(SDS) 5mL가 들어 있는 15mL 배양 튜브로 옮기고 가래가 투명해질 때까지 37°C(흔들림 유무에 관계없이)에서 3-14일 동안 샘플을 투명하게 둡니다. 알림: 청소 시간은 객담 성분에 따라 다르며 일반적으로 흔들면 줄일 수 있습니다. 그러나 샘플 무결성을 방해할 정도로 진탕 속도를 높이지 않도록 주의하십시오. DNA가 풍부한 샘플은 점액이 풍부한 샘플에 비해 투명화하는 데 더 오래 걸립니다.</li>
	<li>8% SDS 용액을 폐기물 수거 용기에 디캔팅합니다. 멸균 핀셋을 사용하여 각 하이드로겔 포매 샘플을 멸균 50mL 코니컬 튜브로 옮깁니다. 50mL 코니컬 튜브 각각에 PBS 10mL를 추가하여 하이드로겔을 세척하고 용액을 30분에서 1시간 동안 그대로 두었다가 디캔팅합니다. 이것을 2배 더 반복하십시오. 알림: 이 세척 단계에는 원심분리가 포함되지 않습니다. 과도한 체액과 상층액은 피펫으로 조심스럽게 제거합니다.</li>
	<li>세척된 시료를 0.01%(w/v)의 아지드화나트륨 및 1x RNase 억제제와 함께 4°C에서 PBS에 보관합니다.</li>
</ol>3. 하이드로겔 형광 in situ hybridization (FISH) 프로토콜
<ol><li>멸균 핀셋을 사용하여 보관 용액에서 하이드로겔 샘플을 제거하고 멸균 표면(예: 유리 슬라이드 또는 페트리 접시)에 샘플을 놓습니다.</li>
	<li>멸균 메스를 사용하여 하이드로겔을 ~ 1mm 두께로 자릅니다.</li>
	<li>하이드로겔 1mm 절편을 멸균 1.5mL 튜브 안에 넣습니다.</li>
	<li>1.5 mL 튜브에 1 mL 혼성 완충액 (25 % 포름 아미드, 0.9 M NaCl, 20 mM Tris-HCl [pH 7.6], 0.01 % SDS, 정제 및 탈 이온화 H2O)을 준비합니다.</li>
	<li>형광 표지된 PseaerA 프로브(150.7nM12)를 혼성화 버퍼에 추가하고 반전으로 혼합합니다. 알림: 포르말린 용액은 화염에서 멀리 떨어져 있어야 합니다. 혼성화 완충액은 고급으로 제조될 수 있고, -20°C에서 부분 표본으로 저장될 수있다 13.</li>
	<li>하이드로겔의 각 ~1mm 섹션에 200-500μL의 혼성화 완충액을 추가하고 하이드로겔 샘플 전체가 잠기도록 합니다.</li>
	<li>PseaerA 프로브가 흔들리지 않고 46°C에서 ~ 18-24시간 동안 어둠 속에 두어 하이드로겔 샘플과 하이브리드화되도록 합니다.</li>
	<li>혼성화 완충액을 폐기물 수거 용기에 디캔팅합니다.</li>
	<li>필터 멸균 세척 완충액 (337.5 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA (에틸렌 디아민 테트라 아세트산) [pH 7.2], 0.01 % SDS)로 샘플을 한 번 헹구고 1.5 mL 튜브 각각에 세척 완충액 1mL를 첨가하여 정제 및 탈 이온화 된 H2O)를 제거한 다음 제거합니다. 알림: 세척 버퍼는 미리 만들어 실온(RT)에서 보관할 수 있습니다.</li>
	<li>1mL의 신선한 세척 완충액을 튜브에 넣은 다음 어두운 곳에서 48°C에서 6시간 동안 흔들지 않고 샘플을 배양합니다.</li>
</ol>4. 하이드로겔 및 Psl0096 항체 결합
<ol><li>1mL 피펫터를 사용하여 세척 버퍼를 멸균 상태로 제거합니다.</li>
	<li>2% BSA 용액 1mL를 추가 및 제거하여 PBS 용액에서 2% BSA(w/v)로 샘플을 헹굽니다.</li>
	<li>하이드로겔 샘플에 2% BSA/PBS 용액 500μL를 첨가하여 비특이적 단백질 결합을 차단합니다. 그런 다음 샘플을 밤새 어두운 곳에서, 흔들지 않고 RT에서 배양합니다.</li>
	<li>1mL 피펫을 사용하여 차단 용액을 멸균 제거합니다.</li>
	<li>항체를 500μL의 신선한 2% BSA/PBS에 최종 농도 0.112μg/mL로 희석하여 Psl0096-Texas Red 항체 용액을 준비합니다.</li>
	<li>하이드로겔 샘플에 500μL 항체 용액을 추가하고 흔들리지 않고 빛으로부터 보호된 상태에서 실온에서 6시간 동안 배양합니다.</li>
</ol>5. DAPI(4′,6′-diamidino-2-phenylindole) 염색
<ol><li>자기 교반 막대가 들어 있는 플라스크에 비이온 밀도 그래디언트 배지 40g, Tween20 30μL, 아지드화나트륨 3μg, PBS 30mL를 첨가하여 굴절률 매칭 용액(RIMS)을 준비합니다. 용액을 자석 교반기에서 15분 동안 또는 완전히 녹을 때까지 저어줍니다.</li>
	<li>10mL 주사기와 멸균 0.2μm 필터를 사용하여 용액을 50mL 원뿔형 튜브로 필터 멸균합니다. 참고: 용액은 4°C에서 몇 달 동안 보관할 수 있습니다.</li>
	<li>멸균 1mL 피펫으로 Psl0096-Texas Red 항체 용액을 제거합니다.</li>
	<li>하이드로겔 샘플을 헹구고 PBS 1mL를 추가한 다음 제거합니다.</li>
	<li>하이드로겔 샘플을 250μL의 RIMS 용액과 10μg/mL의 DAPI로 RT에서 어둠 속에서 밤새 부드럽게 흔들어 배양합니다.</li>
	<li>컨포칼 이미징 전에 샘플을 0.9mm 또는 1.7mm 관류 챔버에 장착하고 유리 커버슬립으로 밀봉합니다. 참고: FISH 및/또는 면역조직화학 검사 후 RIMS에 담그기 전, 객담 점액의 시각화가 필요한 경우 형광 렉틴 염색을 적용할 수 있다12.</li>
</ol>6. 이미징
<ol><li>표준 기술을 사용하여 25x, 40x, 63x 또는 100x 배율에서 컨포칼 레이저 스캐닝 현미경 이미징을 수행합니다.</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+sputum:+Time+from+expectoration+to+processing.">Induced sputum: Time from expectoration to processing.</a> European Respiratory Journal. 19 (4), 706-708 (2002).</li><li>Chao, Y., Zhang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+fixation+methods+for+observation+of+bacterial+cell+morphology+and+surface+ultrastructures+by+atomic+force+microscopy.">Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy.</a> Applied Microbiology and Biotechnology. 92 (2), 381-392 (2011).</li><li>St-Laurent, J., Boulay, M. E., Prince, P., Bissonnette, E., Boulet, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cell+fixation+methods+of+induced+sputum+specimens:+An+immunocytochemical+analysis.">Comparison of cell fixation methods of induced sputum specimens: An immunocytochemical analysis.</a> Journal of Immunological Methods. 308 (1-2), 36-42 (2006).</li><li>Hogardt, M., 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=Specific+and+rapid+detection+by+fluorescent+situ+hybridization+of+bacteria+in+clinical+samples+obtained+from+cystic+fibrosis+patients.">Specific and rapid detection by fluorescent situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients.</a> Journal of Clinical Microbiology. 38 (2), 818-825 (2000).</li><li>Hoffmann, N., 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=Erratum:+Novel+mouse+model+of+chronic+Pseudomonas+aeruginosa+lung+infection+mimicking+cystic+fibrosis.">Erratum: Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis.</a> Infection and Immunity. 73 (8), 5290 (2005).</li><li>Nair, B., 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=Utility+of+Gram+staining+for+evaluation+of+the+quality+of+cystic+fibrosis+sputum+samples.">Utility of Gram staining for evaluation of the quality of cystic fibrosis sputum samples.</a> Journal of Clinical Microbiology. 40 (8), 2791-2794 (2002).</li><li>Howlin, R. P., 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=Low-Dose+Nitric+Oxide+as+Targeted+Anti-biofilm+Adjunctive+Therapy+to+Treat+Chronic+Pseudomonas+aeruginosa+Infection+in+Cystic+Fibrosis.">Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis.</a> Molecular Therapy. 25 (9), 2104-2116 (2017).</li><li>Pestrak, M. J., 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=Treatment+with+the+Pseudomonas+aeruginosa+glycoside+hydrolase+PslG+combats+wound+infection+by+improving+antibiotic+efficacy+and+host+innate+immune+activity.">Treatment with the Pseudomonas aeruginosa glycoside hydrolase PslG combats wound infection by improving antibiotic efficacy and host innate immune activity.</a> Antimicrobial Agents and Chemotherapy. 63 (6), 00234 (2019).</li></ol>

이 프로토콜의 목적은 CF 환자의 객담에서 녹농균 세포의 현장 조직을 엿볼 수 있도록 하는 것입니다. 객담 샘플은 즉시 고정할 수 없는 경우 처리할 때까지 4°C에서 보관해야 합니다. 객담 내 녹농균(P. aeruginosa) 세포 수는 4°C에서 보관할 때 1시간, 24시간 또는 48시간에 처리하면 크게 변하지 않지만, 25°C에서 24시간 또는 48시간 동안 방치하면 박테리아 성장의 결과로 박테리아 세포 수...

이 연구에서 실험은 소아 낭포성 섬유증(CF) 환자의 객담 내에서 녹농균의 생체 내 구조를 시각화하도록 설계되었습니다. 녹농균 감염은 소아 CF 인구의 30-40%에서 만성화됩니다. 만성 감염이 발생하면 제거가 거의 불가능합니다1. 녹농균은 일반적으로 항균제에 더 취약하기 때문에 만성 감염을 예방하기 위해 항슈도몬 항생제로 치료한다2. 안타깝게도, 모든 녹농균 분리가 항생제 치료 후 폐에서 효과적으로 제거되는 것은 아닙니다. 항생제 실패와 관련된 정확한 메커니즘은 완전히 밝혀지지 않았습니다. 이전 연구에서는 생물막 세포 밀도, 응집 및 다당류 생산의 변화가 항생제 효능에 영향을 미칠 수 있음을 보여주었습니다3. 녹농균(P. aeruginosa)은 펠(Pel), 피슬(Psl), 알긴산(alginate)의 세 가지 세포외 다당류를 생산합니다4. 녹농균(P. aeruginosa)의 대부분의 균주는 각 외다당류를 발현할 수 있는 유전적 능력을 가지고 있지만, 종종 한 종류의 다당류가 주로 발현된다5. 엑소다당류 알긴산은 CF 폐의 만성 감염과 관련이 있으며, 점액성 표현형 6,7을 초래합니다. 다당류 Pel과 Psl은 초기 부착 및 생물막 구조 유지를 돕고 항생제 내성을 부여하는 등 여러 기능을 가지고 있습니다8.
조직의 생체 내 구조를 시각화하는 것을 목표로 하는 방법은 다양한 샘플 유형 9,10,11에 대해 개발되었습니다. 최근에는 CF 환자의 객담 내 생체 내 미생물 군집을 시각화하도록 맞춤화되었습니다12. 객담 내 미생물 군집 식별을 위한 조직 투명화 프로토콜의 최적화는 DePas et al., 201612에 의해 개발되었습니다. Pa ssive CLARITY technique 이후 microbial identification의 약자인 MiPACT라는 용어는 CF 가래11,12의 제거를 위해 만들어졌습니다. 조직 투명화 기법의 경우, 표본을 먼저 고정한 다음 투명하게 렌더링하면서 염색 및 현미경 시각화를 위해 고유한 구조를 그대로 유지합니다11. CF 객담 샘플을 고정하고 투명화하면 생물막 구조, 박테리아 세포 밀도, 다균 결합, 병원체와 숙주 세포 간의 연관성과 관련된 질문에 답할 수 있습니다. 객담 내에 보존된 박테리아를 직접 검사하는 것의 장점은 숙주별 맥락에서 분석하고 시각화할 수 있다는 것입니다. 실험을 위해 실험실에서 임상 분리체의 체외 성장은 매우 유익할 수 있지만, 이러한 방법은 CF 폐 환경을 완전히 재현할 수 없으므로 실험실 결과와 환자 결과 사이에 단절이 발생합니다.
여기에 제시된 방법은 CF 환자나 다른 호흡기 감염 환자의 박테리아를 시각화하기 위해 객담을 고정하고 제거하는 데 사용할 수 있습니다. 본 명세서에 기술된 염색 및 현미경 분석의 특정 유형은 형광 in situ hybridization(FISH), 하이드로겔 내 항-PSL 항체 결합, 그리고 컨포칼 레이저 스캐닝 현미경(CLSM)을 통한 후속 분석이다. 조직 투명화 후 다른 면역조직화학 및 현미경 검사법도 적용할 수 있습니다.

<table>
	<tbody>
		<tr>
			<td>29:1 acrylamide bisacrylamide, 30 % solution</td>
			<td>BioRad</td>
			<td>161-0146</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Chambered Coverglass Nunc Lab-Tek</td>
			<td>ThermoFischer Scientific</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerogen2.5L</td>
			<td>Oxid Inc.</td>
			<td>35108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwell perfusion chambers</td>
			<td>Electron Microscopry Sciences</td>
			<td>70326 -12/-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoDenz</td>
			<td>Sigma</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>Protect RNA Rnase Inhibitor</td>
			<td>Sigma</td>
			<td>R7387</td>
			<td></td>
		</tr>
		<tr>
			<td>PseaerA - GGTAACCGTCCCCCTTGC</td>
			<td>Eurofins</td>
			<td></td>
			<td>Order Details: Product: Modified DNA Oligo; Name: PseaerA; Sequence: [Alexa488]GGTAACCGTCCCCCTTGC; Synthesis: 50 nmol; Purification: HPLC; Ship state: Full yield (dry)</td>
		</tr>
		<tr>
			<td>Psl0096-Texas Red</td>
			<td>Medimmune</td>
			<td></td>
			<td>The Psl0096-Texas red antibodies were a gift kindly provided by Medimmune and the company should be contacted for order inquiries.</td>
		</tr>
		<tr>
			<td>VA-044 Hardener</td>
			<td>Wako</td>
			<td>27776-21-21</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of pseudomonas aeruginosa within the sputum of cystic fibrosis patients

낭포성 섬유증 환자의 폐 내에서 녹농균을 조기에 발견하고 근절하면 만성 감염 발병 가능성을 줄일 수 있습니다. 만성 녹농균 감염의 발병은 폐 기능 저하 및 이환율 증가와 관련이 있습니다. 따라서 소아 환자의 약 10-40%에서 발생하는 항생제 요법으로 녹농균을 근절하지 못하는 이유를 밝히는 데 큰 관심이 있습니다. 녹농균(P. aeruginosa)의 숙주 청소율과 항생제 감수성에 영향을 미칠 수 있는 많은 요인 중 하나는 공간 조직(예: 응집 또는 생물막 형성) 및 다당류 생산의 변화입니다. 따라서 CF 환자의 객담 내에서 녹농균의 현장 특성을 시각화하는 데 관심이 있었습니다. 숙주 세포에 대한 3D 구조를 유지하기 위해 샘플을 하이드로겔 매트릭스에 삽입한 후 객담 샘플에 조직 투명화 기술을 적용했습니다. 조직 투명화 후 형광 라벨과 염료를 추가하여 시각화를 가능하게 했습니다. 박테리아 세포의 시각화를 위해 형광 in situ 교잡화를 수행하고, 외다당류의 시각화를 위해 형광 표지된 항-PSL 항체의 결합을 수행하고, 구조적 통찰력을 얻기 위해 숙주 세포를 염색하기 위한 DAPI 염색을 수행했습니다. 이러한 방법을 통해 공초점 레이저 스캐닝 현미경을 통해 CF 환자의 객담 내 녹농균의 고해상도 이미징을 수행할 수 있었습니다.

실험의 전체 설계는 그림 1 과 그림 2에 요약되어 있습니다. 그림 1 은 객담 처리 및 객담 제거 프로토콜에 대한 요약을 제공합니다. 객담 처리 및 청소에는 최대 17일이 소요될 수 있습니다. 그러나 프로토콜은 중단될 수 있으며 샘플은 PFA 고정 후(2일) 또는 조직 투명화 후(투명 시간에 따라 5-17일) 보관할 수 있습니다. <strong class="xf...

Watch this Scientific Journal Video about Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients at JoVE.com

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

이 프로토콜은 낭포성 섬유증 환자의 객담 내에서 박테리아 세포 및 다당류 합성 위치(Psl) 다당류의 시각화 방법을 제공합니다.

낭포성 섬유증 환자의 객담 내 녹농균의 시각화

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

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Immunology and Infection

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

2.9K 조회수.  Hospital for Sick Children. 이 프로토콜은 낭포성 섬유증 환자의 객담 내에서 박테리아 세포 및 다당류 합성 위치(Psl) 다당류의 시각화 방법을 제공합니다.

비디오: Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

공동 문화 모델 녹농균 Biofilms는 라이브 인간 에어웨이 세포에 들어라

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

연구

면역학 및 감염병학

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

흐름 세포 녹농균 및 Saccharomyces cerevisiae의의 Biofilm

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

의 RNA 격리 녹농균 Murine 위장관을 Colonizing

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

위반 항생제 효능을 테스트하는 인공 가래 매체의 사용<em&gt; 녹농균</em&gt; 낭성 섬유증의 폐 관련성이 높은 조건에서

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

장기 만성<em&gt; 녹농균</em마우스의&gt;기도 감염

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

녹농균 유발 폐 손상 모델

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8 transiently Transgenized 마우스 모델<em&gt; In Vivo</em&gt; 염증 반응의 장기 모니터링

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

주문, 녹 농 균 스트레인 PA14 Transposon 삽입 돌연변이의 비중복 라이브러리의 복제

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

방법 감지 세포 독성 Amyloids 다음 감염의 폐 내 피 세포 녹 농 균 에 의해

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

슈도모나스 녹농균의 작은 식민지 변형의 문화와 알긴산의 정량화

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

낭포성 섬유증 환자의 객담 내 녹농균의 시각화

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