이 작품은 보건 보조금 AI62983 국립 연구소 (AYK), AI22535 (GPB)에 의해 재정 지원되었다.

1. P.의 Murine 모델 녹농균이라는 박테리아의 GI의 콜로니 및 전파 <ol><li> C3H/HeN 마우스 (암, 6-8 wks 노인, 할란)는 공생 식물을 고갈 후 이전 4 설명된대로 PA와 함께 모노가 - 식민지에 경구 항생제로 치료하고 있습니다. </li></ol> 2. Murine Cecal Luminal 내용 수확 <ol><li> 액체 질소 욕조에 청소 스테인리스 박격포 젖어. </li><li> 이산화탄소 질식으로 마우스를 안락사. </li><li> 스티로폼 보드와 95 % 에탄올로 샤워에서 보안 마우스 시체. 흉골에서 회음부로 피부를 통해 중간선에 세로 절개를합니다. 피부와 복강을 노출 복막을 반영합니다. </li><li> 전체 맹장을 잘라 내다. 스테인리스 박격포 이상 포셉과 맹장을 잡아. 해부 가위로 맹장의 양쪽 끝이 싹둑. </li><li> 이게 맹장의 근위 끝에 cecal flushate 버퍼 (10 MM TrisHCl, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산) 200 MM NaCl) 5 개 중 1 ML 가득한 P1000 피펫 팁을 넣습니다. 스테인레스 절구에 cecal flushate 버퍼와 cecal luminal 내용을 플러시. cecal flushate 버퍼 1 ML은 cecal 내용을 모두 꼴이지 충분한 것입니다. Cecal flushate 내용은 박격포와 접촉으로 오는 즉시 정지됩니다. </li><li> 멸균 유봉과 함께 cecal flushate 내용을 분쇄. </li><li> 장소 그라운드 드라이 아이스 / 에탄올 목욕탕에 빠져들 50 ML의 폴리 프로필렌 원뿔 관에 cecal luminal 내용을 냉동. </li><li> 반복 각 추가 마우스 2.7을 통해 2.2 단계. </li><li> -80에서 보관 ° C. </li></ol> 3. 박테리아 RNA 격리 <ol><li> 산성 클로로포름 / 페놀의 따뜻한 1 볼륨 (약 3 ML, 두 cecal luminal 내용의 최종 부피에 따라) (5시 1분, V / V)는 65 parafilm에서 보안 뚜껑과 함께 50 ML 오크 릿지의 원심 튜브에 들어있는 ° C의 물 목욕. </li><li> 5 분 50 ML의 폴리 프로필렌 원뿔 관에 포함된 용해 완충액 0.5 볼륨 (2 % SDS, 16 MM EDTA (에틸렌 다이아 민 테트라 초산) 200 MM NaCl) 6 끓여. </li><li> cecal luminal 내용에 끓는 용해 버퍼를 추가합니다. Homogenize. 정기 vortexing 5 분 동안 끓여. </li><li> 추가 100 ° 65 ° C 산 오크 릿지의 원심 튜브에 클로로포름 / 페놀로 C cecal 내용 / 용해 샘플. parafilm로 밀봉 캡. </li><li> 65 품어 ° C 정기 vortexing (매 2 분) 10 분. </li><li> 15 분 동안 4 명이시 2,500g에서 샘플을 원심 분리기. </li><li> 조심스럽게 새로운 50 ML 오크 릿지의 원심 분리기에 수성 단계 (흰색 인터페이스 중 하나를 피하는) 전송. 수성 단계의 볼륨 cecal 내용, 용해 버퍼, 그리고 산성 클로로포름 / 페놀의 전체 볼륨의 약 50 %됩니다. 산성 클로로포름 / 페놀의 동일한 볼륨을 추가합니다. </li><li> 인감 parafilm와 모자와 높은 속도로 vortexing하여 잘 섞는다. </li><li> 15 분 동안 4 명이시 2,500g에서 샘플을 원심 분리기. </li><li> 수성과 유기농 단계 사이에 보이는 흰 인터페이스가 없습니다 때까지 3.9-3.7 단계를 반복합니다. </li><li> 조심스럽게 새로운 50 ML 오크 릿지의 원심 분리기에 수성 단계를 전송합니다. 클로로포름 / isoamyl 알콜의 동일한 음량을 (24:1, V / V) 추가합니다. </li><li> 밀봉 캡 및 vortexing하여 잘 섞는다. </li><li> 15 분 동안 4 명이시 2,500g에서 샘플을 원심 분리기. 15 ML의 폴리 프로필렌 원뿔 관에 수성 단계 (볼륨이 전체 볼륨의 반응 약 50 % 것입니다)를 제거하고 이소프로판올의 동일한 볼륨을 추가합니다. </li><li> 최소한 2 시간 또는 야간을위한 -20 °의에서 품어. </li><li> 45 분 동안 4 명이시 2,500g에서 샘플을 원심 분리기. 펠렛 같은 겔은 튜브의 아래쪽에 형성됩니다. 뜨는 제거합니다. </li><li> 얼음처럼 차가운 70 % 에탄올 1 ML로 펠렛을 씻으십시오. 와동. 5 분 4 명이시 10,000그램에서에서 샘플을 원심 분리기. </li><li> 뜨는를 제거하고 10 분 동안 상온에서 건조 펠렛을 알려주도록 튜브를 반전. </li><li> RNase없는 물 200 μl의 RNA 펠렛을 Resuspend. </li></ol> 4. DNase 처리 (터보 DNA - 무료, 응용 Biosystems) <ol><li> 10X 터보 DNase 버퍼와 2 μl 터보 DNase 20 μl를 추가하고 부드럽게 섞는다. </li><li> 20 분 동안 37 °에서 알을 품다. </li><li> resuspended DNase의 불활 성화 시약 20 μl를 추가하고 잘 섞는다. </li><li> 때때로 혼합, 실온에서 5 분 품어. </li><li> 1.5 분 동안 상온에서 10,000g에서 원심과 새 microfuge 튜브에 뜨는을 전송하기만하면됩니다. </li><li> 페놀 / 클로로포름 감기 (4 °) 산 중 하나가 볼륨을 추가하고 1 분 vortexing하여 철저히 섞는다. </li><li> 2 분 동안 상온에서 1만2천5백g에서 원심 분리기. </li><li> 새로운 튜브로 수성 단계를 전송하고 클로로포름 / isoamyl 알콜 (49:1 V / V), 와동 1 볼륨을 추가합니다. </li><li> 2 분 동안 상온에서 1만2천5백g에서 원심 분리기. </li><li> 새로운 튜브로 수성 단계 (총 반응 량의 약 50 %) 전송합니다. </li><li> 3M의 아세트산 나트륨의 0.1 볼륨, 산도 5.5 및 추가얼음처럼 차가운 100 % 에탄올 2.5 권. 와동. </li><li> 적어도 2 시간 30 분 -80 °에서 -20 °의에서 품어. </li><li> 4 30 분 1만2천g에서 원심 ° </li><li> 뜨는 제거합니다. </li><li> 4 명이시 10,000g에서 5 분 centrifuging하여 얼음처럼 차가운 70 % 에탄올 1 ML로 펠렛을 씻으십시오. </li><li> 10 분 뜨는 공기 건조를 제거합니다. </li><li> RNase없는 물 100 μl의 펠렛을 Resuspend. resuspend 65 ° 물을 욕조에 장소와 펠렛을 해산해야 할 수도 있습니다. </li><li> DNase 처리의 효능을 테스트하려면, ribosomal 단백질 (또는 기타 참조 유전자)의 증폭에 대한 표준 RT - PCR 반응을 더 RT 컨트롤을 사용하지, 처리 및 비 - 처리 샘플을 수행할 수 있습니다. </li></ol> 5. RNA 정리 단계 (Qiagen, RNeasy 키트) <ol><li> Qiagen RNeasy 미니 수첩 (4 번째 에디션 월 2006)의 페이지 56-57을 참조하시기 바랍니다. 로 표시하십시오. </li></ol> 6. 대표 결과 이 프로토콜을 사용하여 복구할 세균성 총 RNA의 양은 두 cecums에서 약 2-3 μg이다. 회수 RNA는 다음 qPCR, 전사 프로 파일링, 및 RNA Seq 실험을위한 충분한 수량과 품질입니다. RNA의 순도는 정기적으로 260nm/280nm 비율에게 샘플 7를 측정하여 평가, 아직이 방법은 RNA 무결성에 대한 정보를 제공하지 않습니다. 애질런트 Bioanalyzer은 사이징, 부량 및 DNA, RNA, 단백질 및 세포의 품질 관리를위한 microfluidics 기반 플랫폼 및 RNA 무결성 번호 (린) 8로 알려진 RNA 무결성 메트 릭을 사용합니다. 이 프로토콜은 1.7에서 2.0와 린 값 ≥ 7.0까지 280분의 260 비율을 생산과 RNA가 추출. 이 프로토콜에 의해 복구 세균성 RNA의 애질런트 Bioanalyzer 분석의 예제는 그림 1에 표시됩니다. <img src="/files/ftp_upload/3293/3293fig1.jpg" alt="그림 1"/> 그림 1. 애질런트 Bioanalyzer electropherogram과 녹농균 총 RNA 샘플의 젤 같은 이미지가 격리되고 murine cecal 내용에서 회복. 린 8.0.

<ol>
	<li>Bodey, G. P., Bolivar, R., Fainstein, V., Jadeja, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+caused+by+Pseudomonas+aeruginosa.">Infections caused by Pseudomonas aeruginosa.</a> Rev Infect Dis. 5, 279-279 (1983).</li><li>Bertrand, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endemicity,+molecular+diversity+and+colonisation+routes+of+Pseudomonas+aeruginosa+in+intensive+care+units.">Endemicity, molecular diversity and colonisation routes of Pseudomonas aeruginosa in intensive care units.</a> Intensive Care Med. 27, 1263-1268 (2001).</li><li>Koh, A. Y. <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+in+vivo+transcription+profiling+for+identifying+Pseudomonas+aeruginosa+genes+needed+for+gastrointestinal+colonization+and+dissemination.">Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination.</a> PLoS One. 5, e15131-e15131 (2010).</li><li>Koh, A. Y., Priebe, G. P., Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Pseudomonas+aeruginosa+in+a+murine+model+of+gastrointestinal+colonization+and+dissemination+in+neutropenia.">Virulence of Pseudomonas aeruginosa in a murine model of gastrointestinal colonization and dissemination in neutropenia.</a> Infect Immun. 73, 2262-2272 (2005).</li><li>Alexander, R. J., Raicht, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+total+RNA+from+human+stool+samples.">Purification of total RNA from human stool samples.</a> Dig Dis Sci. 43, 2652-2658 (1998).</li><li>Fitzsimons, N. A., Akkermans, A. D., de Vos, W. M., Vaughan, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+gene+expression+detected+in+human+faeces+by+reverse+transcription-PCR.">Bacterial gene expression detected in human faeces by reverse transcription-PCR.</a> J Microbiol Methods. 55, 133-140 (2003).</li><li>Manchester, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+UV+methods+for+measurement+of+protein+and+nucleic+acid+concentrations.">Use of UV methods for measurement of protein and nucleic acid concentrations.</a> Biotechniques. 20, 968-970 (1996).</li><li>Schroeder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 3-3 (2006).</li><li>Turnbaugh, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+core+gut+microbiome+in+obese+and+lean+twins.">A core gut microbiome in obese and lean twins.</a> Nature. 457, 480-484 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

RNA 추출 방법은 여기에서 설명한 것은 murine GI 기관을에서 수확 고품질 녹농균의 총 RNA의 충분한 수량의 복구를 허용합니다. 이 방법은 P.에 국한되지 않고 녹농균이라는 박테리아하며 잠재적으로 다른 세균에 적용할 수 있습니다. 소장에서 충분한 미생물 생물의 복구는 생명체에서 생명체로 크게 달라집니다. 우리 murine 모델, P.에서는 녹농균이라는 박테리아는 일반적으로 5 X 10월 7일에서 5일까지 X 10 8 cfu / g 대변 4 사이의 레벨에서 murine GI 넓이를 colonizes. 회수 cecal 내용은 약 0.5 g, P.의 예상 번호이므로 이 cecums에서 발견한 녹농균이라는 박테리아는 5 X 10월 7일에서 5일까지 X 10 8 cfu 사이입니다. 기타 미생물을 사용하는 경우 그것은 식민지 GI 수치를 확인한 후 cfu의 대상 번호를 복구하는 데 필요한 cecums의 수를 계산하기 위해 신중한 것입니다. 이 특정 murine 모델을 사용하는 경우, PA에 감염되지 않은 항생제 - 처리 생쥐들이 cecal 내용에서 격리 RNA에 대한 같지는 금액이 없다는 것을주의하는 것도 중요하다. 녹농균 위장 식민지와 P.의 pathogenesis을 흉내낸 보급 시도 우리의 murine 모델 암 줄기 세포 이식 환자에서 녹농균이라는 박테리아 bacteremia. 이 환자 인구, 공생 식물은 주로 병원성 미생물 (예 : P.의 녹농균이라는 박테리아와 모노 협회)의 전면에 자라난 그 결과 항생제 또는 chemotherapeutic 치료 (GI 공생 식물의 항생제 고갈 등)에 보조 소진 있으며 면역 억제 후에 후속 보급. 이 murine 모델의 장점과 한계는 이미 이전에 네 해결되었습니다. 현재이 연구의 목적은 GI의 넓이로 봐서에서 미생물 총 RNA를 분리를위한 방법론을 제공하는 것입니다. 이 프로토콜은 쉽게 GI 요로 (즉, 공생 식물의 상호 작용, 염증성 장 질환, 세균 등에 효과)에 미생물의 pathogenesis의 다른 측면을 연구 다른 murine 모델 적용할 수 있습니다. 이 방법의 장점 중 하나는 동결 / 해동, 기계 장애 (박격포 / 유봉, 균질로 pulverizing), 끓고, 화학 용해 (예 : SDS)를 포함하여 여러 용해 단계의 결합입니다. 용해 단계의 무리에도 불구하고, 일부 미생물 (특히 그람 양성 세균 및 곰팡이 / 효모) 추가 기계적 중단을 요구할 수 있습니다. 용해 / 산 페놀 - 클로로포름 부화 (3.5 단계), 구슬의 추가 (박테리아와 효모에 대한 / 또는 0.5-0.7 mm 위해 0.1 ㎜)와 이후의 구슬 - 박동 단계 뜨거운 후, 이러한 생물 9 lyse하기에 충분해야합니다 10. murine이게 맹장, 반복 차가운 acid-phenol/chloroform extractions (단계 3.7-3.9)에서 복구 재료의 복잡한 특성상 절대적으로 더 이상 하류 반응에 대한 허용 RNA의 품질과 무결성을 달성하기 위해서 필요합니다. 3-5 추위 acid-phenol/chloroform extractions 사이에 멸망 수성 및 유기 단계 사이의 흰색 인터페이스를하기 전에해야 할 수 있습니다. 마지막으로, DNase 처리 (4 단계)와 RNeasy 정리 프로토콜 (5 단계) 모두의 조합 훼손 DNA와 비 mRNA 소형 (기들과 tRNA)를 제거 필수적입니다. 이전에 언급한 바와 같이,이 프로토콜을 이용하여 회수 RNA는 이후 전사 프로 파일링 3, qPCR (게시되지 않은), 그리고 RNA Seq (게시되지 않은) 실험을위한 충분한 수량과 품질입니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 박격포와 유봉 </td><td> 어부 </td><td> 12-947-1 </td></tr><tr><td> 균 질기 </td><td> 옴니 </td><td> TM - 125 </td></tr><tr><td> 오크 릿지의 원심 분리기 튜브 (50 ML) </td><td> Nalgene </td><td> 3119-0050 </td></tr><tr><td> 산성 페놀 / 클로로포름 </td><td> Ambion </td><td> AM9720 </td></tr><tr><td> 클로로포름 </td><td> 시그마 - 올드 리치 </td><td> C2432 </td></tr><tr><td> Isoamyl 알코올 </td><td> 시그마 - 올드 리치 </td><td> W205702 </td></tr><tr><td> 이소프로판올 </td><td> 시그마 - 올드 리치 </td><td> 190764 </td></tr><tr><td> 디에틸 pyrocarbonate (DEPC) </td><td> 시그마 - 올드 리치 </td><td> 40,718 </td></tr><tr><td> DNase </td><td> Ambion </td><td> AM2238 </td></tr><tr><td> RNeasy 키트 </td><td> Qiagen </td><td> 74,104 </td></tr><tr><td> 3M의 아세트산 나트륨 </td><td> Ambion </td><td> AM9740 </td></tr><tr><td> 100 % 에탄올 </td><td> 시그마 - 올드 리치 </td><td> E7023 </td></tr></tbody></table>

rna isolation of pseudomonas aeruginosa colonizing the murine gastrointestinal tract

이러한 백혈병 환자, 심한 화상 상처, 또는 장기 이식 1로 손상된 면역 체계와 호스트에 중요한 병적 상태와 사망률에 녹농균 (PA) 감염 결과. PA의 혈류 감염을 개발하기위한 높은 위험 환자에서 위장 (GI) 넓이는 식민지 2 주 저수지이지만, 메커니즘하는 asymptomatic colonizing 미생물의 침략, 그리고 종종 치명적인 위해 PA 전환은, 병원체가 명확하지 않습니다. 이전, 우리는 숙주의 면역 상태를 변경하는 동안 세균의 유전자 표현의 변화를 파악하기 위해 쥐를 3 식민지의 cecums에 거주하는 세균 세포에서 PA의 mRNA를 복구하여 생체내 전사 프로 파일링 실험에서 수행했습니다. 모든 전사 프로 파일링 실험과 마찬가지로 속도 - 제한 단계는 고품질의 mRNA의 충분한 양을의 격리에 있습니다. 효소, 파편, 음식 잔류물 및 GI 기관을의 미립자 물질의 풍부한 감안할 때, RNA 분리의 도전은 어려운 것입니다. 여기, 우리는 murine GI 기관을에서 발견한 박테리아 RNA의 신뢰성과 재현성 절연을위한 방법을 제시한다. 이 방법은 PA GI의 식민지와 neutropenia - 유도 보급 4 잘 설립 murine 모델을 활용합니다. PA와 함께 일단 GI 식민지가 확인, 마우스는 euthanized와 cecal 내용 회복과 플래시 냉동입니다. RNA는 다음 기계적 끓고 혼란, 페놀 / 클로로포름 extractions, DNase 처리 및 친화성 크로마 토그래피의 조합을 사용하여 추출됩니다. 수량과 순도​​는 분광 광도법 (Nanodrop 기술)와 bioanalyzer (애질런트 테크놀로지) (그림 1)에 의해 확인됩니다. GI의 미생물 RNA 격리의이 방법은 쉽게뿐만 아니라 다른 세균 및 곰팡이에 적용할 수 있습니다.

Watch this Scientific Journal Video about RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract at JoVE.com

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

의 RNA 격리에 대한 신뢰할 수있는 방법<em&gt; 녹농균</em&gt; murine cecums에서 발견한 것은 설명되어 있습니다. 회수 RNA는 다음 qPCR, 전사 프로 파일링, 및 RNA Seq 실험을위한 충분한 수량과 품질입니다. 이 기술은 다른 장내 미생물의 RNA 분리에 대해 적용할 수 있습니다.

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 ...

rna-isolation-pseudomonas-aeruginosa-colonizing-murine

Research

JoVE Journal

Immunology and Infection

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

19.0K 조회수.  University of Texas Southwestern Medical Center. 의 RNA 격리에 대한 신뢰할 수있는 방법 녹농균 murine cecums에서 발견한 것은 설명되어 있습니다. 회수 RNA는 다음 qPCR, 전사 프로 파일링, 및 RNA Seq 실험을위한 충분한 수량과 품질입니다. 이 기술은 다른 장내 미생물의 RNA 분리에 대해 적용할 수 있습니다.

비디오: RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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 ...

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 ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

마우스 성기 요로 점막에서 림프구의 분리

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

라이트 사이드에와 서: Vivo에서 당뇨병 털 Murine 모델에 만성 상처에서 녹 농 균 Biofilm 감염의 모니터링

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

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 ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

시투 혼성화에서 형광을 사용하여 뮤린 위장에서 칸디다 알비칸스의 시각화

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...